Exam 1 - Chapters 9 and 12

type I kinase inhibitors

ATP-competitive compounds bind in the ATP binding site and H-bond with the hinge region conserved Asp-Phe-Gly (DFG) motif implicated in ATP binding at the N-terminus of the A-loop

glycogen synthase kinase 3 - GSK3

de-phosphorylation of GSK-P activates the kinase phosphorylation of an internal P-Ser residue of GSK3 allows it to occupy the P-Ser binding site, blocking substrate binding in the de-phosporylated active state of GSK3, the internal P-Ser of GSK3 is available to bind the P-Ser of the glycogen synthase substrate, positioning the kinase to phosphorylate neighboring Ser residues in the substrate brings other Ser residues to the active site for phosphorylation

an image of a very small part of one cycle of a454 sequencing run

dNTP added - A, G, T, or C DNA sequences at circled clusters duplicate dNTP incorporation results in 2x brighter flash very slow to do one by one - each dot represents one fragment that needs to be sequenced (doing hundreds at a time)

STAT activation is transient - shut down by multiple regulatory mechanisms

protein inhibitors of activated STATs (PIAS) suppressors of cytokine signaling (SOCS) genes associated with retinoid IFN induced mortality (GRIM) de-phosphorylation by membrane, cytoplasmic, and nuclear phosphatases

phosphodiesterase (PDEs) enzymes

regulate both [cGMP] and [cAMP] cGMP-selective PDE5 activated by cGMP binding and PKG phosphorylation sGC-generated cGMP augments and reduces cellular [cAMP] by inhibiting PDE3 and activating PDE2, respectively inhibiting PDE5 augments [cGMP] from this pool

nuclear receptors and gene expression

regulate gene expression 1. serum binding protein with bound hormone carried to target tissue across plasma membrane and binds to specific receptor in the nucleus 2. binding changes conformation of receptor - homo- or heterodimers with other H-R complexes and binds to hormone response elements (HREs) in the DNA adjacent to specific genes 3. binding regulates transcription of adjacent genes - increases or decreases rate of mRNA formation 4. altered levels of hormone-regulated gene product produce the cellular response to the hormone

neuronal action potentials

regulated by voltage gated ion channels neuronal action potential states: 1. resting state - cytosolic face negatively charged - non gated K+ channel partly open and Na+ channels closed 2. depolarized state - cytosolic face positively charged - voltage-gated K+ channel open and Na+ channels open resting membrane potential (RMP) -> local potential change (graded potential) -> depolarization (opening of voltage gated Na+ channels) -> repolarization (closure of Na+ and opening of K+, voltage gated channels) -> hyper polarization (voltage gated K+ channels remain open after the potential reaches resting level)

protein kinase C activation

regulates numerous downstream pathways in the heart: downstream targets, enzymes, transcription factors, and proteins cellular effects and responses are different for each category phosphorylation of enzymes, transcription factors, and proteins

role of senescence in cancer development and treatment - replicative senescence

replicative senescence (RS) caused by dysfunctional telomeres - telomere erosion or shortening telomerase (hTERT), or alternative lengthening of telomeres (ALT), can prevent telomere erosion or telomere shortening in cancer cells and subsequent induction of cellular senescence telomere shortening and uncapping activates DNA-damage response pathways, inducing apoptosis and senescence since only immortalized and cancer cells have elevated levels of hTERT, it is a target for anti-cancer drugs regulating homeostasis of normal cells - vulnerabilities for cancer cells

mammalian expression vector

replicons - an origin of DNA replication and its associated control elements polycloning site (PCS) - an artificially synthesized nucleotide sequence incorporated in a plasmid that contains multiple cleavage sites for different restriction enzymes antibiotics resistance gene: 1. ampicillin 2. kanamycin-human gene therapy promoter: 1. viral promoter - CMV, cytomegalovirus 2. tissue-specific promoter - albumin gene of your interest: 1. therapeutic gene 2. reporter gene - testing delivery system, then clone the therapeutic gene in the plasmid use of a plasmid - difference in the promoter that is recognized by the mammalian tissue specific - in vivo don't use a complicated gene model

restriction endonucleases

restriction enzymes recognize specific base sequences in double-stranded DNA and cleave both strands of the duplex at specific places characteristics of restriction enzymes: 1. cut DNA sequence-specifically 2. bacterial enzymes - hundreds are purified and available commercially 3. restriction-modification system - bacteria have enzymes that will cleave foreign DNA; hence, "restrict" the entry of viral DNA (to prevent the bacteria's own DNA from being cut, there is a second enzyme that methylates the same sites recognized by the restriction enzyme (modifies that site)) 4. named for bacterial genus, species, strain, and type - example: EcoRI

cloning that involves the use of one restriction enzyme

restriction site - GAATTC (palindromic) 1. restriction enzyme cuts sugar-phosphate backbones and creates sticky ends 2. DNA fragment added from another molecule cut by the same enzyme - base pairing occurs with one possible combination 3. DNA ligase seals the strands - forms recombinant DNA molecule the molecule added to the sticky ends is the gene to be cloned cut with the same enzyme = complementary sequences - all three portions able to combine into one fragment

progesteroner receptor clinical applications

short term and long term uses non-gynecological applications may be used for contraception, endometriosis, pregnancy termination, breast cancer, and more

cloning that involves the use of two restriction enzymes

two different sticky ends are created - two different complementary areas for the gene of interest to complementary base pair too control the orientation of the gene being cloned

interferon receptor signaling

type I interferons (IFNs) bind common receptor on surface of human cells - type I IFN receptor composed of subunits IFNAR1 and IFNAR2, associated with Janus activated kinases (JAKs), tyrosine kinase 2 (TYK2), and JAK1 respectively type II IFN, IFN-γ, binds a distinct type II IFN receptor also composed of two subunits, IFNGR1 and IFNGR2, associated with JAK1 and JAK2, respectively activation of the JAKs associated with type I IFN receptor results in tyrosine phosphorylation of STAT2 and STAT1, leading to formation of STAT1-STAT2-IRF9 (IFN-regulatory factor 9) complexes - ISGF3 (IFN-stimulated gene factor 3 complexes ISGF3 complexes translocate to the nucleus and bind to IFN-stimulated response elements (ISREs) in DNA to initiate the transcription of interferon stimulated genes (ISGs) both type I and type II IFNs also induce formation of STAT1 - STAT1 homodimers that translocate to the nucleus and bind GAS (IFN-γ-activated site) elements in the promoters of ISGs, thereby initiating the transcription of these genes consensus GAS element and ISRE sequences are shown, N, any nucleotide

kinase inhibitor types

type I, type II, and type III inhibitors

cancer cells acquire limitless replicative potential

uncoupling cell's growth program from signals present in its environment: 1. growth signal autonomy 2. insensitivity to antigrowth signals 3. resistance to apoptosis cell-autonomous program limits multiplication: 1. normal cells in culture - finite replicative potential 2. progress to senescence and eventually crisis tumor cells in culture appear immortalized

regulated expression of RecA protein in the bacterial cell

uninduced versus induced induced - RecA grouped around 40,000 bp new band that is enriched - gene of interest

activation of cyclin-dependent protein kinases (CDKs) by cyclins and phosphorylation

when cyclin binds, conformational changes move the T loop away from the active site and reorient an amino-terminal helix, bringing a residue critical to catalysis (Glu51) into the active site phosphorylation of a Threonine residue in the T loop produces a negatively charged residue that is stabilized by interaction with three Arg residues, holding CDK in its active conformation

signal transduction sensitivity: amplification

when enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade signal -> enzyme 1 -> enzyme 2 -> enzyme 3 example: one messenger binds to one receptor -> several G proteins are activated -> each G protein activated an adenylate cyclase -> each adenylate cyclase generates hundreds of cAMP molecules -> each cAMP activates a protein kinase A -> each protein kinase A phosphorylates hundreds of proteins total number of product: 1 -> 10 -> 10 -> 5000 -> 5000 -> 2,500,000 - one messenger molecule leads to phosphorylation of millions of proteins downstream signal transduction cascades serve to amplify responses

using genomics and bioinformatics to study a human genetic disorder

where is the gene located? what is the normal function of the protein encoded by this gene? how does the mutant gene or protein produce the disease phenotype?

Moore's law

year versus cost per genome this project was started about 20 years ago, and it was VERY expensive - typically used for research purposes and the very wealthy (hard to apply clinically due to cost) dramatic price drop since then, so it is much more widely available clinically now

ubiquitin proteasome system (UPS) inhibitors

several drugs targeting the proteasome and components of the UPS currently in clinical and pre-clinical development - ubiquitin activating (E1), conjugating (E2), and ligase (E3) enzymes three enzymes involved - E1, E2, and E3 therapeutic indications

biological membranes contain proteins

1. receptors 2. adhesion molecules 3. transporters 4. ion channels

site-directed mutagenesis

1. recombinant vector - cleave with restriction endonuclease 2. mutation - insert synthetic DNA segment containing mutation synthesize in vitro to introduce a mutation - very difficult

cell cycle and DNA damage checkpoints and cancer

1. response to DNA damage/replication-fork arrest: - DNA damage directly activates ATM and replication-fork arrest activates ATR - ATM and ATR kinase signaling proteins and substrates determine the cell outcome - any genomic instability that ensues can contribute to cellular transformation to cancer 2. mutations leading to cancer development: - if DNA damage is repaired efficiently, the likelihood of tumor development is low - mutations in DNA-damage signaling (sporadic/inherited) leads to more genomic abnormalities - cells with damaged DNA that arrest or do not survive reduces probability of progression to malignancy mutations in apoptosis pathways, DNA-damage, DNA-repair or mitotic-checkpoint pathways permit survival or continued growth of cells with genomic abnormalities, increasing the odds of malignant transformation

three features of GPCR's

1. seven transmembrane domains 2. heterotrimeric GTP-binding protein 3. enzyme or ion channel activation to generate downstream second messengers

coincidence detectors

1. single protein requires phosphorylation on two different residues by two independent signaling pathways to become activated 2. two proteins phosphorylated by two different signaling pathways associate together to form an active signaling complex

uses of DNA-based information technologies

1. studying genes and their products 2. using DNA-based methods to understand protein function 3. genomics and the human story

enzymes used in recombinant DNA technology and their function

1. type II restriction endonucleases - cleave DNAs at specific base sequences (breaks the phosphodiester linkage) 2. DNA ligase - joins two DNA molecules or fragments 3. DNA polymerase I (E. coli) - fills gaps in duplexes by stepwise addition of nucleotides to 3' ends 4. reverse transcriptase - makes a DNA copy of an RNA molecule 5. polynucleotide kinase - adds a phosphate to the 5'-OH end of a polynucleotide to label it or permit ligation 6. terminal transferase - adds homopolymer tails to the 3'-OH ends of a linear duplex 7. exonuclease III - removes nucleotide residues from the 3' ends of a DNA strand 8. bacteriophage exonuclease - removes nucleotides from the 5' ends of a duplex to expose single-stranded 3' ends 9. alkaline phosphatase - removes terminal phosphates from either the 5' or 3' end (or both)

integrin family

24 αβ chain integrin heterodimers 18 α and 10 β subunits multiple extracellular domains α subunits define ligand binding resting state - bent, closed conformation, low affinity for ligand activated state - open head domain in a high-affinity state activation leads to talin binding and tensin, filamin, paxillin and FAK binding

phosphatases regulate kinase signaling

285 phosphatase genes phosphatase classification: 1. PTP - protein-tyrosine phosphatase 2. PPP serine/threonine-specific protein phosphatase 3. PPM - protein phosphatase 2C-like 4. HAD - haloacid dehalogenase-like 5. LP phosphatidic acid phosphatase, inositol monophosphatase and inositol polyphosphate-related phosphatase 6. NUDT NUDIX hydrolase kinase and phosphatase - K adds phosphorylation, P de-phosphorylates

receptors

3 major sub-families - Class A (Rhodopsin-like), Class B (Secretin-like), and Class C (Glutamate Receptor-like) homo- and hetero-dimerization GPCRs and other oligomers

nuclear receptors (NRs)

3 x NR sub-groups based on physiologic ligands and potential functions - endocrine, adopted orphan, and true orphans

G-protein coupled receptors - GPCRs

791 seven transmembrane receptor (7TM-R/GPCR) genes Class A* (Rhodopsin-like), Class B* (Secretin-like), Class C* (Glutamate Receptor-like), and others* (Adhesion(33), Frizzled (11), Taste type-2 (25), unclassified (23))

ion channels

315 genes in the human genome voltage-gated and ligand-gated (extracellular ligand) ion channel super families ATPases pump specific ions to create electrochemical gradients orders of magnitude higher transmembrane rate exhibit some degree of ion specificity not saturable direction of passage dictated by ion's charge and/or the electrochemical gradient voltage-gated - normal membrane potential -> depolarization and opening of the channels

the constructed E. coli plasmid pBR322

4,361 bp ampicillin resistance (Amp^r) - PstI EcoRI BamHI and SalI - tetracycline resistance (Tet^r) PvuII origin of replication (ori) Amp^r and Tet^r produce the enzymes that destroy ampicillin and tetracycline, respectively - selectively grow bacteria that contain this resistance very important implication in antibiotic resistance!

transporters

443 genes in the human genome two major transporter super family's - solute carriers (SLC) and ATP binding cassette (ABC) transporters high specificity substrate binding - Na+, K+, Cl-, Ca2+, etc. transport rate < free diffusion exhibit saturation, like enzymes and receptors

recognition sequences for some type II restriction endonucleases

5'-> 3' direction *indicates the position of the cleaved phosphodiester bond all sequences are palindromic BamHI - G*GATCC ClaI - AT*CGAT EcoRI - G*AATTC EcoRV - GAT*ATC HaeIII - GG*CC HindIII - A*AGCTT NotI - GC*GGCCGC PstI - CTGCA*G PvuII - CAG*CTG Tth111I - GACN*NNGTC (N = any base) appearance frequency = 4^n and can appear multiple times in the DNA sequence

termination of the β-adrenergic signal

1. circulating [epinephrine] concentrations fall below the threshold required for receptor occupancy 2. self inactivation of Gs 3. cAMP degraded by intracellular phosphodiesterase enzymes (PDEs) 4. receptor desensitization by β-arrestin mediated internalization - receptor-arrestin complex enters cell by endocytosis, and arrestin dissociates (receptor returns to cell surface after dephosphorylation)

human kinome

518 putative protein kinase genes - 7 major kinase groups: 1. AGC - PKA, PKG, PKC 2. CAMK - calcium/calmodulin-dependent 3. CK1 - casein kinase 1 4. CMGC CDK, MAPK, GSK3, CLK 5. STE homologs of yeast sterile kinases 6. TK - tyrosine kinases 7. TKL - tyrosine kinase-like 244 kinases map to disease loci or cancer amplicons structural organization of the kinome

schematic illustration of DNA cloning

1. cloning vector is cleaved with restriction endonuclease - plasmid 2. DNA fragment of interest is obtained by cleaving the chromosome with a restriction endonuclease - eukaryotic chromosome 3. fragments are ligated to the prepared cloning vector - DNA ligase 4. DNA is introduced into the host cell - recombinant vector and host cell 5. propagation (cloning) of transformed cell produces many copies of recombinant DNA isolation of the gene using endonuclease - must find one or two enzymes that cut the gene of interest and the cloning vector to form a complementary sequence DNA ligase repairs the phosphodiester linkage bacterial amplification lyse bacteria to release the cloned DNA

ligand-receptor binding complementarity - contributions to pharmacological drug responses

1. confers affinity - directly impacts drug dosing (higher affinity = lower drug dosing) 2. specificity or selectivity of target engagement - influences potential for off-target toxicity and adverse events 3. functional response and/or efficacy - agonist - partial or full - antagonist - inverse agonist - partial or full

introduction of nucleic acids into bacteria

1. conjugation - pilus (from one bacteria to another) 2. transduction - mediated by virus (into bacteria through a virus - thick cell wall) 3. transformation: - physical or chemical means to transiently increase the permeability of bacterial wall - competent cells - electroporation - mediated by electric current

strategies for altering gene sequences to generate altered proteins

1. deletion - truncated proteins (determine if the domain is the motif responsible for function - beneficial or harmful) we can minimize harmful motifs to increase therapeutic benefit/minimize adverse effects 2. fusion - fusion proteins 3. mutagenesis: - site-directed mutagenesis - oligonucleotide-directed mutagenesis

family of RTKs

58 RTKs sub-divided into 20 subfamilies sub-domains of extracellular region indicated

STAT phosphorylation and activation

1. STAT tyrosine phosphorylation RTKs, JAKs, Src, and Tec tyrosine kinases - transcriptional activation 2. STAT serine phosphorylation - JNK and p38 MAPKs, PKC, and PKA - enhances phosphorylation 3. STAT activation is transient - shut down by multiple regulatory mechanisms

effects of post-translational modifications on protein function

1. activate an enzyme activity 2. inactivate an enzyme activity 3. produce a conformation change - create domain docking sites for protein-protein or protein-lipid interactions, etc. - expose or obscure signaling sequences that target proteins to specific sub-cellular compartments 4. target proteins for proteasome degradation

main applications of multiple sequence alignments

1. extrapolation - a good multiple alignment can help convincing you that an uncharacterized sequence is really a member of a protein family 2. phylogenetic analysis - if you carefully chose the sequences to include in your multiple alignment, you can reconstruct the history of these proteins 3. pattern identification - by discovering very conserved positions, you can identify a region that is characteristic of a function in proteins or in nucleic acid sequences 4. domain identification - it is possible to turn a multiple sequence alignment into a profile that describes a protein family or a protein domain; you can use this profile to scan databases for new members of the family 5. DNA regulatory elements - you can turn a DNA multiple alignment of a binding site into a weight matrix and scan other DNA sequences for potential similar binding sites 6. structure prediction - a good multiple alignment can give you an almost perfect prediction of your protein secondary structure for proteins or RNA; sometimes it can also help building a 3-D model 7. PCR analysis - a good multiple alignment can help you in identifying the less degenerated portions of a protein family 8. nsSNP - identify the nsSNP that are the most likely to alter the function gives you a good clue or guidance of what the next step will be - NEVER definitive in protein function, only a prediction

amplification of a DNA fragment by PCR - method 2

1. heat to separate strands 2. anneal primers containing non complementary regions with cleavage site for restriction endonuclease - primers that bind to ends of DNA template that are complementary (must know the sequence) annealing - binding primer 3. replication 4. PCR 5. EcoRI endonuclease 6. clone by insertion at an EcoRI site in a cloning vector additional sequence incorporated into DNA - cloning

studying genes and their products - questions we can answer

1. how to clone a gene (molecular cloning)? 2. how to alter a gene sequence? 3. how to express a gene? 4. how to purify the gene products (proteins)? 5. how to amplify a gene? foundation for other technologies

methods to study protein-protein interactions

1. immunoprecipitation 2. yeast two-hybrid analysis determining which proteins interact with other proteins

introduction of nucleic acids into mammalian cells

1. infection/transduction - viral (mimicking the natural process of the virus) 2. transfection - non-viral

receptor-ligand binding and complementarity

1. ligand 2. protein-ligand binding sites interaction is specific or may be considered selective

factors influencing specificity and selectivity of GPCR signaling

1. ligands - orthosteric agonists, antagonists, and inverse agonists, and allosteric modulators 2. receptors 3. heterotrimeric G-proteins - alpha, beta, and gamma subunits 4. differential receptor expression - cells, tissues, and organs

DNA transformation - chemical method

1. log phase E. coli culture - centrifuge 2. resuspend bacterial pellet in CaCl2 solution 3. chill on ice 4. aliquot competent cells 5. store at -80 degrees C 6. amp^r plasmid DNA 7. chill on ice 8. heat shock - 42 degrees C H2O bath 9. plate on LB + ampicillin - 10^6 to 10^8 amp^r colonies/mcg DNA increasing mobility of the cell wall - different methods used

DNA transformation - electroporation

1. log phase E. coli culture - centrifuge 2. resuspend bacterial pellet in sterile H2O 3. chill on ice 4. centrifuge 5. resuspend bacterial pellet in sterile H2O 6. use competent cells immediately 7. amp^r plasmid DNA - combine with competent cells 8. electroshock 9. transfer to saline buffer 10. plate on LB + ampicillin - 10^8 to 10^10 amp^r colonies/mcg DNA physical method - electrical current

building a cDNA library from mRNA

1. mRNA template is annealed to synthetic oligonucleotide (oligo-dT) primer 2. reverse transcriptase and dNTPs yield a complementary DNA strand - forms an mRNA-DNA hybrid 3. mRNA is degraded with alkali - only DNA remains 4. to prime synthesis of a second strand, an oligonucleotide of known sequence is often ligated to the 3' end of the cDNA 5. DNA polymerase I and dNTPs extend the primer to yield double-stranded DNA same genomic DNA, different gene expression leads to differences in humans interested in what genes are expressed - mRNA isolate all mRNA - poly-A (mRNA) and poly-T (oligonucleotide) sequences that are used to generate dsDNA amplifying library

key components in plasmids

1. origin of replication - two different plasmids cannot function in the same cell if they use the same origin of replication (incompatible) 2. genes that confer resistance to an antibiotics - selectable markers 3. polycloning site (PCS) - does not appear naturally in the plasmid 4. screenable markers (optional) - a reporter gene with a built-in cloning site reside in the bacteria and generate enzymes that increase resistance

oligonucleotide-directed mutagenesis - stepwise process

1. plasmid with gene and target site formation - denature and anneal oligonucleotide primers with mutation (boiling water and cool down for primers to bind) 2. use DNA polymerase to extend and incorporate the mutagenic primers - DNA polymerase -> new dsDNA 3. digest non mutated parental DNA template with methylation-specific nuclease and anneal newly synthesized strands - digest original DNA 4. mutated plasmid with nicked strands - transform dsDNA into cells, cell repairs nicks in mutated plasmid (add new primer to synthesize more new DNA with the mutation) primers are short sequences that are complementary to the target sequence - can still bind if one to two base pairs are mismatched

double stranded DNA

A, C, T, and G - complementary base pairing connected by phosphodiester bonds

Which of the following protein expression systems is least effective in posttranslational modification?

A. E. Coli B. yeast C. insect D. mammalian cells the answer is A

Which of the following libraries would be expected to be essentially the same?

A. genomic libraries made from mouse liver and kidney cells B. cDNA libraries made from mouse liver and kidney cells C. genomic and cDNA libraries made from mouse liver cells the answer is A - mRNA

In order to insert a foreign gene into a plasmid, both must _________

A. have identical DNA sequences B. originate from the same type of cell C. be cut by the same restriction enzyme(s) D. be of the same length the answer is C

In a yeast two-hybrid system, expression of the reporter gene indicates that:

A. one gene regulates the expression of the other gene in the hybrid B. expression of a gene requires the presence of a binding protein C. two proteins have interacted to produce a specific cellular outcome D. both genes in the hybrid are part of an operon the answer is C?

selective androgen receptor modulators (SARMs)

AR - critical role in function of several organs - primary and accessory sexual organs, skeletal muscle, and bone demonstrate osteo- and myo-anabolic activity unlike testosterone or other anabolic steroids, SARMs produce less growth in prostate or secondary sexual organs SARMs - therapeutic opportunities for a variety of diseases: 1. muscle wasting associated with burns 2. cancer 3. end-stage renal disease 4. osteoporosis 5. frailty 6,. hypogonadism

bacterial artificial chromosomes (BACs)

BACs have stable origins of replication that maintain the plasmid at one or two copies per cell BACs include par genes, which encode proteins that direct the reliable distribution of the recombinant chromosomes to daughter cells at cell division allow cloning of very long segments - 100,000~300,000 bp homologous recombination

BCL-2 and inhibitors of apoptosis proteins

BCL-2 proteins mediate the delicate balance between cell survival and death disruption of this balance by cellular alterations that increase the functional activity of anti-apoptotic BCL-2 proteins relative to pro-apoptotic BCL-2 proteins enables the evasion of apoptosis and favors cell survival, promoting the development and progression of cancer inhibitors of apoptosis (IAP) proteins, XIAP, and cIAPs inhibit apoptosis and promote cell survival XIAP BIR2 domain and N-terminal residues in its linker region mediate binding and inhibition of caspases 3 and 7 caspase-9 is inactivated by binding to the XIAP BIR3 domain XIAP also promotes cell survival by regulating important signaling pathways, such as NF-κB activation IAP-binding motif (IBM)-containing proteins like SMAC interact with XIAP BIR2 and BIR3 domains to neutralize its anti-apoptotic activity

intrinsic mitochondrial pathway of apoptosis activation

Bcl-2 family members mediate and control mitochondrial outer membrane permeabilization (MOMP), and cell death is caused by release of apoptotic soluble intramembrane mitochondrial proteins (SIMPs, cytochrome C) or loss of essential mitochondrial functions Bcl-2 family members contain ≥ 1 to 4 relatively conserved Bcl-2 homology (BH) domains are divided into 3 groups anti-apoptotic Bcl-2, Bcl-Xl, Bcl-w, and Mcl-1 proteins promote cell survival, are localized in mitochondria, endoplasmic reticulum, and perinuclear membrane - rescue cells without altering proliferation Bcl-2 proteins preserve mitochondrial membrane status and balance interactions with pro-apoptotic members by sequestering or neutralizing pro-apoptotic BH123 molecules pro-apoptotic "multi-BH domain" (BH123) proteins Bax, Bak, and Bok share three domains (BH1, BH2, and BH3) with Bcl-2 - pro-apoptotic BH3-only proteins, Bid, Bad, and Bim have a short BH3 interaction domain required and sufficient to induce apoptosis both pro-apoptotic groups are required for launching apoptosis BH3-only proteins trigger apoptosis induced by developmental cues, insufficient trophic support, and intracellular damage - damage sensing BH3-only proteins are upstream of BH123 proteins BH3-only and other proteins induce oligomerization of BH123 proteins (Bax and Bak) to insert into the outer mitochondrial membrane and engage either permeability transition PT-dependent or PT-independent MOMP death signals impinge directly or indirectly on mitochondria, inducing the formation of the apoptosome complex promoting the proteolytic maturation of caspase-9, which cleaves and activates effector caspases (caspase-3, -6, and -7) producing apoptosis

cell adhesion molecules (CAMs) and cellular junctions

CAMs participate in cell-cell contacts and cell-extracellular matrix contacts control movement of ions and solutes through and between cells, polarity cell-cell adhesive structures attached to actin and microtubules strong cell-cell attachment mediated by Cadherin interactions with intermediate filaments cells adhere to ECM secreted proteins (fibronectin, collagen, and laminin) via multiprotein receptor complexes connected to the actin, microtubule, keratin, and vimentin cytoskeleton integrins ~ adhesive proteins connected to the cytoskeleton that interact with >150 signaling proteins to alter pathways controlling proliferation, survival, shape, mobility, and gene expression affect the size and shape of the cell

drug discovery and development: CB1-R potentiators, agonists, and antagonists

CB1 stimulation: 1. anxiety 2. pain 3. appetite stimulation 4. suppression of nausea and emesis 5. glaucoma 6. bronchial spasm 7. muscle spasms in MS and spinal injury CB1 inhibition: 1. cognitive enhancement 2. psychosis 3. appetite suppression

cell division cycle 25 (CDC25) phosphatases regulate CDK-cyclin mediated cell cycle progression

CDC25A, B, and C de-phosphorylate CDK-cyclin complexes at all stages of cell cycle CDC25B responsible for activation of CDK1-cyclin B at centrosome contributing to microtubule network reorganization and mitotic spindle assembly nuclear translocation leads to an auto- amplification process of CDC25s that then fire the bulk of CDK1-cyclin B complexes and trigger mitosis

CDK activating kinases (CAKs) and cell division cycle 25 (CDC25) - phosphatases regulate CDK-cyclin mediated cell cycle progression

CDK activity positively regulated by association with cyclins and by phosphorylation of T-loop threonine by CDK activating kinase(CAK) inhibitory phosphorylation of adjacent threonine and tyrosine residues is mediated by dual specific kinases (Wee1 and MYT1) inhibition is released after cell division cycle 25 (CDC25) phosphatases dephosphorylate these residues

increased Ca2+ mobilization

Ca2+ transients measured by the Ca2+ sensitive dye Fura-2 Ca2+ mobilization regulates numerous downstream signaling pathways through the regulation of Ca2+ dependent enzymes, kinases, and ion channels

DNA damage checkpoints

DNA damage checkpoints represent a network of surveillance systems signal transduction systems that interrupt cell cycle progression when genome damage or failure of a previous cell cycle activity has been detected DNA damage checkpoints are associated with biochemical pathways that end, delay, or arrest cell cycle progression checkpoints engage damage sensor proteins in the detection of DNA damage and transduction of signals to Ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), Chk1 and Chk2 kinases Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks), thereby arresting cell-cycle progression

DNA base pairing forms the genetic basis of replication

DNA genetic information coded in the sequence of nucleotides complementary anti-parallel strands of DNA follow base pairing rules A-T and G-C base-paired anti-parallel strands differ in base composition and sequence when read 5' to 3' two polymeric stands form the DNA double helix - deoxynucleotides pair specifically with complimentary deoxynucleotides of opposite strand before cells divide, DNA strands separate and serve as templates for synthesis of new complementary strands, generating two identical double-helical molecules, one for each daughter cell

how to define changes in gene expression patterns

DNA microarrays

immunostaining of DNA polymerase ε, PCNA, and BrdU, and their co-localization

DNA polymerase ε - red PCNA - green BrdU - blue merge of DNA polymerase ε and PCNA - orange/yellow label each antibody with a different fluorescence

DNA to proteins

DNA sequence of genes transcribed into mRNA that is in turn translated into the amino acid sequences of proteins DNA stores the genetic information in the sequence of nucleotides from which all other cellular components are generated 1. DNA sequence of genes transcribed into mRNA sequence 2. mRNA translated into an unfolded polypeptide of linked amino acids 3. folding produces the secondary and tertiary structures required for enzymatic activity/function 4. protein-protein interactions lead to the formation of supramolecular complexes

polymerase chain reaction (PCR)

DNA template a pair of synthetic oligonucleotide primers deoxynucleoside triphosphates (dNTPs) - building blocks of DNA DNA polymerase - heat resistant repeated cycles: 1. denaturing 2. annealing 3. extension automated heating and cooling system allows for DNA amplification - finished amplification in one to two hours

effects of oncogenic RAS on energy metabolism

ERK and PI3K signaling downstream of RAS converge to activate mTOR by inhibiting its negative regulators TSC2, LKB1, and AMPK - TSC2 can be directly phosphorylated by ERK, RSK, and AKT RAF-ERK1/ERK2 signaling disrupts the LKB1-AMPK checkpoint, leading to mTOR-eIF4-dependent translation of hypoxia-inducible factor 1α (HIF1α) - activated RAS also up-regulates HIF1A HIF1α augments multiple steps in glycolytic metabolism - hexokinase upregulation enhances glucose conversion to glucose-6- phosphate used in pentose phosphate pathway-dependent nucleotide synthesis higher levels of phosphofructokinase (PFK) enhance glycolytic flux and pyruvate production, which combined with RAS-dependent increases in lactose dehydrogenase levels, allow glycolysis to persist by regenerating the NAD+ cofactor for glycolytic reactions in addition, pyruvate can enter the tricarboxylic acid (TCA) cycle where its conversion to citrate generates intermediates necessary for the synthesis of fatty acids and non-essential amino acids

two pioneers of the Human Genome Project

Francis S. Collins J. Craig Venter scientists

use of tagged proteins in protein purification

GST and GSH 1. gene for target protein + gene for GST - transcription -> gene for fusion protein 2. express fusion protein in cell 3. prepare cell extract containing fusion protein as part of the cell protein mixture 4. protein mixture is added to column - other proteins flow through column 5. glutathione anchored to medium binds GST tag 6. solution of free glutathione is added to column 7. fusion protein is eluted by glutathione solution tags make it easier to distinguish from other proteins - cleaved by enzymes, which makes purification easier

stages of mitosis

prophase, metaphase, anaphase, telophase during interphase, chromosomes (chromatin) duplicate their chromatids (DNA replication) in S-phase, cells grow in G2 and then enter mitosis

self-inactivation of Gs

Gs protein's intrinsic GTPase activity, in many cases stimulated by RGS proteins, determines how quickly bound GTP is hydrolyzed to GDP, and thus how long the G protein remains active

Gs with GDP/GTP

Gs with GDP bound is turned off - it cannot activate adenyl cyclase contact of Gs with hormone-receptor complex causes displacement of bound GDP by GTP Gs with GTP bound dissociates into alpha and beta-gamma subunits - Gs(alpha)-GTP is turned on and can activate adenyl cyclase GTP bound to Gs(alpha) is hydrolyzed by the protein's intrinsic GTPase - Gs(alpha) thereby turns itself off; the inactive alpha subunit reassociates with the beta-gamma subunit

GPCR heterotrimeric G-protein signaling

Gα and Gβγ subunits activate distinct second messenger systems different Gα subunits modulate distinct downstream second messenger systems: Ca2+, DAG, increase or decrease cAMP, cGMP, cytoskeleton Gβγ subunits modulate distinct second messenger systems: cytoskeleton, PIP3, increase delta V, decrease Ca2+, cAMP, Ca2+, DAG

the discovery of fluoxetine hydrochloride (Prozac)

serotonergic neurotransmission

protein acetylation mediated by histone acetylases (HATs) and deacetylation mediated histone deacetylases (HDACs)

HATs - acetylate histone molecules to increase transcription (more relaxed chromatin) HDACs - deacetylate histone molecules to decrease transcription HDAC inhibitors lead to increased transcription by increasing protein acetylation

IL-6 cytokine family receptor complexes

IL-6 family cytokine receptors signal through a complex of distinct alpha receptor (Rα) subunits and shared gp130 signaling subunits

interleukin-6 (IL-6) signals via classical and trans-signaling

IL-6 multifunctional pro-inflammatory cytokine, part of acute phase response classical IL-6 signaling restricted to IL-6Rα expressing cells trans-signaling - IL-6 binds to sIL-Rα produced by alternative splicing or protease cleaved receptor that and can activate multiple signaling pathways via shared gp130 signaling subunit of IL-6 family receptors hijacked by IL-6 - trans-signaling

regulation of glycogen synthase and glucose transport by insulin signaling

IRS-1 phosphorylated by insulin-INS-R binding activates phosphoinositide 3-kinase (PI-3K) to convert PIP2 to PIP3 PIP3 binding to PKB leads to its phosphorylation and activation by PDK1 activated PKB phosphorylates and inactivates GSK3, thereby maintaining glycogen synthase in the non-phosphorylated active form, leading to increased glycogen synthesis activated PKB also stimulates movement of GLUT4 glucose transporter from intracellular vesicles to the plasma membrane, thereby increasing glucose uptake

Janus tyrosine kinase (JAKs)

Janus - the roman god of gates, doors, doorways, beginnings, and endings FERM (four point one, Ezrin, Radixin, Moesin) domain - binding of the JAK protein to its cognate receptor kinase-like domain catalytically inactive regulator of PTK domain JAKs - JAK1, JAK2, JAK3, and Tyk2 JH2 = kinase-like domain constitutively associated with cytokine receptors ligand binding catalyzes JAK auto-phosphorylation and subsequent cytokine receptor phosphorylation creates docking sites for STAT-SH2 domain binding

nuclear receptor domain structure

N-terminal activation function one surface(AF-1) DNA binding domain (DBD) non-conserved hinge-region ligand binding domain (LBD) C-terminal activation function two surface (AF-2)

nuclear receptor DNA binding and mechanism of action

NR dimerization and DNA binding sequences - arrows indicate consensus NR recognition sequence AGGTCA and variant hormone response element (HRE): 1. homodimeric endocrine receptor - palindrome HRE 2. RXR heterodimers - direct repeat HRE 3. monomeric orphan receptor - half site HRE some NRs activated by ligand-independent PGC-1 coactivator binding and coactivator recruitment without ligand - NRs form a repressive complex between HDACs and SMRT/NCOR corepressors ligand binding induces corepressor dissociation and coactivator recruitment, including HATs and chromatin remodeling complexes

application of PCR

PCR-mediated cloning PCR-mediated mutagenesis PCR-mediated sequencing molecular archaeology and molecular paleontology clinical diagnosis and genetic counseling forensic medicine others

Nobel Prize winners of 2008 in Chemistry

Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien

phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway

PI3K/AKT pathway components are targeted by amplification, mutation, and translocation in many cancers, resulting in pathway activation, PTEN mutations, and deficiencies are also prevalent PI3K/AKT and related pathways mediate the effects of external growth factor receptor tyrosine kinases activated by ligand binding, receptor dimerization and autophosphorylation AKT is activated downstream of PI3K and has many targets and functional consequences mTOR, mammalian target of rapamycin ERK, extracellular signal regulated kinase FKHR, forkhead GDP, guanosine diphosphate IRS, insulin receptor substrate GSK3, glycogen synthase kinase 3 MAPK, mitogen activated protein kinase NF-κB, nuclear factor-κB PKC, protein kinase C PIP2, phosphatidylinositol-3,4-diphosphate PIP3, phosphatidylinositol-3,4,5-triphosphate STAT3, signal transducer and activator of transcription-3

peroxisome proliferator-activated receptor-γ (PPARγ) signaling

PPARγ ligands thiazolidinediones (TZDs) -potent insulin sensitizers highly effective oral medications for type 2 diabetes unique benefits shadowed by the risk for fluid retention, weight gain, bone loss, and congestive heart failure many positive effects on the body

phosphatase and tensin homolog (PTEN) tumor suppressor alterations associated with cancer

PTEN non-redundant phosphatase dephosphorylates phosphatidylinositol- 3,4,5-trisphosphate (PIP3) regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, one of the most critical cancer-promoting pathways also regulates genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration and/or metastasis PTEN mutations and deficiencies prevalent in many cancers - severe PTEN deficiency associated with advanced tumor stage and therapeutic resistance targeting the deregulated PI3K/PTEN-Akt signaling axis is one of the major tenets in anticancer drug development

post-translational modifications that alter protein functions are potential drug targets

PTM enzymes frequently work in pairs: one to add and one to remove 1. phosphorylation/de-phosphorylation 2. hydroxylation/de-hydroxylation 3. acetylation/de-acetylation 4. methylation/de-methylation 5. disulfide bonds 6. lipidation/de-lipidation 7. myristylation/de-myristylation 8. isoprenylation/de-isoprenylation 9. farnesylation/de-farnesylation 10. glycosylation/de-glycosylation 11. sumoylation 12. ubiquitination

three pioneers of the field of DNA-based information technologies

Paul Berg Herbert Boyer Stanley N. Cohen

multiple RTK dimerization mechanisms

RTKs associate into dimers when ligands bind to their extracellular regions bound ligands form all, a portion, or none of the dimer interface, activating receptors by stabilizing specific interactions between two receptor molecules 1. nerve growth factor dimer cross-links 2 TrkA molecules without direct receptor contacts 2. stem cell factor dimer cross links 2 KIT molecules, two Ig-like domains (D4 and D5) reorient upon receptor activation and interact across the dimer interface - KIT combines ligand-mediated and receptor mediated dimerization 3. two fibroblast growth factor receptor (FGFR) molecules contact one another through the Ig-like domain D2 and the accessory molecule heparin or heparin sulfate proteoglycans (white sticks) also contact this domain - additionally, each fibroblast growth factor molecule contacts Ig-like domains D2 and D3 of both FGFR molecules 4. dimerization of ErbB receptors is mediated entirely by the receptor - ligand binds two sites (DI and DIII) simultaneously within the same receptor molecule and drives conformational changes to the epidermal growth factor receptor that expose a previously-occluded dimerization site in domain II

receptor tyrosine kinases (RTKs)

RTKs family of 58 cell surface receptors (20 subfamilies) that regulate critical cellular processes: 1. proliferation and differentiation 2. cell survival and metabolism 3. cell migration and cell cycle control RTKs have a similar molecular architecture - ligand binding region in the extracellular domain - single transmembrane helix - cytoplasmic region contains the protein tyrosine kinase (TK) domain, plus additional carboxy-terminal and juxta membrane regulatory regions ligand binding to RTKs stabilizes a specific relationship between individual receptor molecules in an 'active' dimer or oligomer numerous diseases linked to genetic changes or abnormalities that alter activity, abundance, cellular distribution, or regulation of RTKs - RTK mutations and/or aberrant activation of RTK intracellular signaling pathways causally linked to cancer, diabetes, inflammation, severe bone disorders, arteriosclerosis, and angiogenesis critical for activation - ligand induced dimerization or oligomerization

prototypical GPCR/Serpentine receptors/7TM receptors - complex of Rhodopsin and G protein Transducin

Rhodopsin (red) has seven transmembrane helices embedded in the disk membranes carboxyl terminus on the cytosolic side and its amino terminus inside the disk chromophore 11-cis-retinal (blue space-filling structure) linked to the seventh helix, lies near the center of the bilayer similar location to the epinephrine-binding site in the β-adrenergic receptor several serine (Ser) and threonine (Thr) residues near the carboxyl terminus are substrates for phosphorylation - part of Rhodospin desensitization mechanism cytosolic loops that interact with the G protein transducin are shown in orange three subunits α, β, and γ of G-protein transducin (green) are shown

staggered cleavage

S-OH - recessed 3'-hydroxyl group P-S-N (any base) - 5'-phosphate group extension also known as the sticky end - complementary to each other can ligate them together to form a new DNA strand

dexamethasone in hospitalized patients with Covid-19 — RECOVERY Trial

SARS-CoV-2 (Covid-19) infection associated with diffuse lung damage glucocorticoids may modulate inflammation-mediated lung injury and reduce progression to respiratory failure and death patients hospitalized with Covid-19, dexamethasone reduced 28-day mortality in patients receiving either invasive mechanical ventilation or oxygen alone, but not among those receiving no respiratory support

SNPs, haplotypes, and Tag SNPs

SNPs - single nucleotide polymorphism, variation in a single position in a DNA sequence among individuals (found in chromosome A at three places in 4 individuals) haplotypes - combinations of different alleles from different polymorphisms at the same gene (four found within the SNPs) Tag SNPs - representative single nucleotide polymorphism, which is used to tag a haplotype in a region of the genome (within an SNP) looking at the difference within many nucleotides

STAT family

STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6: 1. cytoplasmic transcription factors regulating cytokine and growth factor gene expression 2. interferons, IFNa/b &IFNg (prototypic activators of STAT1 & STAT2) 3. activated by receptor tyrosine kinases, Janus, Src, and Tec family kinases - STATs 1, 3, and 5 activated by many cytokines - STATs 2, 4, and 6 activated by relatively few

canonical STAT3 signaling pathway

STAT3 signaling initiated by many receptors - growth factors, cytokines, G-protein-coupled receptors, and SRC family kinases IL-6 binds IL6Rα subunit and recruits gp130 subunit - associated JAK becomes auto-phosphorylated and phosphorylates gp130 P-Tyr sites latent cytoplasmic STAT3 recruited to specific P-Tyr docking sites on IL6Rα- gp130 receptor complexes activated by cognate ligands - STAT3 recruitment and docking of mediated thru SH-2 domain interactions with gp130-pY sites STAT3 becomes phosphorylated at Tyr-705 reciprocal interactions between STAT3 SH-2 domains and dimer partner pY-705 triggers - homo-dimerization and translocation into nucleus pSTAT3 dimers bind specific DNA response elements in promoters and enhancers of target genes, activating transcription - promote cell proliferation and survival, inhibit apoptosis, stimulate angiogenesis and metastasis, and hinder anti-tumor immune responses upregulating gene expression

types of small molecule cancer therapies

cytotoxic chemotherapy molecularly targeted drugs issues - kill healthy cells as well as gut bacteria, hair follicles, and bone marrow (development of secondary infections due to immunosuppression)

cooperative binding of O2 to hemoglobin

T state versus R state - four different subunits: alpha1, alpha2, beta1, and beta2 as more O2 molecules bind hemoglobin, it transitions from a low affinity state (T, tense) to a high affinity state (R, relaxed), resulting in a sigmoidal O2 binding curve cooperative binding makes hemoglobin more sensitive to small differences in [O2], it binds O2 efficiently in the lungs (pO2 ~ 13.3 kPa) and releases O2 in tissues (pO2 ~ 4 kPa)

SERMs - clinical benefits and limitations

Tamoxifen used to treat all stages of breast cancer and chemoprevention in women at high risk for breast cancer - beneficial effects on bone mineral density and serum lipids in postmenopausal women Raloxifene approved for prevention and treatment of postmenopausal osteoporosis and vertebral fractures SERMs can also have some potentially serious adverse effects - thromboembolic disorders and uterine cancer for tamoxifen

drugs withdrawn for induced TdP

drug - class, date withdrawn Terfenadine - antihistamine, Feb 1998 Sertindole - antipsychotic, Dec 1998 Astemizole - antihistamine, Jun 1999 Grepafloxacin - antibiotic, Nov 1999 Cisapride - GI Prokinetic, July 2000

cytokinesis

during cytokinesis, cytoplasm of the mother cell is cleaved in half, and the cell membrane finishes its constriction at the middle of the cell to divide the mother cell into two separate daughter cells that proceed into a new interphase

acquired capabilities and hallmarks of cancer

evading apoptosis self-sufficiency in growth signals insensitivity to anti-growth signals tissue invasion and metastasis limitless replicative potential sustained angiogenesis

V-type ATPases

VoV1 H+ ATPase uses ATP to pump protons into vacuoles and lysosomes, lowering their pH only active at low pH

pregnane X receptor (PXR) and constitutive androstane receptor (CAR)

Xenobiotic receptors CAR can be activated by direct ligand binding and indirect mechanisms PXR activation is ligand dependent CAR and PXR share target genes (red box) but also regulate CAR-specific (blue box) and PXR-specific (purple box) targets - phase I and II drug metabolizing enzymes and drug transporters that alter drug clearance potential sites of drug-drug interactions

fusion proteins and immunofluorescence can localize proteins in cells

a fusion protein with green fluorescence protein (GFP) allows direct visualization: 1. straightforward and dynamic observation - easily recognized under a fluorescent microscope (seeing if the protein moves from the cytosol, etc.) 2. altered property - limitation a protein inside cells can also be visualized by immunofluorescence: 1. based on the specific interaction between antibody and antigen - protein of interest (high specificity and diversity) 2. antibody is labeled with fluorescence and can be detected 3. antibody can be targeted to the tag portion of a fusion protein or the protein itself

homologs

a gene related to a second gene by descent from a common ancestral DNA sequence superset of orthologs and paralogs example - frog alpha and beta, chick alpha and beta, and mouse alpha and beta all related genes - umbrella, different species

yeast two-hybrid analysis

a genetic approach to defining protein-protein interactions based on the properties of the Gal4 protein, which activates the transcription of GAL genes in yeast Gal4 has two domains - one that binds to a specific DNA sequence and another that activates RNA polymerase the protein-coding regions of the two genes to be analyzed are fused to the yeast gene for each of the two domains activation of the reporter gene requires the close proximity and interaction of the two proteins studied this method is more complicated, but it is closer to the physiological system

plasmid vector with a polycloning site (PCS)

a plasmid vector with a PCS is more versatile for cloning cleaved with EcoRI and sealed by DNA ligase these plasmids contain a sequence that can be cut by most enzymes - does not naturally occur in the plasmid these plasmids satisfy the needs of many different customers

The Beery twins, Noah and Alexis

a story of Beery twins who benefited from personalized genomic medicine - extremely helpful in their diagnosis born in 1999, parents noticed that the babies were having trouble sleeping at night, but they were fine during the day doctors thought it was colic and that it would go away within two years then the babies developed problems moving at night, but were still fine in the morning - doctors thought it was cerebral palsy through sequencing, a doctor found that they had a deficiency in L-DOPA, which is known as Segawa's Dystonia - each baby was given treatment, which worked well for them both Alexis developed breathing problems - mutation in the gene for SD, which impacted serotonin (given treatment and problems went away)

long QT syndrome and Torsades de pointes

abnormal ventricular repolarization - characterized by long QT interval on ECG congenital QT syndrome: 1. rare cardiac disorder 2. polymorphic ventricular tachycardia - torsades de pointes acquired LQTS due to drugs: 1. block of repolarizing K+ currents 2. stimulation of ICa-l 3. stimulation of INa 4. bradycardia 5. hypokalemia 6. CHF and LVH

role of senescence in cancer development and treatment - accelerated cellular senescence

accelerated cellular senescence (ACS) may play a role in cancer recurrence and drug resistance chemotherapy can lead to induction of senescence in both apoptosis defective or competent cancer cells bypass of ACS may contribute to cancer recurrence drug resistance

two types of cell death: non-programmed and programmed

accidental non-programmed cell death occurs in response to some external insult or injury to the cell - insufficient blood flow to the tissue, heat, mechanical injury, radiation, or chemical exposure programmed cell death is regulated and plays critical roles in development and maintenance of tissue homeostasis

nicotinic acetylcholine ligand gated ion channel

acetylcholine ligand gated ion channel is comprised of five homologous subunits, (α2βγδ), each with 4 transmembrane helices M1 - M4 M2 helices are amphipathic and others have mainly hydrophobic residues: 1. five subunits surround central transmembrane channel - lined with polar sides of M2 helices 2. rings of negatively charged amino acid residues at the top and bottom of the channel model of the acetylcholine receptor based on electron microscopy and x-ray structure determination of related acetylcholine-binding protein from mollusk top view of cross section through center of M2 helices shows 5 Leu side chains (yellow), 1 per M2 helix, protruding into the channel and constricting it to too small a diameter to allow passage of Ca2+, Na+, or K+ ions after both acetylcholine receptor sites (one per α subunit) are occupied, a conformational change occurs - as M2 helices twist slightly, 5 Leu residues rotate away from the channel and are replaced by smaller, polar residues (blue) this gating mechanism opens the channel, allowing passage of Ca2+, Na+, or K+ ions

GPCRs transduce a large number of signals

acetylcholine, extracellular ATP, dopamine, glucagon, glutamate, histamine, melatonin, opioids, serotonin, vasopressin, etc.

cyclin dependent kinase inhibitors (CKIs)

act as brakes to stop cell cycle progression active CDK-cyclin complexes drive cells through cell cycle cell cycle progression is determined by constellation of proteins activated and inactivated by CDK phosphorylation succession of CDKs (4, 6, and 2) and cyclins (D, E, A, and B) expressed as cells progress from G1 through to mitosis CDK4 and CDK6 complexed with cyclin-Ds function early in G1 CDK2 complexed with cyclins E and/A essential for G1 S transition and DNA replication CDC2 complexed with cyclins A and B essential for mitosis cell cycle stages tightly regulated by transcription and degradation of cyclins, and kinase subunit phosphorylation positive and negative feedback loops regulate cell cycle CDK-cyclin partners are positive regulators and accelerators of cell cycle progression cyclin dependent kinase inhibitors (CKIs) are negative regulators and brakes that stop cell cycle progression INK's and KIP's negatively regulate CDK activity by direct association INK4A (p16), INK4B (p15), INK4C (p18), and INK4D (p19) bind to CDK4 and CDK6 blocking association with cyclin-Ds CIP1 (p21), KIP1 (p27), and KIP2 (p57) form heterotrimeric complexes with G1/S CDKs passing the cyclin D and E CDK controlled restriction point irreversibly commits cells to the next cell cycle phase during G1 progression, Rb family members pRB, p107, and p130 are primary substrates of CDK 4, 6, and 2 Rb proteins negatively regulate early G1 cell cycle progression by interactions with E2F transcription factors - regulate expression of cyclin E, dihydrofolate reductase, and thymidylate synthase multiple layers of regulation of the cell cycle

Gq-coupled GPCRs

activate phospholipase C to generate diacylglycerol and inositol 1,4,5-trisphophate (IP3) that mobilizes Ca2+ hormone binds to receptor, causing GDP-GTP exchange on Gq agonist binding to Gq-coupled GPCRs activates phospholipase C (PLC) PLC generates diacylglycerol (DAG) and inositol 1,4,5-trisphophate (IP3) IP3 mobilizes Ca2+ after binding to ER IP3-receptor DAG and Ca2+ bind to and activate protein kinase C protein kinase C phosphorylates downstream targets

IL-6 and oncostatin M

activate the same gp130 canonical STAT3 pathway but via different alpha receptors

extrinsic "death receptor" pathway of apoptosis activation

activated by ligand-binding to death receptors - TNF- TNFR1, FasL-Fas, TRAIL-DR4 or -DR5, and CD95L-CD95 tumor necrosis factor (TNF) ligand binding to cognate type II transmembrane protein homotrimer death receptors activates several functions and apoptosis signaling by preassembled TNF-R1 complexes is masked by binding of the silencer of death domain (SODD) in the un-induced condition ligand binding to death receptors induces formation of the DEATH-INDUCIBLE SIGNALLING COMPLEX (DISC) TNF binding releases SODD from TNF-R1 complexes and death domain-containing adaptor protein TRADD is recruited by homophilic death domain interactions TNF-R1-bound TRADD acts as an assembly platform for TNF receptor-associated factor 2(TRAF2) and death domain-containing receptor-interacting serine-threonine kinase RIP TNFR1 also mediates apoptosis by the recruitment of an adaptor molecule called RAIDD (RIP-associated ICH- 1/CED-3 homologous protein with a death domain) DISC activates pro-caspase-8, which then activates pro- caspase-3 to cleave proteins and activate apoptosis

GPCR agonist activated Gsα activates adenylyl cyclase to elevate cAMP and activate PKA

activation of cAMP-dependent protein kinase (PKA) low cellular [cAMP] two regulatory subunits (R; red) associate with two catalytic subunits (C;blue) inhibitor sequences of R subunits occupy substrate-binding cleft of C subunits in R2C2 complexes, blocking substrate protein binding, and complex is catalytically inactive when cellular [cAMP] rises after GPCR activation, R subunits binds two cAMP molecules and undergo a dramatic conformational shift that pulls the inhibitory sequences away from the C subunits, opening the substrate binding cleft and releasing each C subunit in its catalytically active form cAMP binds to the regulatory subunits of protein kinase A

next-generation reversible terminator sequencing

add blocked, fluorescently labeled nucleotides thymine nucleotide added - fluorescent color observed and recorded remove labels and blocking groups, wash - add blocked and labeled nucleotides adenine nucleotide added - fluorescent color observed and recorded remove labels and blocking groups, wash - add blocked and labeled nucleotides cytosine nucleotide added - fluorescent color observed and recorded remove labels and blocking groups, wash - add blocked and labeled nucleotides guanine nucleotide added - fluorescent color observed and recorded dNTP incorporated - look at the microarray for each specific color sequence assembly program

restriction endonucleases I

cleave DNA at random sites that can be more than 1,000 bp from the recognition sequence cannot precisely control where the DNA will be cut

simplified model of G2/M checkpoint

after DNA damage, cell division cycle 25 (CDC25) proteins are inhibited by various mechanisms - activation of ATM/ATR signaling cascades leading to checkpoint kinase (CHK) activation and CHK-dependent CDC25 degradation, or cytoplasmic sequestration through 14-3-3 binding inhibitory kinases WEE1 and MYT1 are activated by checkpoint kinases (CHKs), and CDK-cyclin complexes are maintained in their inactive state and the cell remains arrested in the G2 to M transition phase

deficiencies in type I interferon (IFN-I) proteins implicated in life-threatening severe responses to COVID-19 infections

after SARS-CoV-2 coronavirus (COVID-19) enters human cells and reaches the endosome, toll-like receptors such as TLR7 and TLR3 recognize viral RNA and activate a pathway leading to the expression of genes encoding type I interferon proteins people with severe COVID-19 have mutations in genes that encode components of this process, and these individuals do not produce interferon normally - 3.5% of patients (23/659) had mutations in 8 genes and were incapable of producing or responding to IFN-I mutated versions of genes encoding TLR7 and interferon receptor (IFNAR1 and IFNAR2) have been reported in patients with severe COVID-19 other individuals with severe COVID-19 have autoantibodies that bind to certain type I interferons (IFN-α & IFN-ω), but not IFN-β, blocking IFN-α & IFN-ω signaling and antiviral gene expression of 987 patients with severe COVID-19, 135 (13.7%) had antibodies that recognized IFN-α2, IFN-ω, or both

cytotoxic chemotherapy

alklylating agents mitotic inhibitors - disrupting mitosis through microtubules: 1. vinca alkaloids - MT disruptors 2. taxanes - MT stabilizers anthracyclines antimetabolites topoisomerase inhibitors

orthosteric and allosteric GPCR ligands

allosteric modulators require orthosteric ligand binding to elicit an effect - use dependency may confer specificity/selectivity benefits orthosteric agonist alone functional response: 1. positive allosteric modulator that increases binding affinity (left-shift) 2. positive allosteric modulator that increases efficacy of functional response 3. negative allosteric modulator that decreases binding (right-shift) 4. negative allosteric modulator that decreases efficacy of functional response positive allosteric - requires less orthosteric agonist concentration for response negative allosteric - requires more orthosteric agonist concentration for response

partial restriction enzyme digestion

allows cloning of overlapping fragments - complete digestion helps delineate the entire sequence

concerted model of cooperative binding

also known as the all or none model assumes that subunits are functionally identical, each subunit exists in a high or low affinity conformation, and all subunits undergo the transition simultaneously both confirmations are in equilibrium and can bind ligand, but with different affinities successive binding to the low affinity confirmation makes the transition to the high affinity conformation more likely

tumor cell microenvironment

also promotes autonomy from growth signaling stromal cells may contribute to tumor growth - tumor associated fibroblasts, endothelial, and immune cell populations heterotypic signaling within tumor - tumor cells co-opt stromal cells to secrete growth factor signals cell-cell and ECM contacts also provide growth signals

cells in 3D experience

altered mechanical, adhesive, and cellular cues, which change biological functions and responses

tumors adaptively respond to therapy to acquire cancer drug resistance

altered target expression accumulation of secondary mutations activation of feedback loops activation of compensatory signaling pathways histologic transformation

GR agonists glucocorticoids

among the most effective and prescribed anti-inflammatory therapies - potent therapies glucocorticoid therapy issues and future: 1. patients develop resistance 2. adverse side effects 3. SEGRAs selective GR agonists

androgen receptor signaling and prostate cancer

androgen action - increases PSA, growth, and survival of prostate drugs targeting mechanisms of castration resistant prostate cancer - work to block the abnormal prostate growth by blocking the production of androgens or the binding to the androgen receptors AR-related mechanism - DNA-based, gene expression, and AR modulators

U2OS-PTH-R beta-arrestin-EGFP

another example of receptor internalization by beta-arrestin 1. GRK phosphorylation 2. b-arrestin binding 3. targeting 4. internalization 5. endosomes -> rapid recycling or slow recycling many Gs-coupled GPCRs exist

anti-IL-6 Rheumatoid arthritis and cancer strategies

anti-IL-6 cancer clinical trials: 1. anti-IL-6 chimeric monoclonal antibody CNTO 328/Siltuximab - metastatic prostate cancer and metastatic CRPC, multiple myeloma, metastatic kidney cancer, Castleman's disease 2. safety - ovarian, pancreatic, colorectal, head and neck, and lung neoplasms anti-interleukin-6 receptor antibody, tocilizumab: 1. Castleman's disease 2. unresectable pancreatic carcinoma 3. recurrent ovarian cancer no small molecules! therapeutic agents targeting IL-6

how do tumor cells evade anti-proliferative signals?

anti-proliferative receptor down regulation anti-proliferative receptor inactivation disruption of Rb and/or Rb signaling pathways - retinal blastoma disruption of CKIs and/or CKI signaling pathways - kinase inhibitors over expression of cMyc oncogene impairs differentiation and promotes growth

cGMP generation

generated by receptor- or soluble-guanylyl cyclase isoforms in response to natriuretic peptides or nitric oxide

programmed cell death - apoptosis

apoptosis - critical in development and maintenance of tissue homeostasis initiated by internal clock, or exposure to extracellular agents - hormones, cytokines, killer cells, viruses, chemicals, and physical cells undergo apoptosis through two major pathways: 1. extrinsic pathway - death receptor pathway 2. intrinsic pathway - mitochondrial pathway morphological characteristics of apoptosis - cell shrinkage, membrane remodeling and blebbing, chromatin condensation, DNA and cell fragmentation cellular apoptosis is genetically determined and tightly controlled by complex regulatory networks de-regulated apoptosis contributes to pathological disorders - developmental defects, autoimmune diseases, neuro-degeneration, and cancer

action potential triggers synaptic vesicle fusion with pre-synaptic membrane and neurotransmitter release

arriving action potentials depolarize the PM at the pre-synaptic terminal, activating voltage-gated Ca2+ channels, which raises intracellular [Ca2+] intracellular [Ca2+] triggers synaptic vesicles containing neurotransmitters to fuse with the PM and release their contents into the synaptic gap

nuclear receptors and diseases

associated with cancer, inflammation, and metabolic diseases synthetic ligands of metabolic disease

depending upon the context however, autophagy may support the growth of aggressive tumors

autophagy is up-regulated in RAS-driven cancers and is also induced in hypoxic tumor regions where it supports tumor cell survival autophagy deficient tumor cells accumulate defective mitochondria and are prone to cell death in hypoxic regions, which can lead to impairment of the growth of RAS-driven and perhaps other cancers

cells

basic structural and functional units of living organisms highly structured and contain organelles with distinct functions cell boundary, nucleus, and organelles are encased in membranes signals (extracellular and intracellular) must traverse at least one membrane to induce a response

phospholipid bilayer

basic structural element of membranes phospholipids contain a glycerol backbone with two fatty acid side chains of varying length and degrees of saturation, and a polar head group linked through phosphate amphipathic nature of phospholipids dictates how they behave in water

liposome

bilayers fold upon themselves into a cell-like 3D sphere with an aqueous cavity

orthosteric ligands

bind directly to the ligand binding site of receptors agonists activate functional responses antagonists block or inhibit agonist-induced responses - have higher affinity/lower kd, so they outcompete the agonist inverse agonists decrease or suppress basal responses

allosteric ligands/modulators

bind sites distinct from ligand binding site require orthosteric ligand binding to elicit an effect alter orthosteric ligand binding affinity and/or efficacy can elicit positive or negative effects

selectins

bind specific polysaccharides on adjacent cells in a Ca2+ dependent manner

allosteric protein binding

binding of a molecule to one site affects the binding properties of another site on the same protein effects can be either positive or negative

cooperative ligand binding - after ligand binds to first subunit

binding stabilizes a high affinity conformation (green), rest of polypeptide assumes a higher affinity confirmation stabilized in the other subunit by protein-protein interactions

distinguishing features of living organisms

bio-signaling and signal transduction is critical 1. high degree of chemical complexity and microscopic organization 2. systems to extract, transform and use energy from the environment 3. defined function for each component and regulated interactions among them 4. mechanisms to sense and respond to alterations in their surroundings 5. capacity for precise self-replication and self assembly 6. capacity to change over time by gradual evolution

how to predict the function of a new gene - protein

bioinformatics

tripartite model of steroid nuclear receptor pharmacology

biological response determined by: 1. steroid nuclear receptor type - endocrine, adopted orphans, and true orphans 2. ligand(s) - endogenous ligand/exogenous small molecule drugs - able to bind drugs (drug metabolism and transporter enzymes) 3. effectors: - co-activators - cell and/or tissue specific expression and distribution - co-repressors - cell and/or tissue specific expression and distribution - direct (HRE) or indirect (tethered) DNA binding to gene specific promoters and enhancers

human genome: protein-coding genes

breakdown of different protein functions - we do not yet fully understand proteins and their functions yet enzymes - 16.2% cytoskeleton - 3% unknown - 37.4% transcription factor - 7% signaling - 2.7% regulation - 4.1% transport - 3.4% receptors - 5.2% nucleic acid binding - 9.8% miscellaneous - 4.5% membrane proteins - 2.4% immune proteins - 1.3% extracellular structures - 3%

protein kinase A regulation

cAMP-dependent activation of PKA leads to the subsequent phosphorylation and activation of enzymes including other kinases - signaling cascade

phosphodiesterase (PDE) inhibitor drugs

cGMP PDE5 inhibitor drugs for erectile dysfunction: Avanafil (Stendra or Spedra), Lodenafil (Phase III), Mirodenafil (Mvix), Sildenafil (Viagra), Tadalafil (Cialis), Vardenafil (Levitra), and Udenafil (Zydena) sildenafil - first studied as potential therapy for angina PDE5 inhibition increases cellular [cGMP], activates PKG mediated protein phosphorylation, and lower [Ca2+] leads to relaxation of vascular smooth muscle erectile dysfunction - PDE5 inhibitors increase blood flow into penis and promote erection

cyclic guanosine 3′,5′-monophosphate (cGMP)

cGMP ubiquitous intracellular second messenger mediating many physiologic processes produced by soluble guanylyl cyclase (sGC) and receptor (rGC) isoforms cGMP exerts its actions through: 1. cGMP-dependent protein kinases 2. cGMP-gated cation channels 3. cGMP-regulation of phosphodiesterases (PDEs) that hydrolyze cyclic nucleotides

GPCR hetero- and homo-dimerization

can alter receptor pharmacology: 1. produce functional dimers 2. result in pharmacological diversity 3. exhibit transactivation and amplification 4. induce internalization and desensitization

oncogenes and tumor suppressors

cancer - evolutionary process involving accumulation of multiple somatic mutations in progeny of normal cells provides a selective growth advantage and leads to uncontrolled proliferation of mutated cells alterations to oncogenes cause gain of function effects tumor-associated oncogenes induce unscheduled proliferation, as well as genomic and chromosomal instability oncogenes not only required for cancer initiation but also for the maintenance of the disease - oncogeneaddiction alterations to tumor suppressor genes that cause loss of function contribute to the malignant phenotype: - tumor suppressor genes are typically negative regulators of growth or affect invasive and metastatic potential - "two hit" hypothesis - both alleles of tumor suppressor genes must be lost to unmask the malignant phenotype

cancer stem cell theory

cancer cell-of-origin/cancer-initiating cell: cell in which first genetic lesion linked to tumor development takes place anywhere within the physiological differentiation pathway - does not require any phenotypic relationship with the final tumor cell phenotype (stem or differentiated) cancer-maintaining cells (cancer stem cells) have the capacity to regenerate the cellular diversity of the tumor and retain broad self-renewal and differentiation potential - initially arise from cancer cell-of-origin and can self-propagate tumoral reprogramming: process by which the initial oncogenic lesion(s) 'reset' the epigenetic and/or transcriptome status of a healthy cell (cancer cell-of-origin), establishing a new pathological differentiation program, ultimately leading to cancer development - oncogenic lesion(s) not required after the initial cancer fate-inducing change has taken place

cancer - basic tenets

cancer cells evolve progressively from normalcy via pre-malignant states into invasive cancers molecular machinery regulating proliferation, differentiation, and cell death are highly conserved: 1. constitute major vulnerabilities 2. cancer cells often defective in regulatory circuits governing cell proliferation and homeostasis tumor cells invariably bear multiple mutations: 1. mutations can produce oncogenes with dominant gain of function 2. mutations to tumor suppressor genes - recessive loss of function many cancers exhibit: 1. age-dependent incidence 2. sex/race/ethnicity incidence

restriction endonucleases II

catalyze the dydrolytic cleavage of particular phosphodiester bonds in the DNA within the recognition sequence itself very specific location of cutting - very important!

non-programmed (accidental) cell death

caused by external factors to cell or tissue - insufficient blood flow, heat, mechanical injury, radiation, or chemical exposure irreversible - inappropriately termed necrosis (necrosis refers to changes secondary to cell death by any mechanism) ischemic cell death - oncosis - caused by failure of the ionic pumps of the plasma membrane - ischemic cell death is accompanied by swelling, hence "oncosis"

extrinsic (death receptor) pathway of apoptosis

cognate ligand binding to death receptors (FAS/TRAIL, TRAILR1, and TRAILR2) recruits death domain containing adaptor proteins (FADD) and procaspases with a death effector domain (8 and 10) to form the Death Inducing Signaling Complex (DISC) elevated active caspase-8 generated by procaspase-8 processing by DISC activates executioner caspases like caspase-3 to induce apoptosis activation of caspase-8 can also cleave the BH3-only protein BID to generate activated BID fragment tBID, which serves to transmit the death signal from the extrinsic to the intrinsic signaling pathway

the potential of drug combination therapy?

combination therapy potential synergistic drug combinations improved efficacy decreased dosage: 1. equivalent or increased efficacy 2. reduced side effects and toxicity control or delay resistance enhanced therapeutic benefit

cellular differentiation and stem cells

cell differentiation is continuous during the development of multicellular organisms from a simple zygote to a complex system of tissues and cell types differentiation is a step-wise process by which less specialized cells become more specialized and involves: 1. proliferation 2. commitment 3. lineage progression 4. differentiation 5. maturation dramatically alters cell's physical characteristics and phenotype: - size, shape, membrane potential, metabolic activity, and signal responsiveness - cells have same genome and almost never involves a change in DNA sequence - highly controlled epigenetic changes in gene expression influenced by trophic factors, location, niche and microenvironment (cell-cell and cell-ECM interactions) adult stem cells divide and create fully differentiated daughter cells: - intrinsic capacity of SCs to secrete cytokines and/or growth factors (trophic mediators) regulated by location, niche and microenvironment (cell-cell and cell-ECM interactions) - SCs are reservoirs for cytokine, chemokine, and ECM production to support SC survival and proliferation - important for tissue repair, homeostasis, and normal cell turnover

adhesion molecules

cell-cell and cell-extracellular matrix (ECM) interactions maintain neighboring cells, tissues, and organs connected information on location, environment, and surrounding matrix

senescence can be a double-edged sword

cellular senescence is a physiological program of terminal growth arrest triggered by various endogenous or exogenous stress signals senescence induced by oncogenic activation can be a barrier to tumorigenesis in response to therapeutic intervention, cancer cells may rapidly undergo apoptosis or enter therapy induced senescence (TIS) TIS cancer cells may be cleared by the immune system, which would be of benefit to patients TIS cancer cells are 'dormant,' but are metabolically active and can have a secretory phenotype if cells emerge from the TIS state, they may contribute to therapeutic resistance and tumor recurrence, which would be detrimental to patients

anaphase

centromeres split, and the sister chromatids separate chromosomes are formed of one chromatid with a centromere sister chromatids move towards opposite poles of the cell - "polar ascension"

major diabetes complications

cerebrovascular disease retinopathy coronary heart disease nephropathy neuropathy peripheral vascular disease in the lower limbs ulceration and amputation for diabetic foot

prophase

chromatin condenses into well defined chromosomes made up of two chromatids joined by a centromere, centrosomes duplicate and transform into asters, nuclear membrane disappears, and achromatic spindle of tubulin fibers forms

telophase

chromosomes de-condense and go back to the chromatin state, asters become centrosomes, new nuclear membrane forms, and spindle fibers break down

metaphase

chromosomes move to the center of the cell and line up along the equator, forming the "equatorial plate"

restriction endonucleases III

cleave DNA about 25 bp from the recognition sequence cannot precisely control where the DNA will be cut

signal transducers and activators of transcription

common STAT domain structure: 1. NH2-terminus STAT dimerization domain (SDD) 2. coiled-coil protein-protein interaction domain (CC) 3. central DNA-binding domain (DBD) 4. Src-homology 2 domain (SH2) 5. conserved tyrosine residue - Y701, Y705, or Y695 (Yp) 6. COOH-terminus transcription activation domain (TAD)

sequence or structural relationship provides information on protein function

comparative genomics orthologs paralogs synteny certain sequences are associated with particular structural motifs

protein-ligand binding sites

complementary size, shape, charge, hydrophobic and/or hydrophilic character

type II kinase inhibitors

compounds bind partially in the ATP binding site, extend past the gatekeeper and into an adjacent allosteric site that is present only in the inactive, or "DFG-out" conformation

type III kinase inhibitors

compounds binding exclusively to less conserved allosteric sites outside the ATP pocket beyond the gatekeeper residue allosteric site formed when the activation loop of the substrate binding cleft that recognizes substrates and influences the catalytic residues to adopt an inactive conformation expected to have superior selectivity profiles and new opportunities for scaffold development

ligand binding and target engagement assays

confirm ligand binding and determine binding affinities, KD: 1. radioligand binding - H3, C14, I125 labeled ligands 2. fluorescent polarization probe binding 3. differential scanning fluorimetry 4. isothermal calorimetry 5. surface plasmon resonance provide additional information on ligand binding sites and binding modes: 1. nuclear magnetic resonance spectroscopy 2. protein X-ray crystallography

precision medicine and cancer drug discovery

connecting genotypes to specific drug responses biomarker identification biomarker validation personalized therapies OMICs profiling, high-throughput drug screening biopsy -> 3D cell cultures - spheroids and organoids diagnostics - sarcoma sub typing

autophagy

conserved pathway that maintains homeostasis by degrading proteins and cytosolic contents of cells clears miss-folded and long-lived proteins, damaged organelles, and invading microorganisms from cells - shuttles them via specialized structures called autophagosomes to the lysosome for degradation stress-induced survival program - provides nutrients and energy in response to cell stressors, such as starvation defective autophagy is linked to a diverse range of disease processes - Crohn's disease, pancreatitis, hepatitis, and cancer

total binding

consists of specific binding plus nonspecific binding together

*shuttle vectors

contain multiple replication origins that permit replication in more than one species

differential expression and distribution of GPCRs within tissues

contributes to specificity parasagittal sections showing the immunocytochemical detection of group 3 mGluR GPCRs in rat brain different GPCRs expressed in different levels in the brain regulating response to glutamate - different signal transduction pathways

cooperative binding

cooperative binding of a ligand to a multimeric protein is a form of allosteric binding

5-hydroxy-tryptamine (5-HT) serotonin and depression

criteria for neurotransmitters: 1. synthesis 2. storage 3. release 4. breakdown 5. uptake serum factor - vasoconstriction smooth muscle distinct and separate 5-HT neurons - catecholamines DA and NE antidepressants: 1. monoamine oxidase inhibitors (MAO) 2. tricyclic antidepressants (TCA) - imipramine and desipramine ~ inhibited uptake of monoamines - Carlsson et al, proposed inhibition of 5-HT uptake basis for mood elevation lower 5-HT in autopsy samples of brains from depressed patients - indicated first connection of serotonin to mood

integrins

critical for embryonic development, tissue maintenance and repair, host defense, and hemostasis

type II restriction endonucleases

cut DNA within the recognition sequence discovered in bacterial species the recognition sequences are usually 4 to 6 bp long and palindromic - same upstream and downstream sequences from 5' -> 3' in either direction generate two types of ends: 1. sticky ends - preferred and easy to ligate 2. blunt ends

cGMP produced by receptor/soluble guanylyl cyclase

cyclic GMP (cGMP) generated by two mechanisms: 1. nitric oxide synthase - soluble guanylate cyclase (sGC) coupled pathway 2. natriuretic receptor-guanylate cyclase (NPR-A/B rGC) pathway both pools of cGMP interact with different effectors

CDK-cyclin complexes are regulated by gene expression, phosphorylation, and proteolysis

cyclin dependent kinases (CDK) inactive without cyclins, but as cyclin protein levels rise, they form active cyclin- CDK complexes - activity of different cyclin-CDK complexes varies with the phase of the cell cycle T-loop of CDKs folds into the substrate binding site and phosphorylation of Tyr-160 in T-loop by CDK activating kinases (CAKs) produces a conformational change that opens the substrate binding site phosphorylation of threonine and tyrosine residues (T14/Y15 in CDC2) by dual specific kinases (Wee1 and MYT1) inactivates cyclin-CDK complexes, which become activated when CDC25 phosphatases dephosphorylates them active cyclin-CDK triggers its own destruction by phosphorylation of destruction box recognizing protein (DRBP), which activates ubiquitination by ubiquitin ligases targeting it for destruction by the 26S proteasome system retinoblastoma phosphoprotein (Rb) is phosphorylated and dephosphorylated during the cell cycle inactive hyperphosphorylated-Rb predominates in proliferating active hypo-phosphorylated-Rb levels are higher in quiescent and differentiating cells Rb function partly involves interactions with E2F family DNA-binding transcription factors - E2F sites in the promoters of many genes important for cell cycle progression are transcriptionally repressed by Rb-EF2 complexes

cyclin-dependent protein kinases - CDKs

cyclin-dependent protein kinases (CDKs) only active when associated with cyclin proteins crystal structure of CDK2 ± cyclin reveals the basis for this activation without cyclin, CDK2 folds, and the T loop obstructs the binding site for protein substrates and inhibits protein kinase activity ATP binding site also near the T loop

SRC kinase activation

de-phosphorylation of the activation loop de-phosphorylation of Src-P activates the kinase when SRC tyrosine kinase is phosphorylated on a specific internal P-Tyr residue, the SH2 domain binds this internal P-Tyr, bringing the SH3 domain near an internal proline rich region and blocks productive substrate binding in active de-phosporylated SRC, the SH2 domain binds a P-Tyr residue of the substrate and the SH3 domain binds a proline rich region of the substrate, lining up the active site of SRC with target Tyr residues of the substrate close proximity of Tyr to the active site for phosphorylation

potential therapeutic applications

decreased frequency of action potential firing -> CNS depression, neuronal conduction disorders, and cognition disorders -> K+ channel inhibitor -> normal neuronal action potential firing increased frequency of action potential firing -> CNS hyper excitability, seizures, pain, ADHD, anxiety, bipolar disease, and schizophrenia -> K+ channel activator -> normal neuronal action potential firing

blunt-end cleavage

deoxyribose and phosphate groups - phosphodiester bonds cleaved 3'-OH and 5'-P ends

green fluorescence protein (GFP)

derived from the jellyfish Aequorea victoria highly fluorescent - lights up when exposed to blue light it has a β-barrel structure, and the fluorophore is in the center of the barrel fluorescent reaction is autocatalytic variants of GFP of other colors are available - ability to test many different proteins at once to determine interactions, etc.

the problem - dismal cancer drug cevelopment success rates!

despite large investments in cancer research, drug discovery and drug development most NCE's fail in clinical trials no therapeutic efficacy/poor bioavailability/unacceptable toxicity rate of new cancer drug approval is ≤ 5% probability of success in oncology clinical trials 3.4% most metastatic cancers remain incurable myriad of factors contribute to low cancer drug development success: 1. genetic heterogeneity, tumor cell differentiation status, and clonal heterogeneity 2. intrinsic and acquired drug resistance 3. aspects of the tumor microenvironment that favor drug resistance

insulin and diabetes

diabetes mellitus - higher than normal blood sugar levels due to abnormal insulin production and action insulin - hormone produced by the pancreas that acts on different tissues to alter glucose utilization when [insulin] is low or does not function properly, it produces high blood sugar (hyperglycemia) type 2 most common form of diabetes - insulin resistance: 1. the body cannot use insulin produced by the pancreas and it is difficult to absorb glucose from the blood 2. initially overcome by the body's ability to produce higher levels of insulin 3. when the body is unable to produce enough insulin, high blood sugar and major complications result

three modes of autophagy

differ in how "cargo" to be degraded is delivered to the lysosome micro-autophagy - nonselective process whereby cytosolic proteins are sequestered by invagination of the lysosomal membrane chaperone-mediated autophagy - selective process by which proteins with defined consensus sequences recognized by molecular chaperones, including Hsp70, delivered to the lysosome macro-autophagy - process whereby bulk proteins and organelles in the cytosol are delivered to the lysosome

normal cells require mitogenic signals to actively proliferate

diffusible growth factors and cognate plasma membrane receptors paracrine and endocrine signals cell interactions with extracellular matrix components or other cells - integrins and/or other cell adhesion molecules

RTK kinase activation mechanisms

dimerization of extracellular regions of RTKs activates intracellular tyrosine kinase domains (TKDs) - TKDs contain C- and N-lobes - dark purple (inactive) or yellow (active) states - activation loop - purple (inactive) or yellow (active) state TKD auto-inhibition (E): 1. activation loop - insulin and FGF receptors 2. juxta-membrane auto-inhibition as in the Kit-like, MuSK, Flt3, and Eph families 3. C-terminal tail inhibition - Tie-1 and Tie-2 allosteric activation of TKDs (F): - EGFR TKD is allosterically activated by direct contacts between the C-lobe of one TKD, the 'activator,' and the N-lobe of another TKD, 'receiver'

thousands of biological signals

discrete number of signal transduction machinery protein components 1. signals and receptors differ - general signal transduction features conserved 2. signal reception - ligand interacts with a receptor 3. integration/adaptation and amplification - activated receptor interacts with cellular machinery - produces a second messenger or altered activity of a cellular protein - phenotype - metabolic activity and/or gene expression profile of target cell changes 4. signal transduction event is terminated

heterotrimeric G-proteins - α, β and γ subunits

distinct Gα and Gβγ subunits activate different second messengers

heterotrimeric G-proteins - 3 subunits

distinct Gα subunits activate different second messengers Gβγ subunits also activate distinct signaling pathways

vasculogenesis, angiogenesis, and vascular remodeling

during development, three major processes form the vascular network in the body to meet tissue requirements for oxygen and nutrients vasculogenesis - de novo blood vessel formation: 1. precursor angioblasts migrate to vascularization sites, differentiate into endothelial cells, and coalesce to form initial vascular plexus angiogenesis - budding of new capillary branches from existing blood vessels vascular remodeling - newly formed vessels increase their luminal diameter in response to increased blood flow and acquire identity as an artery, vein, or capillary processes are completed during postnatal development adult vasculature is stable and does not proliferate under normal physiological conditions in cancer, existing vessels start to grow again to meet the abnormal requirements for oxygen and nutrients of the expanding tumor

cell growth

during embryonic growth and development - cell division occurs in every tissue adult tissues mostly quiescent cell's decision to divide is critical - unregulated cell growth results in cancer normal cell division proceeds in precisely ordered sequence termed the cell cycle assures every daughter cell contains full complement of molecules required for life two phases - interphase and mitosis cell inoculation - using up nutrients in media (stationary phase)

voltage-gated K+ channels

during transmission of action potentials along neurons, the opening of trailing K+ voltage gated channels repolarize the membrane voltage-gated K+ channel activators or inhibitors could be used to increase or decrease the frequency of action potential firing, respectively, depending upon the therapeutic situation

fluoxetine hydrochloride (Prozac)

early member new class of antidepressants selective serotonin reuptake inhibitor (SSRI) - first publications 1974, and took > 16 years to develop US FDA approval 1987 sustained effective treatment of depression - low side-effect profile 2002, 1 year after patent expiration: 1. prescribed to > 40 M patients 2. total sales US $22 billion 3. peak annual sales US $2.8 billion in 1998 hSERT (serotonin transporter) is a Na+ symporter - fluoxetine blocks this

transmembrane electrical potential

electrogenic Na+ K+ ATPase produces transmembrane electrical potential of about - 60 mV (inside is negative) blue arrows show direction ions move spontaneously across the plasma membrane, driven by combination of chemical and electrical gradient chemical gradient drives Na+ and Ca2+ inward, producing depolarization, and K+ outward, producing hyperpolarization electrical gradient drives Cl- outward, against its concentration gradient, producing depolarization

stem cells

embryonic stem cells fetal stem cells adult stem cells - adult somatic cells from the skin induced pluripotent stem cells (iPSCs) stem cells isolated from embryos, fetuses, adult tissues, or by reprogramming (iPSCs) are being investigated and developed for clinical applications in regenerative medicine and replacement therapy

emerging hallmarks and enabling characteristics of cancer

emerging hallmarks: 1. capability to modify and reprogram cellular metabolism to effectively support neoplastic proliferation 2. evade immunological destruction by T and B lymphocytes, macrophages, and natural killer cells enabling characteristics facilitate acquisition of both core and emerging hallmarks: 1. genomic instability and mutability give rise to genetic alterations that drive tumor progression 2. inflammation by innate immune cells that fight infections and heal wounds can sometimes inadvertently support multiple hallmark capabilities of cancer

F-type ATPases

energy coupling factors FoF1 ATPase drives H+ up gradient into mitochondria large proton gradient can drive pump in reverse ~ ATP synthase

stem cells and differentiation

epigenetic processes play crucial roles in regulating the decision to adopt a stem, progenitor, or mature cell fate Lysine-specific demethylase 1 (Lsd1) demethylates histone H3on Lys4 or Lys9 (H3K4/K9), an indispensable epigenetic governor of hematopoietic differentiation Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism of hematopoietic maturation epigenetic changes like DNA methylation and histone modification, alter gene expression but not the underlying DNA sequence regulated by microenvironment and cytokines and growth factors

β-adrenergic signaling pathway: integration, cascades and amplification

epinephrine activation of the GPCR expressed in liver cells initiates a cascade of signaling events involving activation of adenylyl cyclase, increased cAMP levels, activation of PKA and downstream kinases that elevates the production of glucose from glycogen signal transduction pathways are rarely linear and more typically exist as complex 3-dimensional pathways with multiple potential positive and negative inputs or outputs with opportunities for cross talk more than one functional response altered by epinephrine GPCR activation

ligand binding equations and graphical representations

equilibrium expression for reversible binding of a ligand (L) to a protein (P): P +L -(ka)><(kd)- PL association (slope increases) and dissociation curves (slope decreases) Ka is the association constant at equilibrium Kd is the dissociation constant at equilibrium ka and kd are rate constants, respectively Kd = [P] x [L]/[PL] = kd/ka when [L] = Kd, half the ligand binding sites are occupied slope = -1/Kd

JAK-STAT transduction mechanism for the erythropoietin receptor

erythropoietin (EPO) binding causes EPO receptor dimerization, which allows JAK, a Tyr kinase, to phosphorylate it on several Tyr residues SH2 domain of STAT5 binds to P-Tyr residues on the receptor, bringing it into proximity with JAK after phosphorylation of STAT5 by JAK, two STAT5 molecules dimerize, each binding the other's P-Tyr residue STAT5 dimerization exposes a nuclear localization sequence (NLS) targeting the dimer for transport into the nucleus in the nucleus, STAT5 binds to specific DNA sites to turn on the expression of EPO-controlled genes in another pathway, after EPO binding and autophosphorylation of JAK, the adaptor protein Grb2 binds P-Tyr residues in JAK and triggers the MAPK cascade (like insulin) two different pathways

ATPases

establish ion gradients across membranes 1. P-type 2. V-type 3. F-type electrochemical gradients provide energy for secondary active transport

yeast two-hybrid analysis - diagram

example 1: Gal4p DNA-binding domain on the Gal4p binding site - protein X attached reporter gene downstream protein Y attached to Gal4p activation domain comes in and binds to protein X/RNA polymerase, leading to increased transcription of reporter gene must be close together to activate transcription of reporter gene example 2: yeast strain 1 with Gal4p DNA-binding domain fusions and yeast strain 2 with Gal4p-activation domain fusions mate to produce diploid cells plate on medium requiring interaction of the binding and activation domains for cell survival survivors from colonies - sequence fusion proteins to identify which proteins are interacting activation and survival

active transport

expends energy of ATP to move solutes against an electrochemical gradient

comparison of different expression systems

expression system: cell growth, med. complexity, cost, expression bacteria: rapid (30 min), minimum, low, high yeast: rapid (90 min), minimum, low, low-high baculovirus: slow (18-24 h), complex, high, low-high mammalian: slow (24 h), complex, high, low-medium post translational modification differences as well bacteria is the most efficient expression system

plasmids

extrachromosomal molecules of DNA (1kb~20kb) found in a variety of bacterial species circular double-stranded DNA evolved a variety of mechanisms to maintain a stable copy number in the hosts and to partition accurately to daughter cells are dependent on the enzymes and proteins encoded by the host for their replication and transcription frequently contain genes for enzymes that are advantageous to the bacterial host, such as antibiotic resistance

signaling pathways that trigger programmed cell death - apoptosis

extrinsic - receptor mediated intrinsic - mitochondria mediated caspase-2-dependent caspase-independent - perforin/GrA-mediated

transporters

facilitate movement of solutes into and/or out of cells

DNA microarray experiment

fertilized egg and tadpole -> mRNA -> reverse transcriptase ->cDNA -> DNA microarray -> removal of unhybridized cDNAs isolate mRNAs from cells at two stages of development - each mRNA sample represents all the genes expressed in the cells at that stage convert mRNAs to cDNAs by reverse transcriptase, using fluorescently labeled deoxyribonucelotide triphosphates add the cDNAs to a microarray - fluorescent cDNAs anneal to complementary sequences on the microarray each fluorescent spot represents a gene expressed in the cells yellow - unchanged expression green - higher expression at singe-cell stage red - higher expression at tadpole stage studying which DNA is expressed - genomic DNA doesn't help, isolate the mRNA microarrays depict huge amounts of data - very powerful tool

cell differentiation

fertilized egg zygote divides and differentiates into all cell types in the body to create a new organism zygote cells omnipotent or totipotent - can become any cell type - first 3 cell divisions of the zygote give rise to totipotent cells as embryonic cells continue to divide - daughter cells differentiate and become progressively specific early mammalian embryo made of extra-embryonic cell layers - trophoblast and inner cell mass (ICM) cells in the ICM - no longer omnipotent but are pluripotent and have a committed embryonic fate (become any cell but trophoblast) pluripotent - cannot become totipotent ICM differentiates into three germ layers - ectoderm (external), mesoderm (middle), and endoderm (internal) each germ layer follows a specific developmental destiny - cells differentiate along ever-specifying path into distinct organs specialized cell types express subset of genes in genome - each cell type is defined by a specific pattern of gene expression transition from one cell type to another during differentiation involves a switch from one pattern of gene expression to another cellular differentiation during development is the result of a gene regulation network - epigenetics

kinase drug discovery - circa 2016

first small-molecule kinase inhibitor Imatinib - approved for clinical use only 15 years ago 33 kinase inhibitor drugs have received FDA approval for the treatment of various cancers - discovery and development of kinase inhibitors has increased exponentially - 231 inhibitors targeting 38 different protein and lipid kinases in clinical use or under clinical investigation

kinase drug discovery

first small-molecule kinase inhibitor Imatinib approved for clinical use 2001 2016 - 33 kinase inhibitor drugs approved by FDA for the treatment of a variety of cancers discovery and development of kinase inhibitors has increased exponentially - 231 inhibitors targeting 38 different protein or lipid kinases are either in clinical use or under clinical investigation

how to detect the intracellular location of a protein

fluorescent fusion protein immunofluorescence

plasmid applications

gene carriers - amplification, maintenance, and storage act as a "filing cabinet" expression vector: 1. bacterial system - recombinant proteins (must have a promoter that is recognized by the bacteria) 2. mammalian system - recombinant proteins and gene therapy (protein based therapy) example: coagulation factor 9 to treat hemophilia

epigenetic modifications: histone acetylation, DNA methylation, and gene transcription

gene on - active open chromatin with unmethylated cytosines and acetylated histones gene off - silent condensed chromatin with methylated cytosines 7 and deacetylated histones only change which genes are transcribed or expressed

Na+ gradient

generated by the Na+ K+ ATPase - used to drive secondary active transport of solutes 1. passive transport goes down a concentration and electrochemical gradient until equilibrium is achieved 2. primary active transport utilizes the energy from the hydrolysis of ATP to move solutes against a concentration or electrochemical gradient directly 3. secondary active transport uses the energy from the hydrolysis of ATP to create a concentration or an electrochemical gradient and then harnesses these to drive the co-transport of a solute transport via a symporter or antiporter carrier

tumor heterogeneity and clonal evolution

genetic heterogeneity - mutations, copy number alterations, tumor suppressor inactivation epigenetics and gene regulatory networks tumor cell differentiation - cancer stem cells, Epithelial to mesenchymal transition not all tumor cells are identical

deoxyribonucleic acid (DNA) provides genetic foundation of reproduction

genetic information must be: 1. maintained in a stable form 2. expressed accurately as gene products 3. reproduced with a minimum of errors deoxyribonucleic acid (DNA): 1. genetic information stored and encoded by nucleotide sequence 2. basis of generation of all other cellular components 3. template for production of identical DNA molecules to be distributed to progeny during cell division

major obstacles to HNC cancer drug development

genetic inter-tumor heterogeneity of cancer cell differentiation state and clonal heterogeneity cancer drug resistance - intrinsic and acquired tumor cell microenvironment

significance of genome sequencing

genome sequencing informs us about our humanity genome comparison helps locate genes involved in disease genome sequences inform us about our past and provide opportunities for the future

DNA libraries are specialized catalogs of genetic information

genomic library: 1. isolation and partial digestion of genomic DNA 2. BAC or YAC is often used as a cloning vector cDNA library: 1. isolation of mRNAs 2. building of a cDNA library from mRNA diagnosis of rare diseases plasmid - only small DNA fragments, not entire genomes

receptor tyrosine kinases (RTKs) and domains

growth factor receptors that signal through Tyr kinase activity have a Tyr kinase domain on the cytoplasmic side of the plasma membrane (blue) extracellular domain is unique to each type of receptor, reflecting the different growth-factor specificities extracellular domains are typically combinations of structural motifs such as cysteine- or leucine-rich segments and segments containing one of several motifs common to immunoglobulins (Ig-like domains)

pharmacological effects of compounds on hERG

hERG channel studied by command-voltage protocol after depolarization from a holding potential of -80 mV, currents develop and rapidly inactivate (C- type) after cell is rapidly repolarized to -40mV, channel recovers to open state and tail currents ensue pharmacological effects on tail currents measured - classical patch system

hERG inwardly rectifying voltage gated K+ channel

hERG subunits form channels that mediate IKr inward rectification due to voltage dependent C-type inactivation, more rapid than activation amplitude of IKr decreases as external [K+] decreases

biological membranes

have very diverse functions 1. define external boundaries of cells 2. divide intracellular space - discreet compartments, segregate organelles, processes, and components 3. regulate molecular traffic and signaling across or through cells 4. central cell-to-cell communication 5. organize complex reaction sequences 6. central to energy conservation 7. flexible - shape changes; growth and movement 8. self sealing - fission and/or fusion; endocytosis, exocytosis, mitosis 9. selectively permeable to polar solutes

orthosteric ligand binding site

mediates functional responses to epinephrine target of β-blocker drugs prescribed for hypertension, cardiac arrhythmia, glaucoma, anxiety, and migraine headache isoproterenol is an agonist of epinephrine, and propranolol is an antagonist of epinephrine

natriuretic peptides and heart disease

heart muscle contracts cyclically and continuously - mechanical force for blood circulation to modulate biochemical and metabolic homeostasis heart muscle must adapt to acute and chronic stress - signaling cascades, modulators of protein function, and organ remodeling - adrenergic stimulated myocardial cAMP generation stimulates function and/or growth atrial, brain, and C-type natriuretic peptides - endocrine/autocrine regulatory factors - ANP, BNP, and CNP activate NPR-A and NPR-B membrane receptor guanylyl cyclases - cGMP activates PKG and cGMP-PDE-5 interactions act as counter-brake to myocardial stressors - organo-nitrites and NPs that stimulate cGMP synthesis used to treat clinical heart failure - cGMP-selective PDE5 inhibitors sildenafil and tadalafil ameliorate cardiac pressure, volume overload, ischemic injury, and cardiotoxicity clinical trials for PDE-5 inhibitors for dilated cardiomyopathy and heart failure with a preserved ejection fraction

hematopoiesis in the bone marrow

hematopoietic stem cells (HSCs) in bone marrow medulla produce all mature blood cell types and tissues: 1. myelocytes - monocytes, neutrophils, eosinophils, basophils, and erythrocytes 2. lymphocytes: T and B cells - maturation, activation, and proliferation of lymphoid cells occurs in secondary lymphoid organs (spleen, thymus, and lymph nodes) hematopoietic growth factors and cytokines initiate signal transduction pathways to activate transcription factors and alter gene expression, which regulates blood cell differentiation cell determination also dictated by location and microenvironment factors that determine differentiation path - cell-cell and cell-ECM interactions, etc. hematopoietic microenvironment prevails upon some cells to survive and proliferate, while others undergo apoptosis

endocrine nuclear receptors

high affinity (Kd ~nM) NRs for fat-soluble hormones and vitamins: 1. steroid hormones, thyroid hormone receptor (TR), vitamins A (retinoic acidreceptors, RAR) and D (VDR) 2. essential for homeostatic control of endocrine system 3. steroid NRs function as homodimers 4. TR, VDR, and RAR form heterodimers with the Retinoid X Receptor(RXR) 5. endocrine NRs very successful drug targets - ligands widely used clinically

prostate cancer - sex, race/ethnicity, and age

higher incidence of breast cancer in females, prostate cancer in males (black most common) higher incidence of melanoma in white people median age at diagnosis is 66, median age at death is 80

adaptation of metastatic cells to foreign environments

homing and colonization of tumor cells (TCs) to a distant organ/metastatic site is a complex process mechanical trapping - TCs may become lodged in the capillary beds of specific organs due to size site-specific adhesion or chemoattraction - TCs may express specific adhesion molecules, enabling them to adhere to micro-vessels in specific organs or may respond to chemo-attractive gradients produced by tissues pre-metastatic niche - TCs may preferentially home to organs where pre-metastatic niches create microenvironments favoring survival quiescence - TCs exiting the blood-system may undergo period of quiescence or dormancy so that they can adapt to the new microenvironment micro metastasis - dormant cells may progress to micro-metastatic deposits (recruited by appropriate stroma or due to enhanced proliferative responses to microenvironment signals) where tumor size is regulated by a balance between proliferation, apoptosis, and phagocytosis by tissue immune cells macro metastasis - TCs recruit an adequate blood supply (required beyond 1-2mm) to become macro-metastasis

cadherins

homophilic binding to cadherins on adjacent cells

neural cell adhesion molecule

homophilic binding to glycoproteins expressed on neurons, glia, skeletal muscle, and natural killer cells

thermostable DNA polymerase

hot springs at Yellowstone owe their vibrant colors to heat-loving microorganisms Thermus aquaticus in an outflow channel of a hot spring at Yellowstone this led to the development of heat-resistant DNA polymerase in PCR to make it more efficient

using DNA-based methods to understand protein function

how to construct genomic and cDNA library how to predict the function of a new gene (protein) how to detect the intracellular location of a protein how to define protein-protein interaction how to define changes in gene expression patterns

three types of senescence

human cells experience diverse forms of stress in their lifetimes that, if left unchecked, could be detrimental to the cells and the organism one mechanism that cells may use to keep the damage to DNA in-check is by inducing cellular senescence sepending on the stress, stimuli cells may activate pathways to undergo: 1. replicative senescence (RS) 2. oncogene-induced senescence (OIS) 3. accelerated cellular senescence (ACS)

synteny in the human and mouse genomes

human chromosome 9 - EPB72, PSMB7, DNM1, LMX1B, CDK9, STXBP1, AK1, and LCN2 mouse chromes 2 - Epb7.2, Psmb7, Dnm, Lmx1b, Cdk9, Stxbp1, Ak1, and Lcn2 group of genes *co-localized together mirror sequence with the mouse - easier to understand protein function in mice by genetic manipulation

nuclear receptor super family

human nuclear hormone receptor super family endocrine receptors: 1. steroid and heterodimeric receptors 2. high affinity ligands Kds nM (endogenous ligand identified) adopted orphan receptors: 1. lipid sensors and enigmatic orphans 2. lower affinity ligands Kds μM orphan receptors: 1. ligands unknown

amphipathic nature of phospholipids

hydrophobic fatty acid side chains cluster with each other polar hydrophilic head groups interact with water mixing different phospholipid:water ratios produces different structures: 1. micelle 2. bilayer 3. liposome

micelle

hydrophobic fatty acids sequestered in the interior of the sphere

signal transduction sensitivity: integration/adaptation

if two signals have opposite effects on a cellular state, the net change determines the eventual response cells may require multiple signal proteins coincidentally to induce responses - multiple signals may use integrator proteins which require >1 signal input to have a downstream output coincidence detectors cross talk produces unified response

mechanisms to evade apoptosis

impaired receptor signaling pathway disrupted balance of Bcl-2 family of proteins increased expression of IAPs reduced expression of caspases defects and mutations in p53

disruption of Rb and/or Rb signaling pathways

in G1 phase and low level of CKIs, active CDK/cyclin complexes trigger hyper-phosphorylation of Rb, releasing the dimeric transcription factor E2F/DP transactivation of E2F target genes - regulators of growth, cell cycle, nucleotide, and DNA synthesis disruption of Rb signaling pathway liberates E2Fs, allowing cell proliferation and insensitivity to anti-growth factors that block G1 passage two broad classes of CKIs regulate cyclin-dependent kinase (CDK) driven progression through the cell cycle - INKs and KIPs INK4 family (p15/p16/p19) specific for CDK4/Cyclin D CIP/KIP family (p21, p27) - all CDK-Cyclin complexes transforming growth factor beta (TGFb) is an anti-proliferative signal in some cells - induces INK and KIPs expression to block CDK4/6-Cyclin D phosphorylation of Rb and release of EF2 to activate genes required for DNA synthesis in S phase

G-protein activation - GTP/GDP bound

inactive G proteins (Ras/Gα) interact with GTP-GDP exchange factors, GEFs, or activated GPCRs to become activated by GTP binding (GTP bound) Gα proteins activate downstream effector enzymes GTPase activator proteins (GAPs) and regulators of G protein signaling (RGSs) modulate the GTPase activity of G proteins, converting GTP to GDP and inactivating the G protein (GDP bound)

allosteric modulators

induce conformational changes that interconvert more-active and less-active forms of the protein homotrophic - ligand and modulator identical heterotrophic - ligand and modulator different

structural adaptation

induced fit model lock and key

autophagolysosome formation and regulation of autophagy

induction of autophagy - isolation membrane/phagophore elongates and engulfs cell contents sealing of the tips leads to completion of the double-membrane autophagosome outer membrane then fuses with the lysosome, resulting in degradation of its contents key elements are the autophagy-related (ATG) 12-ATG5-ATG16L complex, which recruits light chain (LC3) to the membrane and ATG4, which controls lipidation and recycling of LC3 mammalian complex ULK1/2-mAtg13-FIP200 is required for autophagy during starvation, mTORC1 is released from the complex, resulting in dephosphorylation of ULK1 and ULK2, ubiquitin-like conjugation systems that play roles in the early steps of autophagy glucagon, starvation, and reactive oxygen species (ROS) activate autophagy inhibitors of autophagy include insulin and amino acids (AAs) gives the cell energy to survive in stressful situations

oncogenic RAS and angiogenesis

induction of pro-angiogenic growth factors VEGFA and FGF2 by RAS in tumor cells - RAS also increases the stability of VEGFA mRNA and augments its translation the release of proteases by tumor cells cleaves components of the ECM and releases bound VEGFA and FGF2, which induce neo-proliferation and sprouting of micro-vessels towards the tumor site recruitment of macrophages by tumor cells through RAS-induced NF-κB-dependent production of IL-6 and IL-8 cytokines and subsequent promotion of endothelial proliferation and sprouting by newly recruited macrophages

bacterial expression systems - disadvantages

ineffective posttranslational modification - complicated PTM most of proteins of interest form inclusion bodies (insoluble cellular precipitates) and have to be refolded contamination of bacterial components - LPS (endotoxin that generates a severe inflammatory response, basis of septic shock)

initiation and progression of cancer: traditional oncogenesis

initiating genetic alteration occurs that is required for the immortalization of a committed/differentiated target cell over time, the cell acquires more genetic hits that increases deregulation of differentiated cells, leading to the clinical features of cancer human cancer genetic defects act on cells already committed to a differentiation program tumor phenotype derived from and reflects the initial differentiated target cell that was the normal cell of origin

definition of molecular cloning

inserting a piece of DNA molecule (of interest) into a DNA carrier (vector) to generate multiple copies in a host cell, such as bacteria purposes: 1. separate a gene from others 2. amplification of modified forms of genetic materials - very small amount of DNA that needs amplified 3. manipulation of DNA for further experiments vector (DNA carrier): 1. plasmids 2. bacterial artificial chromosome (BAC) 3. yeast artificial chromosome (YAC) 4. bacteriophage 5. virus

effects of insulin on different tissues

insulin reduces blood glucose levels activates glucose transport and utilizing pathways in cells downregulates glucose forming pathways stimulates glucose uptake in muscle and adipose tissue stimulates: - glycolysis, glycogenesis, and protein synthesis inhibits: - gluconeogenesis and lipolysis

regulation of gene expression by insulin through a MAP kinase cascade

insulin regulates specific gene expression through a cascade of protein kinases, each of which activates the next in the pathway INS-R is a Tyr-specific kinase - other kinases phosphorylate protein Ser/Thr residues insulin-INS-R binding induces conformational change, enabling autophosphorylation of β- subunit COOH-terminal domains Tyr residues autophosphorylation further activates the Tyr kinase domain, which phosphorylates other target proteins INS-R phosphorylates Tyr residues on IRS-1 Grb2 binds P-Tyr of IRS-1 and Sos binds to Ras, causing GDP-GTP exchange activated Ras binds and activates Raf-1, which phosphorylates two Ser residues on MEK MEK is a mitogen-activated, dual-specificity kinase, which phosphorylates Thr and Tyr residues on ERK (extracellular regulated kinase) ERK tranlocates into the nucleus and phosphorylates Elk1 transcription factor P-ELK-1 binds SRF (serum response factor) to modulate gene transcription

activation of the insulin-receptor tyrosine kinase by autophosphorylation

insulin-binding region of the insulin receptor lies outside the cell and comprises two α subunits and extracellular portions of two β subunits, intertwined to form the insulin binding site insulin binding is communicated through the single transmembrane helix of each β subunit to paired Tyr kinase domains inside the cell, activating them to phosphorylate each other on three Tyr residues in the inactive form of the Tyr kinase domain, the activation loop sits in the active site, and none of the critical Tyr residues are phosphorylated - conformation stabilized by H-bonding between Tyr1162 and Asp1132 Tyr kinase activation allows each β subunit to phosphorylate three Tyr residues (Tyr1158, Tyr1162, and Tyr1163) on the other β subunit introduction of three highly charged P-Tyr residues forces a 30 Å change in the position of the activation loop, away from the substrate-binding site, which becomes available to bind and phosphorylate a target protein

signaling networks regulate the operations of cancer cells

integrated circuits of normal cells reprogrammed to regulate hallmark capabilities in cancer cells separate sub-circuits are specialized to orchestrate the various hallmark capabilities - proliferation, motility, viability, cytostasis, and differentiation considerable crosstalk between sub-circuits each cancer cell exposed to a complex mixture of signals from its microenvironment and signals originating from other cells in the tumor microenvironment three-dimensional pathways - crosstalk, positive, and negative feedback mechanisms

cell adhesion molecules - CAMs

integrins, cadherins, selectins, and neural cell adhesion molecule

types of tumor heterogeneity

interpatient heterogeneity - example: same cancer, but tumor metastasis to different organs in different patients intratumor heterogeneity intermetastatic heterogeneity intrametastatic heterogeneity

four phases of the cell cycle

interphase (G1, S, and G2) and mitosis (M) S phase - DNA is replicated to produce copies for both daughter cells G2 phase (G = gap between divisions) - new protein and RNA synthesis occurs and cell doubles in size (G2 and M = two copies of DNA, fluorescence doubled) mitosis (M phase): - maternal nuclear envelope breaks down - mitotic spindle formed - paired chromosomes pulled to opposite poles of the cell - two daughter cells formed by cytokinesis after mitosis cells pass into G1, another growth phase where new protein and RNA synthesis occurs - G1 = one copy of DNA rapidly proliferating cells pass through G1 into S and cell division cycle begins again cells that stop dividing like terminally differentiated cells may enter a quiescent phase G0 - cells in G0 that re-enter the cell cycle do so in G1

mitochondrial (intrinsic) pathway initiates apoptosis through caspase 9

intracellular signals (DNA damage and endoplasmic reticulum stress) converge on mitochondria, inducing MMP MMP causes the release of proapoptotic factors, including cytochrome c (Cytc), which induces the apoptosis protease-activating factor 1 (APAF-1) and ATP/dATP to assemble the apoptosome apoptosome promotes the proteolytic maturation of caspase-9, which cleaves and activates effector caspases producing the apoptotic phenotype DNA damage signals may also activate caspase-2, which acts upstream mitochondria to favor MMP

caspases - initiators and effectors of apoptosis

intrinsic mitochondrial example caspase family of 14 proteins are some of the main effectors of apoptosis caspase activation is a hallmark of apoptosis, and they cleave substrates at Asp-Xxx bonds synthesized as inactive zymogens, caspases are cleaved to active enzymes after apoptosis induction 3 caspase groups - inflammatory, apoptotic initiator, and apoptotic effector caspases caspases are signaling mediators orchestrating apoptotic execution pathways by cleaving subsets of (>100) cellular proteins: 1. mediators and regulators of apoptosis 2. structural proteins 3. cellular DNA repair proteins 4. cell cycle-related proteins extrinsic and intrinsic apoptotic pathways converge to activate effector caspases (caspase-3 -6, &-7), which cleave death substrates

acomplia (Rimonabant)

investigational agent for the management of obesity after successful clinical trials in overweight and obese subjects, Sanofi-Aventis plans to file an NDA with the FDA during the first half of 2005 and, if successful, hopes to bring the drug to market in early 2006 developed by Sanofi-Aventis (formerly Sanofi-Synthelabo), Acomplia(rimonabant) selective CB1 endocannabinoid receptor inverse agonist drug blocks endogenous cannabinoid binding to neuronal CB1 receptors activation of CB1-R by endogenous cannabinoids, such as anadamide, increases appetite approved by the EU in 2006 for the treatment of obesity - never approved by the FDA potential smoking cessation therapy - endocannabinoids involved in tobacco dependence approval by EU withdrawn on 16 January 2009 - risk of serious psychiatric problems, including increased suicide incidence

neuronal transmission

involves both voltage and ligand gated ion channels initially, plasma membrane (PM) of pre-synaptic neurons are polarized due to the Na+ K+ ATPase stimulation of pre-synaptic neurons induces an action potential moving along the axon away from the cell body towards the synapse, causing voltage gated Na+ channels to open and depolarize the PM PM depolarization triggers adjacent voltage gated Na+ channels to open, followed by the opening of K+ voltage gated channels that repolarize the PM directionality ensured by a brief refractory period after the opening of voltage-gated Na+ channels when depolarization wave reaches synaptic terminal, voltage-gated Ca2+ channels raise intracellular [Ca2+], triggering synaptic vesicles to fuse with PM and release neurotransmitters into the synaptic gap neurotransmitters in the synaptic gap bind to GPCRs and ligand-gated ion channels in the PM of the post-synaptic neuron acetylcholine actives ligand-gated nicotinic ion channels that allow Na+ and Ca2+ to enter the cell and depolarize the post-synaptic neuron, thereby initiating an action potential to propagate the signal

NR transcriptional regulation

involves recruitment and interactions of multiple protein complexes in the absence of ligand, NRs form a repressive complex with HDACs (histone deacetylases) via corepressors SMRT or NCOR ligand binding induces corepressor dissociation and coactivator recruitment, including histone acetyl-transferase (HATs) and chromatin remodeling complexes

guanosine nucleotide-binding protein (G-Protein) coupled receptors (GPCRs)

membrane receptors with seven transmembrane helices - Serpentine receptors, 7TM receptors, GPCRs guanosine nucleotide-binding Protein (G-Protein) - cycles between GTP-bound active ↔ GDP-bound inactive effector enzyme or ion channel modulated by the G-protein that generates a second messenger

normal cardiac action potential and re-polarization

involves several ion currents: 1. Na+ current - inward 2. Ca2+ current - L and T type (inward) 3. transient outward current - ITO1 and ITO2 (outward) 4. delayed rectifiers - IKS, IKR, IKur (outward) 5. ICl or IKp - outward 6. inward rectifier - IK1 (inward and outward) 7. pacemaker current - If (inward) 8. Na+-Ca2+ exchange - inward and outward 9. Na+ K+-ATPase - outward phase 0 - depolarization; phase 1 - rapid repolarization; phase 2 - plateau; phase 3 - repolarization; phase 4 - resting membrane potential cardiac action potentials mediated by the coordinated action of several ion channels and currents

impaired ion channel diseases and drugs

ion channel family and the associated disease with impairment current ion channel drugs that are indicated for specific diseases disregulation of ion channels causes disease

DNA microarrays reveal RNA expression patterns and other information

is based on DNA/DNA specific hybridization - Southern Blot revealing RNA expression patterns offers help in identifying candidate genes for further study serves as a biomarker to aid diagnosis, individualized treatment, and follow-up of therapeutic response very simple - based on base pairing (determining the right treatment for a patient)

DNA cloning - summary of the procedure

isolate DNA from the organism use restriction enzymes to cut the DNA and a cloning vector, and then ligate the DNA fragment into the cloning vector transform recombinant DNA into a host, which will replicate the DNA and pass copies to all progeny

immunoprecipitation

it is based on the antibody-antigen (Tag/ligand) specific interactions - Tag/ligand not as specific as antibody interaction, but it is the same principle/premise a specific antibody (immobilized on a supporting system, such as glass beads) can bind to and pull down a protein and its associated proteins under physiological ("non-disruptive") conditions pull down - precipitate from solution, making sure not to disrupt the protein-protein complex formed during cell lysing the associated proteins can then be dissociated from the complexes under disruptive conditions and be subjected to further analysis - analyze each individual component

DNA ligases

join two DNA molecules or fragments that are cut by the same one or two restriction endonucleases catalyze the formation of new phosphodiester bonds in a reaction that uses ATP a critical step in the cloning is the identification of one or two restriction endonucleases that can cut both the gene sequence to be cloned and the vector!!!

molecularly targeted drugs

kinase inhibitors HSP90 inhibitors proteasome inhibitors HDAC inhibitors DNA methyltransferase inhibitors aromatase inhibitors hormone therapies, anti-estrogens, anti-androgens immunotherapies

SAPK/JNK and ERK MAPK signaling pathways

kinase signaling cascades are complex 3-dimensional pathways with multiple potential positive and negative inputs or outputs with opportunities for cross talk

verification of PCR product on agarose or separide gel

ladder - acts as a guide for the PCR fragments PCR fragments - show up at different base pair lengths and increase with intensity at higher concentrations confirming amplification of desired product

DNA polymerases ensure fidelity of DNA replication

large number and diversity of transactions involved in DNA synthesis must faithfully replicate and stably maintain genome despite constant challenges from cellular metabolism and external environment cells contain multiple DNA polymerases with: 1. DNA extension capabilities 2. proofreading abilities 3. exonuclease activity

milestones within DNA-based information technologies

late 1960s - Werner Arber and Hamilton Smith discovered restriction endonuclease 1969 - Herbert Boyer isolated restriction enzyme EcoRI 1972 - David Jackson, Robert Symons, and Paul Berg successfully generated rDNA molecules 1973 - for the first time, S. Cohen and H. Boyer developed a recombinant plasmid 1976 - first prenatal diagnosis by using gene specific probe 1979 - insulin synthesized by using rDNA 1985 - PCR development 2003 - human genome project completed

differentiation of mesoderm cells

mesenchymal stem cells (MSCs) exhibit extensive diversity in differentiation, trophic mediator production (cytokines and growth factors), and interactions with the environment adult hMSCs differentiate into cellular phenotypes capable of fabricating bone, cartilage, muscle, marrow, tendon or ligaments, adipocytes, and connective tissue hMSCs produce large quantities of bioactive factors, which provide molecular cueing for regenerative pathways, as well as affecting the status of intrinsic tissue cells intrinsic capacity hMSCs to secrete trophic mediators defined by their in vivo location, niche, and microenvironment (cell-cell and cell-ECM interactions) hMSCs are reservoirs for producing cytokines, chemokines, and ECM, which support stem cell survival and proliferation

comparative genomics

level of homology in a given sequence is the key in this diagram looking for a motif - unique for a given function determining if a gene belongs to a family

sequential model of cooperative binding

ligand binding can induce a change of conformation in a single subunit, and this makes a similar change in an adjacent subunit and the binding of a second ligand more likely

ligand

ligand molecules bind reversibly to proteins receptors, enzymes, or antibodies

death-receptor (extrinsic) pathway initiates apoptosis through caspase 8

ligand-induced activation of death receptors induces assembly of DISC inside the plasma membrane DISC promotes caspase-8 (and maybe caspase-10) activation which in turn cleaves effector caspase-3, -6, and -7 caspase-8 can also proteolytically activate Bid, promoting mitochondrial membrane permeabilization (MMP) and linking the extrinsic to the intrinsic apoptotic pathway

typical biological Kd values

ligand-receptor binding interactions depend on: 1. concentration of the molecules present 2. physical environment - temperature, pressure, and solvent 3. kinetics and thermodynamic consequences of binding highest affinity reaction - 1x10^-15, avidin (egg white, protein) to biotin (ligand)

nonspecific binding

linear vs [Ligand] usually determined in presence of large excess (> 500-fold) of un-labeled ligand

medical marijuana

long history of cannabinoid consumption for medical and recreational use approved therapeutics - Dronabinol (THC, Marinol) and Nabilone (Cesamet): - appetite stimulants indicated for nausea and vomiting associated with cancer chemotherapy and AIDS patient anorexia/cachexia syndrome - anti-emetic, analgesic, anti-convulsant, and lower intra-occular pressure psychoactive effects, tolerance, and abuse potential: - euphoria, relaxation, and distortions of both hearing and vision - impaired memory, altered time sense, and decrements in reaction time, learning perception, motor coordination, and attention toxicity - shrinkage of neuronal cell bodies and nuclei, DNA fragmentation indicating apoptosis

ion channels

maintain cell membrane potential - critical for neuronal & cardiac action potentials

Na+ K+ ATPase active transport system - ICF

maintains intracellular [Na+] and [K+] to generate membrane potential critical for neuronal action potential and transmission critical for heart muscle contraction

Na+ K+ ATPase active transport system

maintains intracellular [Na+] and [K+] to generate membrane potential for every ATP, Na+ K+ ATPase moves 3 Na+ out and 2 K+ into the cell membrane potential across the cell is central to neuronal signaling - action potential Na+ gradient used to drive secondary active transport of solutes membrane potential = -50 to -70 mV ECF or blood plasma - [K+] = 140 mM and [Na+] = 145 mM cytosol - [K+] = 140 mM and [Na+] = 12 mM inside concentrations: [Na+] low, [K+] high, [Ca2+] low, and [Cl-] low (opposite outside)

cell cycle checkpoints

major goal of cell division is to accurately pass mutation free parental DNA to daughter cells 'cell-cycle checkpoints' - mechanisms to actively halt cell cycle progression until DNA replication and mitosis completed cells that don't meet conditions required at checkpoints arrest, preventing duplication with major genetic errors G1/S checkpoint (restriction) point - end of G1 before S phase: if environment or DNA is unsuitable for DNA synthesis cells can arrest the process and/or go into G0 G2-M checkpoint - ensures that DNA has been copied without major errors spindle checkpoint - ensures that chromosome separation has occurred correctly cells experiencing problems at checkpoints (DNA damage or inappropriate number ofchromosomes) either repair the mistake, or if that is not possible, the cell arrests or undergoes apoptosis - controlled programmed cell death

multiple GPCR sub-types

may respond to the same ligand - activate different signaling pathways, depending on the coupled G protein 5-HT receptors -> 5-HT (1-7) 5-HT1 -> 1A, 1B, and 1D 5-HT2 -> 2A-2C dopamine: 1. D1-like - Gq -> phospholipase C and A2 - Gs -> increases adenylyl cyclase 2. D2-like - Gi - decreases adenylyl cyclase - Go - K+-channel

cancer cell metastasis

metastasis responsible for ≥ 90% of cancer-associated mortality phase I - physical translocation of cancer cells from primary tumor to distant organ phase II - colonization of the translocated cells within that organ metastatic dissemination, primary tumor cancer cell executes sequence of steps: 1. intravasation - locally invades surrounding tissue, enters microvasculature of lymph and/or blood systems 2. survives and translocates largely through the bloodstream to microvessels of distant tissues 3. extravasation - exits the bloodstream 4. colonization - survives and adapts to the foreign microenvironment of distant tissues in ways that facilitate cell proliferation and the formation of macroscopic secondary tumors

DNA microarrays

microarray is a rectangular grid of spots printed on a glass microscope slide, where each spot contains DNA for a different gene monitor the expression of a large group of genes: 1. which gene is turned on or off in a particular biological condition? 2. is this on/off state different between two biological conditions? more genes in sample = more green

intrinsic (mitochondrial) pathway of apoptosis

mitochondrial outer membrane permeabilization (MOMP) releases cytochrome C and other apoptogenic factors - cytosol apoptosome formation activates caspase-9 and downstream apoptosis-inducing caspase cascade interactions among BCL-2 family proteins play critical roles in mediating MOMP and consequent apoptosis BH3-only proteins (activators like BIM and tBID, and sensitizers like BAD) relay apoptotic signals to the mitochondria through activation of BAX/BAK, main effectors of intrinsic apoptosis anti-apoptotic BCL-2 proteins (such as BCL-2) inhibit apoptosis by blocking BAX/BAK activation

estrogen NR signaling - selective estrogen response modifiers (SERM)

molecular networks influence SERM action in tissues structure of ligands direct ERα or ERβ complexes to become an estrogenic or anti-estrogenic signal ER complex (ERC) tissue context influences response through numbers and balance of co-repressors (CoR) to coactivators (CoA) expression of estrogenic action involves ERC binding to estrogen-responsive gene promoters and dynamic assembly and destruction of CoA complexes ERC and core CoAs like SRC3 influenced by phosphorylation core CoAs assemble multiprotein complexes of specific co-co- activators (CoCo) with enzymatic activity that is activated later CoCo activators trigger complex dissociation via methylation or acetylation ubiquitinylation targets CoAs and ER for proteasome destruction regimented cycle of assembly, activation, and destruction occurs based on the preprogrammed ER complex co-activators like SRC3 can regulate and amplify ligand- activated triggers further by modulating genes that can consolidate and increase ERC stimulatory tissue responses target tissues are programmed to express a spectrum of responses from full estrogenic to anti-estrogenic action based on ligand structure and sophistication of tissue-modulating network

EGFR signaling and drug therapy

monoclonal antibodies (Mabs) - bind to ligand, receptor, or domain to block EGFR - anti-EGF - anti-EGFR (receptor) small molecules -tyrosine kinase inhibitors (TKIs)

bacterial expression systems - advantages

most commonly used - cost effective and cheap the expression plasmids and the hosts are well understood high yield easy to purify

oligonucleotide-directed mutagenesis

mostly done by polymerase chain reaction (PCR) the mismatch of a single base pair in 30 to 40 bp does not prevent annealing the first round of amplification generates the first strand DNA that contains the mutation subsequent steps amplify the mutation-containing DNA confirmed by DNA sequencing

tumorigenesis

multi-step process and succession of genetic alterations progressive conversion of normal cells to cancer cells cancer involves dynamic changes to the human genome drives progressive transformation into highly malignant derivatives confers some form of growth advantage

signal transduction features: protein modularity

multiple domains in signaling proteins allows for recruitment and assembly of specific protein complexes cells mix and match signaling proteins to create a variety of signaling complexes scaffold proteins assemble cascades of signaling proteins into signal-somes - input signal -> cascade of reactions on the scaffold protein -> output signal adaptor, scaffold, kinase, phosphatase, ras signaling, transcription, signal regulation, and phospholipid second-messenger signaling different binding domains have different binding properties - proline-rich protein or membrane lipid PIP3, Tyr-P, Tyr-P, PIP3, phospholipids (Ca2+-dependent), DNA, transcriptional activation, and carboxyl-terminal domain marking protein for attachment of ubiquitin

protein kinase G (PKG)

natriuretic peptide receptor-coupled cGMP activates PKG signaling cardiac stress regulation by PDE5 inhibition mostly controlled by PKG targeting PDE5 inhibition enhances PKG signaling that stimulates several pathways and transcription factors

true orphan nuclear receptors

natural or synthetic ligand has not been identified

endocannabinoids as neurotransmitters

neurotransmitter criteria: 1. synthesis - action potential triggers phospholipid remodeling or de novosynthesis 2. storage - phospholipid membrane 3. release - action potential 4. uptake - selective anandamide transporter 5. breakdown - fatty acid amidohydrolase activating - synthesis, storage, release desensitization process - uptake and breakdown

DNA sequences in a typical E. coli expression vector

ori gene encoding repressor that binds O and regulates P bacterial promoter (P) and operator (O) sequences ribosome-binding site polylinker transcription-termination sequence selectable genetic marker - antibiotic resistance

integrins - α and β subunit heterodimer CAMs

non-covalent α and β subunit heterodimers with 1 TM helix extracellular domains bind ECM proteins - fibronectin, collagen, and laminin or counter receptors on adjacent cells cytoplasmic tails linked to cytoskeleton - permit bi-directional force transmission across plasma membrane outside-in signaling - transmit external chemical signals into the cell: 1. information on location, local environment, adhesive state, and surrounding matrix 2. control cell migration, survival, differentiation, and motility responses 3. provides context for responding to other inputs transmitted by growth factor receptors, GPCRs, etc. inside-out signaling/activation - alter affinity for extracellular ligands: 1. conformational changes of extracellular domains produced by signals impinging upon their cytoplasmic tails integrins have key roles in a diverse range of diseases - cancer, infection, thrombosis, and autoimmune disorders

normal cells versus tumor cells - different responses to pro- and anti-growth signals

normal cells receiving growth and survival signals - survive and proliferate normal cells receiving growth inhibition and death signals - cell death and no cell division tumor cells exhibit: 1. self-sufficiency for growth - growth inhibition cues ignored (creating their own signals of survival and proliferation) 2. insensitivity to anti-growth signals - death cues ignored

defects in autophagy

normally, autophagy is tumor suppressive; however, defects in autophagy contribute to tumor initiation normally autophagy, either basal or stress-induced, prevents the accumulation of oncogenic proteins such as p62, as well as damaged proteins and organelles in autophagy defective tissues, p62 and damaged proteins and organelles accumulate and activate oncogenic nuclear factor erythroid 2-related factor 2 (NRF2) and nuclear factor-κB (NF-κB) signaling pathways that promote survival but may eventually be overwhelmed by sustained oxidative stress, resulting in reactive oxygen species (ROS) production, chronic tissue damage, inflammation and genome instability this creates a tumor-initiating and tumor-promoting environment

tumor cells acquire insensitivity to anti-growth signals

numerous anti-proliferative signals keep normal cells quiescent and maintain tissue homeostasis soluble growth inhibitors, cell-ECM, and cell-cell contacts signals transduced by cell surface receptors primarily cell cycle associated signaling pathways cells are forced to exit active proliferation into the G0 quiescent phase of the cell cycle terminally differentiated cells relinquish their proliferation potential and enter post-mitotic states tumor cells evade these anti-proliferative signals

cancer stem cells (CSCs)

oncogenic lesions may reprogram early stem cells or precursor cells towards oncogene-specific differentiated tumor cell fates CSCs exhibit traits such as motility, invasiveness, and self-renewal, which are central to malignancy may reflect the actions of elusive CSC subpopulations within larger populations of neoplastic cells CSCs may represent a tiny fraction of the total cellularity of individual tumors but may be critical drivers of malignant progression

role of senescence in cancer development and treatment - oncogene-induced senescence

oncogene-induced senescence (OIS) acts as a barrier to neoplastic transformation regulated by various pathways - most commonly p53 and Rb pathways primary cells that acquire activating mutations and/or overexpression of oncogenes and/or loss of tumor suppressor genes undergo OIS - cells must bypass OIS to become immortalized and eventually transformed OIS may be induced by other mechanisms - secretion of senescence inducing proteins, autophagy, and miRNAs oncogenic stimuli that induce a senescence response may have the potential to initiate tumor promotion

RAS oncogene effects on proliferation

oncogenic RAS establishes independence from extracellular growth factors and growth inhibitors promoting exit from the G0 phase of the cell cycle, progression through G1, and entry into S phase. RAS induces increased transcription of growth factors and interferes with transforming growth factor-β (TGFβ) signaling through inhibition of TGFβ receptor expression or downstream signaling by down regulating the expression of SMAD3 and nuclear accumulation of SMAD2 and SMAD3 RAS upregulates expression of Cyclin D1 and suppresses cyclin-dependent kinase inhibitor (CDKI) p27 cyclin D1 associates with and activates CDK4 and CDK6, leading to phosphorylation of RB and dissolution of RB-E2F complexes - E2F transactivates several genes required for cell cycle progression, including cyclin E (CCNE) and cyclin A (CCNA) that induce transition through the G1/S checkpoint hyperproliferative cues from RAS oncogene activation can result in replicative stress, leading to DNA damage and activation of DNA damage checkpoints to transiently arrest and restore the integrity of the genome, enter a state of irreversible arrest (senescence), or undergo apoptosis - inaccurate DNA damage repair can lead to mutations and chromosome aberrations, contributing to tumorigenesis

RAS oncogene effects on apoptosis

oncogenic RAS may have pro-apoptotic and/or anti-apoptotic functions, depending on the status of RAS effector pathways and apoptotic machinery oncogenic RAS signaling through the RAF pathway engages an apoptotic response mediated by p53 and other effectors (RASSF1, NORE1, and JNK) activating BAX, BAK1, caspase 3, and apoptosis acquisition of a tumorigenic phenotype is marked by the suppression of RAS-induced apoptosis mediators and in this context, the anti-apoptotic activity of RAS prevails, mediated by several signaling pathways, including the PI3K pathway which down regulates BAK1 and up-regulates inhibitors of apoptosis (IAPs), and the RAF pathway, which down regulates the pro-apoptotic PAR4 repressor while up-regulating the anti-apoptotic proteins BCL-2 and ARC both pathways phosphorylate and inactivate the pro-apoptotic protein BAD1

Which host cell expression system?

prokaryotic: 1. E. Coli - very commonly used and very efficient eukaryotic: 1. yeast 2. insect cells/baculovirus 3. mammalian cells 4. cell free - for research purposes

orthologs

orthologs are genes in different species that evolved from a common ancestral gene result of a speciation event normally, orthologs retain the same function in the course of evolution example: 1. alpha chain gene of early globing gene - frog alpha, chick alpha, mouse alpha 2. beta chain gene of early globing gene - frog beta, chick beta, mouse beta same/similar sequence and functions

orthosteric ligands: interact directly with ligand binding site of receptors

orthosteric ligand binding site mediates functional effects of epinephrine the target of β-blocker drugs prescribed for hypertension, cardiac arrhythmia, glaucoma, anxiety, and migraine headaches

orthosteric and allosteric ligands

orthosteric ligands bind to the ligand binding site: 1. agonists - activate functional responses 2. antagonists - block/inhibit agonist-induced responses 3. inverse agonists - decrease/suppress basal responses allosteric ligands/modulators bind to sites distinct from ligand binding sites: 1. require orthosteric ligand binding to elicit an effect 2. alter orthosteric ligand binding affinity and/or efficacy 3. can elicit positive or negative effects 4. use dependency may confer specificity/selectivity benefits

paralogs

paralogs are genes related by duplication within a genome paralogs evolve new functions, even if these are related to the original one example - mouse alpha and beta genes new genes developed within the genome

cancer mono-therapy - issues

partial patient responses emergence of resistance narrow therapeutic index: 1. dose limiting toxicities 2. reduced quality of life 3. poor patient adherence

cell cycle subject to multiple regulatory mechanisms

passage of cells from one stage of the cell cycle to another is tightly regulated by multiple mechanisms acting on: 1. transcription of cyclin genes 2. degradation of cyclins 3. modification of (Cdk) kinase subunits by phosphorylation (kinases) and/or dephosphorylation (phosphatases) ubiquitin-proteasome system (UPS) functions as an ATP-dependent proteolytic system that requires polyubiquitination via lysine residues of the target protein prior to its degradation by the 26S proteasome polyubiquitination involves the concerted action of three enzymes that confers substrate specificity: 1. E1 - ubiquitin- activating 2. E2 - ubiquitin-conjugating 3. E3 - ubiquitin ligase 26S proteasome is a large, multi-catalytic protein complex composed of 19S regulatory complex and 20S proteasome damaged, misfolded, or mutant proteins degraded by 3 major peptidase activities - chymotrypsin-like, trypsin-like, and caspase-like

mammalian expression vector - example

pcDNA3.1/Zeo 5.0 kb Pcmv BGH pA f1 SV40 ori Zeocin SV40 pA pUC ori ampicillin + or - T7

molecular complementarity

permits tight protein binding via multiple noncovalent interactions complementary size and shape of ligands and their binding sites ionic/charge-charge interactions hydrogen bonding interactions hydrophobic (non-polar) and/or hydrophilic (polar) interactions fewer non covalent interactions = less stable complex

oncogene activation of the extracellular signal-regulated kinase (ERK) mitogen activated protein kinase (MAPK) cascade

persistent activation of the ERK MAPK cascade occurs in many different human cancers epidermal growth factor receptor (EGFR) overexpression and/or activating mutations of EGFR cytoplasmic kinase domain mutationally activated B-Raf and/or Ras activated ERKs translocate to the nucleus, where they phosphorylate and regulate transcription factors, leading to changes in gene expression ERK-mediated transcription can result in the upregulation of EGFR ligands, such as TGFα, thus creating an autocrine feedback loop that is critical for Ras-mediated transformation and Raf-mediated gene expression changes

intrinsic and acquired cancer drug resistance

pharmacokinetic (PK) and pharmacodynamic (PD) factors influence drug resistance - absorption, distribution, metabolism, excretion (drug influx and efflux within the cancer cell) downstream processes that influence drug resistance - DNA damage and enzyme inhibition

CDK activating kinases (CAKs) and cell division cycle 25 (CDC25)

phosphatases regulate CDK-cyclin mediated cell cycle progression CDK activity positively regulated by association with cyclins and by phosphorylation of T-loop threonine by CDK activating kinase (CAK) inhibitory phosphorylation of adjacent threonine and tyrosine residues is mediated by dual specific kinases (Wee1 & MYT1) inhibition is released after cell division cycle 25 (CDC25) phosphatases dephosphorylate these residues orange - negative/inhibitory phosphorylation site

STAT serine phosphorylation - JNK and p38 MAPKs, PKC, and PKA

phosphorylation of single carboxy-terminus serine residue (S727) enhances STAT transcriptional activation post tyrosine phosphorylation

example of fluorescence

promoter -> cDNA -> GFP cDNA and GFP make up the reporter construct transcription occurs from the promoter on this results in fluorescence transduction or transfection - know this terminology

STAT tyrosine phosphorylation RTKs, JAKs, Src, and Tec tyrosine kinases

phosphorylation of single carboxy-terminus tyrosine (Y701/Y705 /Y695) STATs form homo- or hetero-dimers via reciprocal SH2 domain interactions dimerization uncovers nuclear localization signal and dimers translocate to nucleus STAT dimers bind specific 9 base pair DNA sequences (5'TTCCNGGAA3') in target gene regulatory regions - promoters and enhancers

cooperative ligand binding - no ligand

pink segments are flexible and few confirmations facilitate ligand binding green segments are stable in low affinity state

bilayer

polar head groups surface exposed to water and acyl side chains directed towards each other in the interior

What's wrong with agarose gels?

poor precision low sensitivity short dynamic range < 2 logs low resolution non-automated - cannot be incorporated into one large automatic system with PCR size-based discrimination only results are not expressed as numbers ethidium bromide staining is not very quantitative

introns and exons

post-translational modification mRNA transcript - 5'UTR -> exon 1 -> intron A -> exon 2 -> intron B -> exon 3 -> intron C -> exon 4 -> intron D -> exon 5 -> 3'UTR splicing of introns A, B, C, and D - introns removed to form mature RNA information about what genes are transcribed spliced mRNA contains only exons 1-5 research and clinical applications

intrinsic resistance factors - cancer drug resistance

pre-exist in sub-populations of tumor cells to impart drug resistance coexistent genetic alterations to drug targets mutations in other signaling pathway genes inactivation of pro-apoptotic pathways patient and or drug specific PK/PD features - ADME properties, drug efflux transporter expression, drug metabolism, and drug-drug interactions

immune checkpoint cancer therapies

prevent immune cells from attacking tumor cells - turn this off through medication to kill cancer cells

CB1 and CB2 cannabinoid receptors

primary active constituent of the Hemp plant - Cannabis sativa is D9-tetrahydrocannabinol synthetic and endogenous cannabinoid ligands synthesis of high affinity cannabinoid ligands led to cloning and characterization and cannabinoid GPCR's endocannabinoids - "the cannabinoids within"

principle of immunofluorescence

primary antibody, secondary antibody with fluorescent dye antibodies interact with the protein of interest and fluorescence grows stronger with more interaction signal that can be amplified

FRET example

probe, fluorophore, and quenching molecule 1. fluorophore signal is quenched when the probe forms a semi stable hairpin - hairpin structure brings the two closer together 2. probe binds preferentially to target DNA - fluorophore is separated from the quenching molecule and the fluorescence signal increases (hairpin structure is let go) - quenching - farther away, signal increases quantifies the amount of product very accurately PCR cycle number versus signal (arbitrary units) - baseline -> plateau exponential phase - sample 1, 2, and 3

bio-signaling and signal transduction

process how cells receive and respond to signals in the microenvironment outside the plasma membrane information received by receptors transduced into altered cellular responses alterations and changes can be positive or negative: 1. altered gene expression - mRNA and/or protein levels 2. enzyme or metabolic activity - activate or inhibit 3. changed sub-cellular localization - expose or conceal targeting signals 4. protein-protein interactions - establish or disassemble PPI complexes

effects of protein phosphorylation

protein kinases use ATP as a substrate to phosphorylate Tyr, Ser, and Thr residues of proteins to regulate their activity activate an enzyme activity inactivate an enzyme activity create domain docking sites for protein-protein interactions induce translocation to a sub-cellular localization target proteins for proteasome degradation protein phosphatases regulate and terminate kinase signaling by de-phosphorylating proteins

kinases regulate protein phosphorylation and activity

protein kinases use ATP as a substrate to phosphorylate tyrosine (Tyr), serine (Ser), and threonine (Thr) residues of proteins to regulate their activity effects: 1. activate an enzyme activity 2. inactivate an enzyme activity 3. create domain docking sites for protein- protein interactions 4. induce translocation to a sub-cellular localization 5. target proteins for proteasome degradation

phosphatases regulate protein phosphorylation and activity

protein phosphatases regulate/terminate kinase signaling by de-phosphorylating proteins effects: 1. activate an enzyme activity 2. inactivate an enzyme activity 3. create domain docking sites for protein- protein interactions 4. induce translocation to a sub-cellular localization 5. target proteins for proteasome degradation

human genome: DNA sequence types

protein-coding genes - 1.5% introns - 25.9% miscellaneous unique sequences - 11.6% long repetitive sequences (centromeres, telomeres) - 8% segment duplications - 5% simple-sequence repeats - 3% other transposons - 11.2% SINEs - 13.1% LINEs - 20.4%

induced fit model

proteins are flexible ligand binding induces conformation changes - protein and/or ligand may change conformation - changes may be subtle or dramatic - active site can change the shape to fit with substrate structural adaptation/induced fit - allows for tighter binding of the molecule - may increase affinity of protein for a second ligand more widely accepted theory than lock and key model

next-generation pyrosequencing

pulse in dATP - no flash of light, dATP degraded by apyrase pulse in dGTP - base is incorporated, pyrophosphate is released sulfurylase converts pyrophosphate into ATP in the presence of ATP, luciferase reacts with luciferin - flash of light DNA sequencing by PCR amplification powerful approach - increasing light with increasing the sequence fireflies!

How to purify the gene products (proteins)?

purifying proteins is very difficult 1. gel filtration - separate based on molecular weight 2. ion-exchange - differences of surface charge 3. affinity - based on the principle of specific binding

real-time PCR

real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

signal transduction feature: desensitization/adaptation

receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface downstream effects proportionally termination - negative feedback discontinues the effector function feedback loops used to turn off signal

growth factor receptor tyrosine kinases (RTKs)

receptors are the first substrates RTKs phosphorylate: - auto-phosphorylation of sites in RTK tyrosine kinase domains (TKDs) have important regulatory roles - phosphorylation of insulin receptor activation loop increases catalytic efficiency 50-200 fold (self-phosphorylation) additional tyrosine's are then phosphorylated in other parts of the cytoplasmic regions of RTKs: - P-Tyr's create specific docking sites for the assembly of downstream signaling molecules recruited to receptors activated by growth factor binding - IRS (insulin response signaling) proteins fulfill this function for the insulin receptor

amplification of a DNA fragment by PCR - method 1

region of target DNA to be amplified is identified 1. heat to separate strands 2. add synthetic oligonucleotide primers and cool 3. add thermostable Taq DNA polymerase to catalyze 5' -> 3' DNA synthesis 4. repeat steps 1 and 2 5. DNA synthesis (step 3) is catalyzed by the thermostable DNA polymerase, which is still present 6. repeat steps 1-3 7. after 20 cycles, the target sequence has been amplified about 10^6-fold different steps occur at different temperatures - high temperature denatures polymerase, which is why thermostable DNA polymerase must be used

role of telomeres and telomerase in cancer

reverse transcriptase telomerase adds new DNA to ends of chromosome telomeres contain kilobases of TTAGGG nucleotide repeats and Shelterin protein - form special t-loop structures to protect chromosome ends from end-to-end fusions and degradation, and prevent recognition as ssDNA and dsDNA breaks normal cells progressively lose telomeres during cell division until a few short telomeres become uncapped and undergo a form of growth arrest termed "senescence" - senescent cells without genomic alterations remain quiescent but produce different proteins than young quiescent cells specific genetic and epigenetic alterations allow normal cells to bypass replicative senescence and proliferate until many telomere ends become uncapped, leading to crisis cells in crisis with critically shortened telomeres continuing to divide lead to apoptosis and/or ongoing genomic instability on rare occasions, cells that express telomerase maintain short stable telomeres and escape crisis, progressing to cancer activation of telomerase is a mechanism to slow down the rate of genomic instability due to dysfunctional telomeres telomerase does not drive the oncogenic process - rather it is permissive, required for sustained growth of most cancers since telomerase is not expressed in most normal human cells, its a target for cancer drug clinical development - inhibitors of telomerase

reverse transcriptase PCR (RT-PCR)

reverse transcription - converting mRNA into DNA (forming dsDNA to enter PCR) creating dsDNA from an mRNA template - placing DNA product into PCR: 1. to amplify 2. to quantify 3. to identify translated into a function protein

P-type ATPases

reversibly phosphorylated during transport cycle sarcoplasm/endoplasmic reticulum Ca2+ ATPase (SERCA)

specific binding

saturable at increased [Ligand] specific binding = total - non-specific

scaffolding proteins and signal transduction

scaffold proteins play essential roles in assembling and controlling signaling cascades localize signaling molecules to specific locations in cells and elevate effectiveness protect signaling proteins from deactivation and/or degradation regulate thresholds and dynamics of signaling by controlling positive and negative feedback signals 1. amplification - positive feedback 2. inhibition - negative feedback determine the nature and/or duration of the signaling output analogue (graded) signal, digital (switch-like) signal, sustained or transient signal, and oscillatory signal

cooperative ligand binding - second ligand binding to second subunit

second ligand binding occurs with higher affinity than binding of the first molecule, giving rise to positive cooperativity

membrane fusion during release of neurotransmitter at a synapse

secretory vesicle membrane contains v-SNARE synaptobrevin (red) target PM contains t-SNAREs syntaxin (blue) and SNAP25 (violet) local increase in [Ca2+] triggers release of neurotransmitter v-SNARE, SNAP25, and t-SNARE interact, forming a coiled bundle of 4 α helices, pulling 2 PMs together and disrupting the local bilayer joining the inner leaflets of the two PMs leads to hemifusion, then to complete membrane fusion and neurotransmitter release

inactivation of anandamide

selective anandamide reuptake transporter fatty acid amidohydrolase mediated degradation of anandamide and 2-Arachidonyl glycerol

voltage gated neuronal Na+ channels

selectivity filter - pore region activation gate inactivation gate voltage sensor Na+ channels of different tissues and organisms have a variety of subunits, but only the principal subunit α is essential α subunit is a large protein with four homologous domains I to IV, each containing six transmembrane helices (1 to 6): 1. helix 4 (blue) is the voltage sensor 2. helix 6 (orange) is the activation gate 3. segments between helices 5 and 6, pore region (red), form the selectivity filter 4. segment connecting domains III and IV (green) is the inactivation gate

cellular senescence

senescence is irreversible growth arrest - characterized by altered morphology, gene expression pattern and chromatin structure, and activated DNA damage response senescence has a dual role in tumor development - prevents the proliferation of seriously damaged cells through activation of ATM, p53 and the DNA damage response (DDR) genetic stress from oncogene activation induces senescence using similar downstream components - DNA damage activation, altered gene expression, and altered chromatin structure senescence is a powerful tumor suppressor protecting cells expressing activated oncogenes from becoming neoplastic the presence of oncogene induced senescent cells in early pre-malignant tumor stages suggests that senescence must be overcome during tumorigenesis to progress to malignancy

receptors

sense extracellular environment to receive and transduce signals downstream signaling induces molecular changes in the cell

adopted orphan nuclear receptors

sequence homology to endocrine NRs and "de- orphanized" by identification of naturally occurring ligand identification of vitamin A derivative, 9-cis retinoic acid as an endogenous high-affinity ligand for RXR - first true adoption of an orphan NR low affinity NRs (Kd = μM range) for dietary lipids and xenobiotics form heterodimers with RXR NRs regulate lipid and/or glucose homeostasis controlling uptake, synthesis, storage or clearance "enigmatic" adopted orphans - ligand identified but the nature of ligand-dependent regulation in physiology has not been established

signal transduction features

signal -> reception -> integration/adaptation -> amplification -> target -> desensitization 1. affinity/specificity/selectivity - complementarity of ligand:receptor interaction - restricted receptor/target expression by tissue or cell type 2. sensitivity - cooperativity, integration, amplification 3. desensitization and termination of signaling

coordinated assembly of multi-protein complexes in RTK

signaling provides branching points in signaling network fibroblast growth factor (FGF) receptor substrate-2 (FRS2α) docking protein forms a complex with activated FGF-Rs (or nerve growth factor receptors, NGF-Rs) through phospho-tyrosine (Tyr-P) binding domains (PTBs) activated RTK phosphorylates multiple Tyr's on FRS2α, recruiting multiple Grb2 and Shp2 molecules, and bringing a second docking protein Gab1 to the complex Gab1-Tyr-P recruits additional signaling proteins, including phosphoinositide 3-kinase (PI3K) PI3K generates PtdIns (3,4,5) P3 (PIP3), initiating a positive feedback loop where more Gab1 is recruited, leading to further PI3K activation multiple domains of phospholipase C-γ (PLCγ) cooperate to integrate multiple signals at the plasma membrane N-terminal PLCγ SH2 domain responsible for complex formation with activated RTKs C2 and PH domains cooperate with SH2 domain targeting of PLCγ to plasma membrane PH domains specifically recognize products of RTK- activated PI3K RTK-mediated phosphorylation of PLCγ Tyr-P-783 intra-molecular binding of C-terminal SH2 domain stimulates PLCγ hydrolysis of PtdIns (4,5) P2 (PIP2) to produce Ins(1,4,5) P3 (IP3) and diacylglycerol(DG) cascade of downstream signaling

tumor microenvironments

solid tumor microenvironment - parenchyma and stroma of tumors contain assemblies of distinct cell types and subtypes that collectively enable tumor growth and progression tumor associated immune inflammatory cells can be either tumor-promoting or tumor-killing subclasses multiple stromal cell types create a succession of tumor microenvironments that change as tumors invade normal tissue and seed and colonize distant tissues abundance, histologic organization, and phenotypic characteristics of the stromal cell types and ECM evolve during progression, thereby enabling primary, invasive, and metastatic growth abundance and characteristics of surrounding normal cells of primary and metastatic sites likely also affect the character of the various neoplastic microenvironments

protein kinase inhibitor challenges

specificity and selectivity are major issues - kinase active site is highly conserved not very selective kinase inhibitors - ATP competitive inhibitors Tyr-kinase - leukemia disease phenotype competes for active site binding - ATP binding site

types of microarrays

spotted (cDNA) - robotic transfer of cDNA clones or PCR products synthetic (oligo): 1. direct oligo synthesis on solid microarray substrate 2. uses photolithography (Affymetrix) or ink-jet printing (Agilent) all configurations assume the DNA on the array is in excess of the hybridized sample, thus the kinetics are linear and the spot intensity reflects that amount of hybridized sample labeling can be radioactive, fluorescent (one-color), or two-color - visualizing the DNA

organization of MAPK kinase signaling cascades

stimulus -> MAPKKK -> MAPKK -> MAPK -> biological response different signaling pathways exist that lead to different biological responses

yeast artificial chromosomes(YACs)

suitable for maintaining eukaryotic chromosomes a yeast origin of replication - do not replicate in bacteria (no ori that is recognized, so must introduce a new origin, aka shuttle vector) two selectable markers specialized sequences derived from the telomeres and centromere that are needed for stability and proper segregation of the chromosomes at cell division

cancer therapy options

surgical resection: 1. tumors that are confined locally and accessible 2. outcome is stage dependent radiation therapy: 1. tumors that are confined locally and accessible 2. typically combined with surgery and chemotherapy chemotherapy: 1. often combined with surgery and radiation 2. mono-therapy with cytotoxic compounds 3. mono-therapy with molecularly targeted agents 4. drug combination therapy immunotherapy - typically combined with chemotherapy

biosynthesis of anandamide

synthesis and release upon demand stimulus-induced remodeling of phosphoglycerides de-novo synthesis routes

commonly used protein tags

tag protein/peptide - molecular mass (kDa), immobilized ligand protein A - 59, Fc portion of IgG (His)6 - 0.8, Ni2+ glutathione-S-transferase (GST) - 26, glutathione maltose-binding protein - 41, maltose beta-galactosidase - 116, p-Aminophenyl-beta-D-thiogalactoside (TPEG) chitin-binding domain - 5.7, chitin these do not naturally exist in proteins - introduce tags to the protein of interest

GPCRs play key roles in normal physiology and disease

targeted by around 30-40% of all marketed drugs 50% of all recently launched drugs are targeted against GPCRs annual worldwide sales exceeding $30 billion in 2001 among the 100 top-selling drugs, 25% target GPCRs

µ, δ & κ opioid receptors - GPCR's

targets of anti-pain medication and drugs of abuse opiate analgesic drugs - codeine, oxycodone, etc. drugs of abuse - heroin, fentanyl heroin antidote - naloxone gamma-aminobutyric acid chief inhibitory neurotransmitter GABA regulates the amount of release of dopamine - decreases the release into the synaptic cleft morphine and heroin turn off GABA - increase dopamine release

partial digestion

terminate the reaction before completion a useful strategy in the construction of the genomic library: 1. a given sequence may appear in many DNA fragments of different sizes 2. overlapping fragments help to delineate the entire genomic sequences partial restriction enzyme digestion allows cloning of overlapping fragments - complete digestion and many DNA copies (DNA cut at the individual size) we don't know the alignment of the sequences - random digestion, which helps to determine the alignment

targeted cancer drug therapy

the Achilles heel of cancer? clinical experience with molecularly targeted drug monotherapy - modest efficacy with responses that are neither rapid nor durable

molecular cloning

the general scheme of cloning restriction endonucleases, DNA ligases, and others (tool box) three commonly used gene vectors: 1. plasmids 2. bacterial artificial chromosomes (BACs) 3. yeast artificial chromosomes (YACs) plasmids and BACs are maintained and replicated in bacteria as the host cell

purify your DNA fragments

the insert of interest that you want to clone into your plasmid needs to be separated from the other DNA you can separate your fragment using gel electrophoresis - separation based on size you can purify the DNA from the gel by cutting the band out of the gel and then using a variety of techniques to separate the DNA from the gel matrix

RAS GTPase family oncogenes

three RAS genes encode four distinct but homologous RAS proteins - HRAS, NRAS, KRAS4A, and KRAS4B couple cell surface receptors to intracellular signal transduction pathways RAS proteins cycle between GTP-bound 'on' and GDP-bound 'off ' conformations - guanine nucleotide exchange factors (GEFs) promote RAS activation by stimulating GDP-GTP exchange GTPase-activating proteins (GAPs) accelerate RAS mediated GTP hydrolysis and inactivation inactivation of RAS activity by GAPs - highest incidence of somatic mutations in oncogenic variants of RAS alleles persistence of the GTP-bound state of RAS and incessant activation of a multiple RAS-dependent signaling pathways RAS GTPase oncogenes impact multiple cell pathways and functions: 1. tumor cell proliferation 2. apoptosis 3. energy metabolism 4. angiogenesis

tumor microenvironment: angiogenesis and immunosuppression

tissue microenvironment critical in angiogenesis stroma of solid cancers show signs of inflammation and infiltration by many leukocyte populations: 1. endothelial cells, fibroblasts, neutrophils, eosinophils, basophils, monocytes/macrophages, dendritic cells, natural killer cells, and lymphocytes 2. tissue microenvironment influences angiogenesis, immunosuppression, and metastasis although many of these leukocytes are potentially capable of killing tumor cells, experimental and clinical evidence suggest that in many cases they can contribute to tumor progression - myeloid derived suppressor cells (MDSCs), immunosuppressive regulatory T cells (Tregs), etc. cancer immunity mediated by cytotoxic T-cells (CTLs) suppressed by molecules secreted by tumor associated immune and stromal cells, along with cell surface expression of molecules (CD80-CTLA-4 &PD-L1-PD-1) that contribute to energy and exhaustion of anti-cancer immune cells

DNA repair mechanisms maintain genetic stability

to maintain genetic stability cells, have developed extensive DNA repair mechanisms correct any damage that occurs arising from cellular metabolism and/or the external environment direct repair corrects alkylated bases to a normal base without any intervening steps or DNA strand breaks other repair mechanisms involve DNA strand breaks: 1. base excision repair (BER) 2. nucleotide excision repair (NER) 3. mismatch repair (MMR)

ligand binding

total binding, specific binding, and nonspecific binding Kd dissociation constant at equilibrium = measurement of affinity Bmax = number of binding sites available

the use of epitope tags to study protein-protein interaction

transcription - promoter -> cDNA -> epitope tag 1. express tagged protein in a cell 2. make cell extract 3. immunoprecipitate - precipitate tagged protein with specific antibody (pull down all associated proteins - A, B, C, etc. in non-disruptive conditions) 4. separate precipitated proteins - PCR (size markers, pure tagged protein, and immunoprecipitate) 5. identify new proteins in immunoprecipitate limitation - artificial system that does not occur naturally in the cell, which may confer different protein interactions

β-adrenergic signaling pathway

transduction of the epinephrine signal epinephrine (E), binding to it's GPCR, activates adenylyl cyclase (AC) by a stimulatory G protein (Gs) hormones that induce GTP binding to Gs activate adenylyl cyclase, resulting in higher cellular [cAMP] 1/2. EPI binds to receptor, causing replacement of GDP bound to Gs by GTP, activating Gs 3/4/5. Gs moves to adenylyl cyclase and activates it, catalyzing the formation of cAMP, which activates PKA 6. PKA phosphorylates cellular proteins, causing cellular response to EPI 7. cAMP is degraded, reversing the activation of PKA

fluorescence resonance energy transfer (FRET)

transfer of excited state energy from one fluorophore to another transfer from CFP -> YFP - quantified by excitation and emission spectra plotting wavelength (nm) versus relative fluorescence emission wavelengths overlap with second probe distance plays a role - closer for more efficient energy transfer

simplified model of G1/S transition and checkpoint induction

transition from G1 to S phase initiated by mitogen-stimulated cyclin D/Cdk4/6 phosphorylation of pRb and E2F transcription factor release activates positive feedback loop, making cell cycle progression independent of mitogenic stimulation - restriction point (R-point) up-regulation ofcCyclin E in complex with Cdk2 further stimulates E2F release and transcription of genes necessary for S phase after double-stranded DNA break (DSB) induction, two parallel checkpoint pathways target the activity of cyclin/Cdk complexes slower pathway involves the stabilization of p53 and transcriptional up-regulation of p21, which binds and inhibits cyclin/Cdk complexes faster pathway - activated Chk2 inactivates Cdc25, which can no longer remove the inhibitory phosphates of the cyclin E/Cdk2 complex

ion channels and transporters

transporters and channels carriers - secondary active transporters, uniporters, primary active transporters SLC series: coupled transporter, exchanger, passive transporter, vesicular transporter, and mitochondrial transporter other transport proteins: water channel, ion channel, pump, ABC transporter

tumor cells acquire a self sufficiency for growth signals

tumor cells exhibit a reduced dependence for growth stimulation exposed to elevated concentrations of extracellular growth signals synthesize and secrete growth factors to create an autocrine/paracrine positive feedback signaling loop oncogenes may mimic growth signaling pathways over-expression of growth factor receptors mutations of growth factor receptors conferring constitutive activity altered integrin expression biased to those transmitting growth signals mutations activating intracellular growth signaling pathways

tumors acquire sustained angiogenesis

tumor growth is angiogenesis dependent antingiogenic therapy targets the genetically stable microvascular endothelial cell many extracellular and intracellular molecules modulate angiogenesis - growth factors and receptors, adhesion molecules, ECM proteins, remodeling and guidance molecules, matrix-degrading proteinases, signaling molecules, transcription factors/inhibitors, and homeobox gene products adult vasculature stable and rarely proliferates under physiological conditions in cancer - existing vessels start to grow (neo-angiogenesis) again in response to Hypoxia Inducible Factor-driven tumor vascular endothelial growth factor (VEGF) expression newly formed vessels provide oxygen and nutrients to expanding tumors

acquisition of the metastatic phenotype

tumors - heterogeneous populations of stromal cells and cancers cells in different differentiation stages, including cancer stem cells (CSC) poised to complete metastasis intrinsic CSCs in early tumorigenesis - oncogenic derivatives of normal-tissue stem/progenitor cells epithelial-to-mesenchymal transition (EMT) is a trans-differentiation program critical to embryonic morphogenesis - driven by EMT-inducing transcription factors, EMT deployed during several critical morphogenesis steps enables cells of epithelial phenotype to generate mesenchymal derivatives induced CSCs may arise because of EMT carcinoma cells initially recruit stromal cells (fibroblasts, myofibroblasts, granulocytes, macrophages, mesenchymal stem cells, and lymphocytes) to create a reactive microenvironment releasing factors (Wnt, TGFb, and FGF) that induce cancer cells to undergo EMT and acquire CSC-like characteristics CSCs promote metastasis