Exam 1

oncogenes and tumor supressors

-cancer-- evolutionary process resulting from accumulation of somatic mutations in the progeny of normal cells --provides a selective growth advantage in mutated cells and leads to uncontrolled proliferation -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 (oncogene addiction) -alterations to tumor supressor genes cause loss of function effects that contribute to the malignant phenotype --tumor supressor genes are typically negative regulators of growth of affect invasive and metastatic potential --"two hit" hypothesis- both alleles of the gene must be lost to unmask the malignant phenotype

passage through the plasma membrane

-diffusion down a concentration and/or electrical gradient - large amounts of energy required to remove hydration shell and to move solutes to the plasma membrane but transporters lower this energy -electrically neutral solutes diffuse down a concentration gradient -electrically charged solutes diffuse down an electrochemical gradient - when concentration and charge are equal, the system is at equilibrium -Relative to simple diffusion, a transporter protein reduces the free energy cost of activation required for removal of the hydration shell and passage of a hydrophilic solute through the hydrophobic core of the membrane. Electrically neutral solutes diffuse down the concentration gradient. Charged solutes diffuse down an electrochemical gradient comprised of both the electrical potential Vm and the chemical concentration.

cell growth

-during embryonic growth and development- cell division occurs in every tissue -most adult tissues are quiescent -cell's decision to divide is critical to the organism because unregulated cell growth results in cancer -normal cell division proceeds in a precisely ordered sequence of events termed the cell cycle -assures every daughter cell contains full complement of molecules required for life

acquired capabilities of cancer

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

desmosomes (macula adherens)

-focal adhesion junction between adjacent cells -desmosomes provide strong attachment between cells-- prominent in tissues subject to stress e.g. skin and cardiac muscle -multiprotein complexes, where adhesion is provided by transmembrane catherine proteins, desmogleins, and desmocollins -desmosomes interact with intermediate filaments rather than actin or microtubules

gap junctions

-focal adhesion junction between adjacent cells -formed between adjacent cells to allow low molecular weight substances (<1000 kDa amino acids, sugars, ions, and chemical messengers) to pass between cells, permitting metabolic and electric coupling (e.g. cardiomyocytes)

tight junctions (zonula occludens)

-focal adhesion junction between adjacent cells -integral membrane proteins claudine and occludens hold cells together-- formed at apical sides of cells to provide a regulated barrier to the movement of ions and solutes through and between cells: 24 claudine differentially expressed in different cell types regulate paracellular transport -confer polarity to cells acting as a gate between the apical and bass-lateral membranes to precent diffusion of membrane lipids and proteins

adherents junctions

-focal adhesion junction between adjacent cells -multiprotein intercellular adhesive structures, prominent in epithelial tissues e.g. fascia adherents in cardiac muscle -attach to actin microfilaments and microtubules within cells via multiple additional proteins -apical sides of epithelial cells, zonula adherents attaches to circumferential actin stress fibers

basement membrane adhesion

-focal adhesions and hemidesmosomes -cells adhere to non-basal lamina ECM via secreted proteins such as fibronectin and collagen -cells adhere to basal lamina proteins via multi protein complexes (e.g. keratin or vimentin) -integrins are the key adhesive proteins which change shape upon binding to ECM, a process called "outside-in" signaling -integrins interact inside cells with the cytoskeleton and complex array of over 150 proteins that influence intracellular signaling pathways affecting proliferation, survival, shape, mobility and gene expression

General Types of Signal Transduction

-gated ion channel -receptor enzyme -serpentine receptor -receptor with no intrinsic enzyme activity -steroid receptor -adhesion receptor

cellular differentiation

-process by which less specialized cells become more specialized -continuous during development of a multicellular organism from a simple zygote to a complex system of tissues and cell types -dramatically alters a cell's size, shape, membrane potential, metabolic activity, and responsiveness to signals -adult stem cells divide and create fully differentiated daughter cells during tissue repair and normal cell turnover -cell differentiation is largely due to highly controlled modifications in gene expression --cellular differentiation almost never involves a change in the DNA sequence itself --cells have very different physical characteristics despite having the dame genome

Cancer therapy options

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

RAS Oncogene

-three RAS genes encode four distinct but homologous RAS proteins: HRAS, NRAS, KRAS4A, and KRAS4B --couple vell 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 -Inactivation of RAS activity by GAPs --highest incidence of somatic mutations in oncogenic variants of RAS alleles --persistance of the GTP-bound state of RAS and incessant activation of a multitude of RAS-dependent signaling pathways

cancer cells acquire limitless replicative potential

-uncoupling cell's growth program from signals in it's environment --growth signal autonomy --insensitivity to antigrowth signals --resistance to apoptosis -cell-autonomous program limits multiplication --normal cells in culture have a finite replicative potential afterwards they progress to senescence and eventually crisis --tumor cells in culture appear immortalized

Factors Affecting the Sensitivity of Signal Transduction

1) Affinity (lower the Kd the higher affinity) 2) Cooperativity (oxygen-hemoglobin binding) 3) Amplification (enzymes activate enzymes, the number of affected molecules increase geometrically in an enzyme cascade ex. secondary messengers and G-proteins) 4) Protein Modularity (Multiple domains in signaling proteins allows for assembly into multiple complexes. Cells mix & match signaling proteins to create variety of signaling complexes.) 5) Desensitization (receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface) 6) Integration (when two signals have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm the regulatory outcome results from the integrated input from both receptors)

Termination of beta-adrenergic signal

1) Circulating epinephrine concentrations fall below threshold required for receptor occupancy 2) self inactivation of Gs 3) cAMP degraded by intracellular phosphodiesterase enzymes 4) Receptor desensitization by beta-arresting mediated internalization

DNA replication

1) Helices unwind the parental double helix 2) single-strand binding proteins stabilize the unwound parental DNA 3) The leading strand is synthesized continuously in the 5'-->3' direction by DNA polymerase 4) The lagging strand is synthesized discontinuously. Primase synthesizes a short RNA primer, which id extended by DNA polymerase to form an okazaki fragment. 5) After the RNA primer is replaced by DNA (by another DNA polymerase) DNA ligase joins the Oakazki fragment to the growing strand. A variety of enzymes orchestrate and participate in DNA replication including, DNA polymerases, helicases, single strand binding proteins, primases, and DNA ligases. DNA polymerases ensure the complementary of base pairing that conserves the base sequence of DNA.

Features of Living Organisms

1) High degree of chemical complexity and microscopic organization. 2) Systems to extract, transform & use energy from the environment 3)Defined function for each component & regulated interactions among them 4) Mechanisms to sense & respond to alterations in their surroundings 5) Capacity for precise self-replication & assembly 6) Capacity to change over time by gradual evolution

human kinome

518 putative protein kinase genes 7 major groups: AGC- PKA, PKG, PKC CAMK- calcium calmodulin-dependent CK1- casein kinase 1 CMGC- CDK, MAPK, GSK3, CLK STE- homologs of yeast sterile kinases TK- tyrosine kinases TKL- tyrosine kinase-like 244 kinases map to disease loci or cancer amplicons there is genetic evidence that kilometers mutations cause disease

Nrs associated with metabolic diseases and their ligands

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P-type ATPases

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

Selectivity and Specificity of GPCR Signaling

-Receptor 791 GPCRs -- 3 major sub-families -- homo and herero dimerization of GPCRs and other oligomers -Heterotrimeric G-proteins - alpha beta and gamma subunits --distinct Galpha subunits activate different second messengers --G beta gamma subunits also activate signaling pathways -Ligands --orthosteric (ligand binding site) &/or allosteric modulators -differential receptor expression --cells, tissues and organs

DNA damage checkpoints

Checkpoints can be seen as a network of surveillance systems, i.e., signal transduction systems, that interrupt cell cycle progression, when damage to the genome or failure of a previous activity in the cell cycle is detected. DNA damage checkpoints are associated with biochemical pathways that end, delay or arrest cell cycle progression. In the simplifed version these 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.

Caspase-independent pathway

Caspase-independent, GRANZYME A (GrA)-mediated pathway. After being delivered into the target-cell cytosol through Ca2+-dependent, perforin-mediated pores, GrA triggers a pathway that is characterized by the formation of single-stranded DNA nicks and the appearance of apoptotic morphology. The endonuclease involved in the formation of DNA strand breaks in this system is GAAD (GrA-activated DNase), also known as NM23-H1. GAAD activity is inhibited by its specific inhibitor IGAAD, also known as the SET complex, which is located in the endoplasmic reticulum (ER). This complex contains an inhibitor of protein phosphatase 2A (pp32), the nucleosome assembly protein SET, HMG2 and Ape1 (apurinic endonuclease-1, also known as redox factor-1; Ref-1). In this pathway, GrA cleaves SET, HMG2 and Ape1, but not pp32, to release and activate GAAD. Active GAAD translocates to the nucleus to induce DNA strand breaks.

Fluid Mosaic Model of Plasma Membranes

Fatty acyl side chains in the interior of the membranes form a fluid hydrophobic region. Integral membrane proteins are immersed in the lipid held by hydrophobic interactions with their non-polar amino acids side chains. Both proteins and lipids are free to move laterally in the plane of the membrane bilayer, but movement from one leaflet (side) of the bilayer to the other is restricted. Carbohydrate moieties attached to some proteins are exposed at the external surface of the membrane.

PKA activation

GPCR agonist activated Gsaplha activates adenylyl cyclase to elevate cAMP and activate PKA Inactive PKA regulatory subunits: empty cAMP sites Catalytic subunits: substrate-binding sites blocked by auto inhibitory domains of R subunits Regulatory subunits: autoinhibitory domains buried Active PKA Catalytic subunits: open substrate-binding sites

GPCR Hetero- and Homo-Dimerization

GPCR dimerization can alter receptor pharmacology 1) produce functional dimers 2)Result in pharmacological diversity 3)Exhibit transactivation and amplification 4)Induce internalization and desensitization

Mu, delta, kappa opioid receptors

GPCR's targets of anti-pain medication and drugs

Interaction between cells and the extracellular matrix

Integrins, integral proteins expressed in the plasma membrane of cells mediate binding to the ECM comprised of collagen, fibronectin, laminin, and proteoglycans.

purine and pyrimidine bases and corresponding nucleotides of DNA and RNA

DNA and RNA are composed of deoxyribonucleotides and ribonucleotides that are composed of purine and pyrimidine bases attached to deoxy-ribose or ribose respectively.

DNA to Proteins

DNA is Transcribed into mRNA and that is Translated into Proteins. DNA stores the genetic information its sequence of nucleotides from which all other cellular components are generated. DNA sequence of genes transcribed into mRNA. mRNA translated into an unfolded polypeptide of linked amino acids. Folding produces the secondary and tertiary structures required for enzymatic activity/function. Protein-protein interactions lead to the formation of supramolecular complexes.

S phase

DNA synthesis doubles the amount of DNA in the cell. RNA and protein also snythesized

Oncogenic RAS and Angiogenesis

Oncogenic RAS and angiogenesis: a. The induction of pro-angiogenic growth factors VEGFA & FGF2 by RAS in neoplastic cells is shown. RAS also increases the stability of VEGFA mRNA and augments its translation. b. The release of proteases by neoplastic cells cleaves components of the extracellular matrix (ECM) and releases VEGFA and FGF2, which are trapped in the ECM. This induces neo-proliferation and sprouting of microvessels towards the tumour site. c. The recruitment of macrophages by neoplastic cells (through RAS-induced nuclear factor-κB (NF-κB)-dependent production of the cytokines interleukin-6 (IL-6) and IL-8) and subsequent promotion of endothelial proliferation and sprouting by newly recruited macrophages is shown.

SAPK/JNK and ERK MAPK signaling pathway

Kinase signaling cascades are actually complex 3-dimensional pathways with multiple potential inputs and outputs with opportunities for cross talk. not linear; activation and feedback loops for signal transduction

Caspase-2-dependent pathway

The activation of pro-caspase-2 by DNA damage leads to the release of cytochrome c and apoptosome formation (as shown for the intrinsic pathway).

DNA Base Parings

The complementary anti-parallel strands of DNA follow base pairing rules; the base- paired anti-parallel strands differ in base composition and also differ in in sequence when each chain is read 5' to 3'. The linear sequence of deoxynucleotides arranged in a precise sequence encodes the genetic information. Two of the polymeric stands form the DNA double helix in which each deoxynucleotide in one strand pairs specifically with a complimentary deoxynucleotide in the opposite strand. Before the cell divides, the two strands separate and each serves as a template for the synthesis of a new, complementary strand, generating two identical double-helixal molecules, one for each daughter cell.

intrinsic mitochondrial pathway of apoptosis activation

The most important turning point in the course of the intrinsic apoptotic process occurs in the mitochondria. A pivotal event in the mitochondrial pathway is mitochondrial outer membrane permeabilization (MOMP). MOMP is mainly mediated and controlled by Bcl-2 family members. Once MOMP occurs, it precipitates cell death through either the release of molecules involved in apoptosis, or the loss of mitochondrial functions essential for cell survival. The 20 Bcl-2 family members can be divided into at least three groups. They all contain at least one of four relatively conserved Bcl-2 Homology (BH) domains. Anti- apoptotic members, including Bcl-2 and four relatives (Bcl-Xl, Bcl-w, A1 and Mcl-1) promote cell survival. The other two groups of pro-apoptotic members instead elicit cell death. The "multi-BH domain" Bax, Bak, and Bok share three domains (BH1, BH2 and BH3) with Bcl-2. The eight or more diverse BH3-only proteins, such as Bid, Bad, and Bim, posses only the short BH3 interaction domain, which is necessary and probably sufficient for the induction of apoptosis. Both pro-apoptotic groups seem to be required for launching apoptosis. The damage-sensing BH3-only proteins clearly lie upstream of Bax/Bak, because they cannot kill cells lacking both Bax and Bak. Pro-survival Bcl-2 is localized in the mitochondria, endoplasmic reticulum and perinuclear membranes. Bcl-2 family proto-oncogenes rescue cells that are destined to die without affecting the proliferation rate of the cell. Bcl-2 is involved in maintaining homeostatsis, including the mitochondrial membrane status and balance of the interactions between the pro-apoptotic members of the Bcl-2 protein family (e.g., Bax). Bcl-2 family members can inhibit cell death by sequestering or neutralizing the pro-apoptotic BH123 molecules. The BH3-only proteins trigger apoptosis in response to developmental cues, insufficient trophic support, and intracellular damage. They play distinct roles in these different death stimuli-induced apoptotic pathways. Death signals activate some BH3-only members of the Bcl-2 protein family and other proteins, which in turn induce oligomerization of the pro-apoptotic, BH123 proteins like Bax and Bak to insert into the outer mitochondrial membrane (OMM). The BH123 proteins engage either PT (permeability transition)- dependent or PT-independent mechanisms for MOMP. MOMP results in the cell death through two mechanisms including release of soluble mitochondrial intramembrane proteins (SIMPs), such as cytochrome c (cyt c) and disruption of essential mitochondrial functions Death signals function directly or indirectly on the mitochondria, resulting in the formation of the apoptosome complex. This cell death pathway is controlled by Bcl- 2 family proteins (regulation of cytochrome c release), inhibitor of apoptosis proteins (IAPs)(inhibition of caspases), and second mitochondrial activator of caspases (Smac), and Omi120 (negative regulation of IAPs). The apoptosome function is also regulated by the oncoprotein pro-thymosin- (Pro-T) and the tumour suppressor putative HLA-DR-associated protein (PHAP)121. The intrinsic pathway might also operate through caspase- independent mechanisms, which involve the release from mitochondria and translocation to the nucleus of at least two proteins, apoptosis inducing factor (AIF) and endonuclease G (EndoG). The nuclear location of AIF is linked to chromatin condensation and the appearance of high-molecular-mass chromatin fragments, whereas the role of EndoG is still unclear.

Drugs withdrawn for induced TdP

Terfenadine (antihistamine) Sertindole (antipsychotic) Astemizole (antihistamine) Grepafloxacin (antibiotic) Cisapride (GI Prokinetic)

signaling pathway through the phosphatidylinositol-3-kinase (PI3K)/AKT pathway and PTEN alterations associated with cancer

The PI3K/AKT and related pathways are important in internalizing the effects of external growth factors and of membrane tyrosine kinases. Activation of membrane kinases including epidermal growth factor receptor (EGFR) by external growth factors initiates receptor dimerization and subsequent events to activate these intracellular pathways. AKT is activated downstream of PI3K and has multiple targets. AKT and the cellular energy sensors LKB1 (STK11) and AMP-activated protein kinase (AMPK) exert opposing effects on mammalian target of rapamycin (mTOR), which is activated by AKT. ERK, extracellular signal regulated kinase; FKHR, forkhead; GDP, guanosine diphosphate; IRS, insulin receptor substrate; GSK3, glycogen synthase kinase 3; MAPK, mitogenactivated protein kinase; NF-κB, nuclear factor-κB; PIP2, phosphatidylinositol-3,4-diphosphate; PIP3, phosphatidylinositol-3,4,5- riphosphate; PKC, protein kinase C; STAT, signal transducer and activator of transcription. Numerous components of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway are targeted by amplification, mutation and translocation more frequently than any other pathway in cancer patients, with resultant activation of the pathway. PTEN mutations and deficiencies are prevalent in many types of human cancers

Molecular Organization of Cells

The organelles and other large structures of the cell are composed of supramolecular complexes (DNA & proteins/ Lipids & proteins) that are in turn made up of smaller macromolecules (Proteins, DNA, & lipid bilayers) made from smaller monomers (amino acids, nucleotide & lipids).

regulatory mechanisms of the cell cycle

The passage of cells from one stage of the cell cycle to another is tightly regulated by multiple mechanisms that act on the transcription of cyclin genes, the degradation of cyclins, and the modification of the kinase subunits by phosphorylation and/or dephosphorylation. The 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: E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase), the latter affording substrate specificity. The eukaryotic 26S proteasome is a large, multi-catalytic protein complex composed of the 19S regulatory complex and the 20S proteasome. Damaged, misfolded, or mutant proteins are degraded by three major peptidase activities (chymotrypsin-like, trypsin-like, and caspase-like).

Lectin-ligand interactions in leukocyte recruitment/migration during inflammation

Transient interactions between P-selectin on the surface of capillary endothelial cells (ECs) and glycoproteins on the leukocyte (LCs) cell surface lead to tethering and rolling of LCs on the EC surface. Near the site of inflammation stronger interactions develop between the integrins on the LC surface and glycoprotein ligands on the EC surface leading to slower rolling, arrest and adhesion. Under the influence of inflammatory cytokines and chemokines the LC begins extravasation through the capillary cell wall to the site of inflammation.

Type IV Integral membrane proteins

Transmembrane helices from multiple polypeptides form a channel

hSERT

a Na+ symporter, serotonin transporter

reentry point

a cell returning from G0 enters at early G1 phase

restriction point

a cell that passes this point is committed to pass into S phase

Phospholipid Bilayer

basic structural element of membranes. Amphipathic lipids cluster together-- hydrophobic moieties in contact with each other & hydrophilic groups interacting with the surrounding water. Membranes impermeable to polar/charged solutes Membranes permeable to nonpolar compounds. The polar hydrophilic head group and hydrophobic fatty acid side chains dictate how phospholipids behave in water. In micelles the hydrophobic fatty acids are sequestered in the interior of the sphere. In bilayers the polar side chains are at the surface exposed to the water and the acyl side chains are directed towards each other. In some cases bilayers fold upon themselves to form a 3-D liposome sphere with an aqueous cavity in the center.

adhesion receptor

binds molecules in extracellular matrix, changes conformation, thus altering its interaction with cytoskeleton

Tripartite model of steroid nuclear receptor

biological response determine by: -steroid nuclear receptor type -ligand(s) -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/enhancers

Cocaine

blocks DA reuptake transporter leading to elevated synaptic [DA]

SRC Kinase activation

de-phosphorylation of activation loop de-phosphorylation of Src-P activates the kinase Protein kinases use ATP to phosphorylate Tyr, Ser & Thr residues of substrate proteins directly regulating their activity. When the SRC tyrosine kinase is phosphorylated on a specific internal P-Tyr residue, the SH2 domain binds this internal P-Tyr bringing the SH3 domain in close proximity to an internal proline rich region and blocking productive substrate binding. In the dephosporylated active state of 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.

accidental non-programmed cell death

ccurs in response to some external insult or injury to the cell - insufficient blood flow to the tissue, heat, injury, radiation, or exposure to chemicals. -caused by factors external to the cell of tissue --insufficient blood flow, heat, injury, radiation, or chemical exposure --not reversible -commonly used term necrosis is inappropriate --necrosis refers to changes secondary to cell death by any mechanism -ischemic cell death-oncosis --caused by failure of ionic pumps of the plasma membrane --Ischemic cell death is accompanied by swelling hence "oncosis"

preclinical models to evaluate anticancer drugs

cell lines-->xenograft mice-->genetically engineered mouse models-->phase I studies Models: monolayer cultures, 3D cultures (including spheroids and multilayers)-->tumor injected subcutaneously, orthopedic models--> increase or lack of activity to a specific gene, tissue specific, temporary window--> first time a drug is administered in humans, identification of the dose based on the toxic effects observed Objective: target identification, probe of efficacy, mechanism of action-->testing of activity, biomarker identification, evaluation of toxicity--> mechanism of action biomarker validation--> biomarker studies, validation of a biomarker, probe of target inhibition

Immunoglobulin superfamily (CAMs)

cell-cell adhesion molecule can be hemophiliac or heterophilic

impaired ion channel

certain diseases and drugs can impair ion channel functions

karyolysis

chromatin dissolution by DNAases and RNAases

targeted cancer drug therapy

clinical experience with molecularly targeted drug mono therapy: modest efficacy with responses that are neither rapid nor durable -EGFR inhibitors= sustaining proliferative signaling -cyclin-dependent kinase inhibitors= evading growth suppressors -immune activating anti-CTLA4 mAb= avoiding immune destructions -telomerase inhibitors=enabling replicative immortality drugs -selective anti-inflammatory drugs= tumor promoting inflammation -inhibitors of HGF/c-MET=activating invasion and metastasis -inhibitors of VEGF signaling=inducing angiogenesis -PARP inhibitors=genome instability and mutation -proapoptotic BH3 mimetics=resisting cell health =aerobic glycolysis inhibitors= deregulating cellular energetics

nuclear receptors regulate gene expression

co-activators--> recruit basal transcription factors 1) Hormone (H), carried to the target tissue on serum binding proteins diffuses across the plasma membrane and binds to its specific receptor protein (Rec) in the nucleus 2) Hormone binding changes the conformation of Rec; it forms homo- of heterodimers with other hormone-receptor complexes and binds to specific regulatory proteins called hormone response elements (HREs) in the DNA adjacent to specific genes 3) Binding regulates transcription of the adjacent gene(s), increasing or decreasing the rate of of mRNA formation 4) Altered levels of the hormone-regulated gene product produce the cellular response hormone

collagen

collagen-coated glass (2D) a) soluble gradients absent b) forced apical-based polarity c) continuous layer of matrix d) unconstrained spreading and migration in x-y e) adhesions restricted to x-y plane f) high stiffness (GPa range) collagen gel (3D) a) soluble gradients present b) no prescribed polarity c) discrete matrix fibers d) spreading and migration statically hindered e) adhesions distributed in all three dimensions f) low stiffness (kPa range)

signals and specificity

complementarity between signal and receptor molecules confers specificity -non-covaltent receptor binding interactions similar to enzyme-substrate and antibody-antigen -receptor and intracellular target expression for a given signal pathway restricted by cell type -hormone-receptor interaction typically have Kd values 10^-9 to 10^-11= very tight binding

Type 2 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

glycogen synthase kinase 3 (GSK3)

contains a phosphoserine instead of phospholipasetyrosine de-phosporylation 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 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

Nucleus (eukaryotes) or nucleoid (bacteria)

contains genetic material-- DNA an associated proteins. Nucleus is membrane-bounded.

defects in autophagy

contribute to tumor initiation a) Autophagy, either basal or stress-induced, prevents the accumulation of oncogenic proteins such as p62, as well as damaged proteins and organelles. B) In autophagy defective tissues, p62 and damaged proteins and organelles accumulate. This is associated with the activation of oncogenic signalling pathways (nuclear factor erythroid 2-related factor 2 (NRF2) and nuclear factor-κB (NF-κB)) that promote survival but that are probably eventually overwhelmed by sustained oxidative stress. This leads to reactive oxygen species (ROS) production, chronic tissue damage, inflammation and genome instability, creating a tumour-initiating and tumour- promoting environment.

Cytoplasm

aqueous cell contents and suspended particles and organelles.

Phospholipids

are composed from a glycerol backbone with two fatty acid side chains of varying length and degree of saturation at two positions and a polar head group linked through a phosphate at the third position.

cardiac action potentials

are mediated by the coordinated action of several ion channels and currents. Re-polarization is also effected by this. Phase 0 (depolarization) sodium current Phase 1 (rapid depolarization) potassium current Phase 2 (plateau) calcium current Phase 2 (repolarization) potassium currents Phase 4 (resting membrane potential) potassium current

Cytoskeleton

cytoplasm is organized by this, highly dynamic. Made up of fibers, microtubules and intermediate filaments comprised of actin, tubulin and keratins help to maintain the organization of the cytoplasm, enable organelle and vesicle movement within the cytoplasm, help define the shape of cells, and undergo remodeling during cell movement and replication.

typer of small molecule cancer therapies

cytotoxic chemotherapy: -alkylating agents -mitotic inhibitors --vinca alkaloids-MT disruptors --taxanes-MT stabilizers --antracyclines -antimetabolites -topoisomerase inhibitors molecularly targeted drugs: -kinase inhibitors -HSP90 inhibitors -proteasome inhibitors -HDAC inhibitors -DNA methyltransferase inhibitors -aromatase inhibitors -horomone therapies, anti-estrogens, anti-androgens -immunotherapies

insulin receptor signaling

dimer of alpha and beta subunits stimulates a cascade of protein phosphorylation Insulin regulates both metabolic enzymes and gene expression. The insulin receptor (INSR) is a dimer of alpha-beta monomer subunits. Insulin binding between the two dimers activates autophosphoryltion of the TKD activation loop and other Tyr's on the beta-subunits. The activated INSR phosphorylates the Insulin receptor substrate-1 (IRS-1) which becomes a nucleation point for a complex of proteins that transduce the message to several target proteins in the cytosol and nucleus.

Type I and II Integral membrane proteins

single transmembrane helix differs in location of the amine and carboxyl group

telomeres and telomerase in aging and cancer

telomerase is a cellular reverse transcriptase (molecular motor) that adds new DNA onto the telomeres that are located at the ends of chromosomes. Telomeres consist of many kilo bases of TTAGGG nucleotide repeats and an associated protein complex, termed Shelterin. The sheltering complex protects chromosome ends doom end-to-end fusions and degradation forming special t-loop like structures and thus masking the linear ends of chromosome from being recognized as single and/or double stranded DNA breaks. Certain reproductive cells and embryonic stem cells retain full or almost full telomere length due to expression of telomerase activity. Pluripotent stem cells have regulated telomerase activity and thus they lose telomeres throughout life but at a reduced rate. Most somatic cell do not express telomerase activity and thus lose telomere length with each division at a faster rate until the cells uncap a few of their telomeres and undergo a growth arrest called replicative senescence. In the absence of cell cycle checkpoints (e.g. p53/pRB pathway), cells bypass senescence until they reach crisis. In crisis telomeres are so short that chromosome end fusions occur and there is increased genomic instability (probably due to chromosomal, breakage, fusion, bridge cycles). A rare cell that escapes crisis almost universally does so by reactivating telomerase and this cell can now become a cancer cell with limitless potential to divide. Almost all cancer cells have short telomeres and thus inhibitors of telomerase should drive such cancer cells into apoptotic cell death. While most tumor cells have short telomeres there is heterogeneity such that some cells may have slightly longer telomeres than others. After standard radiotherapy or chemotherapy a subset of residual cancer cells often re-grow and become resistant to the initial therapy. In this setting, adding a telomerase inhibitor in the maintenance or consolidation phase of treatment may prolong progression free survival.

G0 phase

terminally differentiated cells withdraw from cell cycle indefinatly

cells

the basic structural & functional unit - highly structured and contain membrane bound nucleus and organelles with distinct functions

RTK Kinase activation mechanisms

dimerization of extracellular regions of RTKs activates the intracellular tyrosine kinase domains (TDKs), which contain a C-lobe, N-lobe, and an activation loop insulin receptor like (activation loop inhibition) KIT-like (junta-membrane inhibition) Tie1-like (C-terminal tail inhibition) EGFR TKD is allosterically activated by direct contacts between the C-lobe of one TDK, the activator and the N-lobe of another TKD the receiver Ligand binding and receptor dimerization lead to activation of the tyrosine kinase domain (TKD) by one of several different mechanisms: TKD auto-inhibition by the activation loop - insulin and FGF receptors; Juxta-membrane auto-inhibition as in the MuSK, Flt3 & Eph families; Auto-inhibition by C-terminal sequences - Tie-1 & Tie-2; Allosteric activation of TKDs as for the EGFR family.

cAMP-dependent activation of PKA

eads to the subsequent phosphorylation and activation of enzymes including other kinases - signaling cascade

secondary active transport

electrochemical gradients provide energy for secondary active transport

programmed cell death

plays critical roles in development and maintenance of tissue homeostasis. -apoptosis plays and important role in development and maintenance of tissue homeostasis --initiated by an internal clock, or extracellular agents such as hormones, cytokines, killer cells, or a variety of chemical, physical and viral agents -cells undergo apoptosis through two major pathways --extrinsic pathway (death receptor pathway) --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 is tightly controlled by complex regulatory networks -deregulated apoptosis contributes to pathological disorders --developmental defects, autoimmune diseases, neuron-degeneration and cancer

Selectins

extracellular domains bind to specific polysaccharides on adjacent cells in a Ca+2 dependent manner have lectin domain (binds to carbohydrates) heterophilic P selection + counter-receptor PSGL-1, glycosylated

Uniporter GLUT 1

facilitated diffusion of glucose A) 12 transmembrane alpha helices of GLUT1: 3-4 amino acids per turn; B) wheel diagram of polar & non-polar at surface of helical segment; C) side by side association of four amphipathic helices, polar residues facing central cavity forms a transmembrane channel lined with polar and charged residues that for H-bonds with glucose as it moves through. D. GLUT1 exists in 2 states; T1 glucose binding site exposed to outside of cell & T2 glucose binding site exposed to inside of cell; 1) glucose in blood plasma binds to GLUT1 in the T1 state and 2) lowers the activation energy for a conformation shift from T1 to the T2 state effecting the transmembrane passage of glucose, 3) glucose is released from T2 into the cytoplasm and 4) GLUT1 returns to the T1 state.

modular domain structure of signaling proteins

facilitates binding interactions and complex formation The reversible phosphorylation of Tyr, Ser & Thr residues creates docking sites for protein-protein interactions that can have indirect effects on proteins downstream in the signaling pathway. Signaling proteins interact with phosphorylated proteins or phopholipids in many permutations to form integrated signaling complexes.

Kinesins and Dyneins

power the movement of organelles, vesicles, and protein cargos along microtububules. Kinesins and cytoplasmic dynein move organelles, vesicles and protein cargos along microtubules and maintain the intracellular organization of the cytoplasm. Kinesins typically move cargo from the cell interior towards the periphery of the cells, anterograde transport. Dynein moves cargo from the cell periphery to towards the cell interior, retrograde transport.

therapeutic agents targeting IL-6

for cancer and rheumatoid arthritis

amphitropic membrane proteins

found both in the cytosol and associated with membranes

receptor tyrosine kinases (RTKs)

important targets for drug discovery -RTKs family of 58 cell surface receptors (20 subfamilies)-regulate critical cellular processes --proliferation and differentiation --cell survival and metabolism --cell migration and cell cycle control -RTKs have similar molecular architecture --ligand binding region in extracellular domain --single transmembrane helix --cytoplasmic region contains protein tyrosine knase (TK) domain plus additional carboxyl-terminal and juxtaposition membrane regulatory regions -numerous diseases linked to genetic changes or abnormalities that alter activity, abundance, cellular distribution, or regulation of RTKs -RTK mutations and aberrant activation of their intracellular signaling pathways causally linked to cancer, diabetes, inflammation, severe bone disorders, arteriosclerosis and angiogenesis 58 RTKs sub-divided into 20 subfamilies. Sub-domains of the extracellular region indicated.

Cell adhesion molecules and cellular junctions

homophilic interactions between cadherins and Immunoglobulin superfamily (CAMs) are major groups of adhesion molecules adherents junctions contains actin fibers attached to cadherins by a plaque this former between adjacent cells basement membrane adhesion consists of focal adherents junction (actin fibers, plaques, and integrins) and hemidesmosomes (intermediate microtubules e.g. keratin, vimentin and plaques and interns)

hypothesis driven approaches

human cancer-->oncogenic alteration--> identification of molecular alterations--> in vitro and in vivo validation (efficacy and safety)-->targeted drug combinations-->clinical studies

allosteric GPCR ligands****

increases or decrease signaling depending on whether it is a positive or negative modulator neutral allosteric ligands unaltered signalling

Molecular motors

interact with elements of the cell cytoskeleton to organize and maintain cells. There are three major motor proteins in cells; myosin is associated with the actin cytoskeleton, and kinesin and dynein are associated with microtubules. Together these motors and protein filaments/fibers maintain the organization of the cytoplasm, enable organelle and vesicle movement within the cytoplasm, help define the shape of cells. There are also involved in cell attachment and cell movement.

receptor with no intrinsic enzyme activity

interacts with cytosolic protein kinase, which actives a gene-regulating protein (directly or through a cascade of protein kinases), changing gene expression.

receptor enzyme

ligand binding to extracellular domain stimulates enzyme activity in intracellular domain

M phase

mitosis (nuclear division) and cytokinesis (cell division) yield two daughter cells

G alpha subunits

modulate a variety of second messengers

GPCR sub-types

multiple GPCR sub-types respond to the same ligands specificity is maintained by differential expression and distribution within tissues

Type III Integral membrane proteins

multiple transmembrane helices in the same polypeptide

Insulin signaling pathway

not linear pIRS-1 activates the Ras-Raf-MEK-ERK MAPK kinase cascade that leads to pERK translocation into the nucleus where it phosphorylates and activates transcription factors. pIRS-1 also binds and activates the phosphoinositide 3-kinase (PI3K) which converts PIP2 to PIP3 which leads to the binding of PKB/AKT to PIP3 and its subsequent phosphorylation and activation by PDK1. Activated PKB/AKT inactivates GSK3 by phosphorylation keeping glycogen synthase in it's non-phosphorylated and active form thereby increasing glycogen synthesis. Activated PKB/AKT also stimulates the movement of GLUT4 from intracellular vesicles to the plasma membrane thereby increasing glucose uptake.

MAPK Kinase signaling cascades

not linear stimulus-> MAPKKK-> MAPKK-.> MAPK-> biological response

Protein binding interactions mapped to integral cytoplasmic tails

outside-in and inside-out signaling require dynamic, and spatially and temporally regulated assembly and dis-assembly of multi-protein complexes that form around the cytoplasmic tails of integrins There is a network of 156 components (linked via 690 interactions) that make up the integrin 'adhesome'.

scaffolding proteins

play an essential role in controlling the processes of many signaling cascades. With two or more components of a signaling pathway, scaffold proteins can help to localize signaling molecules to a specific location in the cell or elevate the signaling pathway's effectiveness. Scaffold proteins regulate the thresholds and dynamics of signaling reactions by positive and negative feedback signals - controlled positive and negative feedback signals, and protection from phosphatase or protease deactivation. are involved in SAPK/JNK signaling

Drug Targeting for 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

macroautophagy

process whereby bulk proteins and organelles in the cytosol are delivered to the lysosome

selective progesterone receptor modulators (SPRMs)

progesterone receptor signaling clinical applications: breast cancer and endometriosis

protein phosphorylation

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

DNA

provides the genetic foundation of reproduction -genetic information must be: --maintained in a stable form --expressed accurately in the form of gene products --reproduced with a minimum of errors -deoxyribonucleic acid-DNA --genetic information stored and coded in the nucleotide sequence from which all other cellular components are generated -provides a template for production of identical DNA molecules to be distributed to the progeny when a cell divides

peroxisome proliferator-activated receptor-gamma (PPAR gamma) signaling

thiazolidinedions (TZDs) are potent insulin sensitizers that act through PPAR gamma highly effective oral medications for type 2 diabetes. However, their unique benefits are shadowed by the risk for fluid retention, weight fain, bone loss, and congestive heart failure

integral membrane proteins

tightly associated with the lipid bilayer, extracted with detergents Six classes of integral membrane protein classified by the position of the NH- and COOH-terminus, the number of transmembrane helicies, the number of polypeptides involved, and the surface that the lipid anchor is attached to. Palmitoylation, farnesylation and myristoylation are post translational modifications that can direct proteins to the inner surface of the plasma membrane. Glycosyl phosphatididylinositol anchored proteins are always on the external surface.

Plasma membrane

tough, flexible lipid bilayer. Selectively permeable to polar substances. Includes membrane proteins that function in transport, in signal reception, and as enzymes.

anticancer drug disovery

traditional cytotoxic chemotherapies: compounds from different sources-->evaluation of anti tumor activity--> study mechanism of action molecular targeted drugs--> know ontogenetic pathway--> identification of compounds--> evaluation of anti tumor activity-->study of the mechanism of action

cancer stem cell theory

(A) Cancer cell-of-origin (or cancer-initiating cell): the cell where the first genetic lesion linked to the development of the tumor takes place might be located anywhere within the physiological differentiation pathway. It does not need to have any phenotypic relationship with the final phenotype of the tumor cells (either stem or differentiated). (B) CSC (cancer-maintaining cell): those cells that have the capacity to regenerate all the cellular diversity of the tumor. They retain broad self-renewal potential and differentiation potential. They arise initially from the cancer cell-of- origin, and then they can self-propagate. (C) Tumoral reprogramming: the process by which the initial oncogenic lesion(s) can 'reset' the epigenetic and/or transcriptome status of an initially healthy cell (the cancer cell-of-origin), therefore establishing a new, pathological differentiation program ultimately leading to cancer development, where the oncogenic lesion(s) does not need to be present anymore once the initial cancer fate-inducing change has taken place.

Nuclear Receptor Domain Structure, DNA binding and mechanism of action

(A) Common domain structure of NRs; N-terminal activation function 1 (AF-1), DNA binding domain (DBD) consisting of two zinc fingers (ZF), non-conserved hinge-region (Hinge), ligand binding domain (LBD), and C-terminal AF-2 helix. (B) Schematic diagrams for NR dimerization and DNA binding sequences; homodimeric endocrine receptor (Palindrome HRE), RXR heterodimers (Direct Repeat HRE) and monomeric orphan receptor (Half Site HRE). Arrows: the consensus NR recognition sequence AGGTCA or a variant. Typical NR activation mechanisms: In the absence of ligand, NRs form a repressive complex with HDACs (histone deacetylases) via corepressor SMRT or NCOR. Ligand binding induces a dissociation of corepressors and a recruitment of coactivators including HAT (histone acetyl-transferase) and chromatin remodeling complexes. Some nuclear receptors are activated by ligand-independendent binding of the PGC-1 coactivator and subsequent recruitment of additional coactivator complexes.

Integrin Drug Development

-1st integrin successfully targeted was the platelet alphaIIbbeta3 integrin involved in platelet aggregation --reduce the risk of ischemic events in patients with acute coronary syndromes and undergoing percutaneous coronary intervention -3 IV alphaIIbbeta3 integrin inhibitors were approved (1990s) -- antibody fragment Abciximab -- small-molecule inhibitors Eptifibatide and Tirofiban - attempts to develop oral antagonists for more convenient administrations were unsuccessful- use of these agents restricted to high-risk patients -clopidogrel, an orally active ADP receptor antagonist, filled the market expectationsted for alphaIIbbeta3 integrin antagonists- became the second biggest-selling drug globally -Natalizumab (Tysabri) -- monoclonal antibody (mAb) that bings to the alpha 4 integrin subunit, was approved for the treatment of multiple sclerosis and Chrohn's disease -Efalizumab (Raptiva) --mAb that targets the alpha l beta 2 integrin (LFA1) was approved for the treatment of moderate to severe psoriasis -both agents were associated with a potentially fatal side effect progressive multifocal leukoencephalopathy (PML) --thought to be related to their immune suppressive properties -Initial reports of PML lead to the withdrawal of natalizumabin 2005 -- due to hits high efficacy in reducing he rate of relapses in multiple sclerosis and the medical need, it was reintroduced in 2006 with a black-box warning on the drug label and risk management strategy -Efalizumab was withdrawn from the market in 2009 -Integrin targeted drugs are in development for cancer, infection and osteoporosis

Kinase Drug Discovery

-1st small-molecule kinase inhibitor Imatinib was approved for clinical use only 15 yrs ago -33 more kinase inhibitor drugs have received FDA approval for the treatment of a variety of cancers -discovery and development of kinase inhibitors has increases exponentially -231 inhibitors targeting 38 different proteins and lipid kinases in clinical use or under clinical investigation

Membrane Transporters and Drug Development

-ATP binding cassette (ABC) and solute carrier (SLC) transporter families -impact on pharmacokinetics of drugs --absorption, distribution, and excretion of drugs -drug-drug interactions --substrate or inhibitor of transporters -clinically relevant polymorphisms --significantly impact individual patient dosing a) intestinal epithelia has one P-gp and MRP2, 6 inputs b) hepatosites MRP 2,3,4,6 and P-gp, 6 inputs c) kidney proximal tubules MRP 2,4 and P-gp, 7 inputs d) blood brain barrier P-gp, MRP 4, 5 only two inputs transporters into the cell and that is why it is hard for drugs to pass the blood brain barrier P-gp and MRPs pump drugs out of cells SLC and ABS transporteras also affect absorption and distribution of drugs selected human transporter mediated drug-drug interactions**

Type 1 kinase inhibitors

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

Long QT Syndrome

-Abnormal ventricular repolarization --characterized by long QT interval on ECG -Congenital QT syndrome --rare cardiac disorder --polymorphic ventricular tachycardia- tornadoes de pointes -Accquired LQTS due to drugs --block of re polarizing K+ currents -- stimulation of I ca-i --stimulation of I na --hypokalemia --CHF and LVH there are many drugs associated with tornadoes de points &/or QT prolongation

DNA sequence transcribed into mRNA sequence that is translated into the amino acid sequence of proteins

-DNA sequence of genes transcribed into mRNA sequence -mRNA translated into an unfolded polypeptide of linked amino acids -folding produces the secondary and tertiary structures required for enzymatic activity/function -protein-protein interactions lear to the formation of supramolecular complexes The process of transcription is the process of converting DNA to its complementary mRNA strand which can then be read as codons, three nucleotides, which encode for a specific amino acid which makes up a protein. The genetic code specifies which amino acid is encoded for by each codon. However, each amino acid is not always specified by one codon. Often, more than one closely related codons may specify the same amino acid. The DNA sequence is transcribed by the RNA polymerase into a complimentary mRNA sequence. The transfer RNA's (tRNA) that transport amino acids to the ribosomes during protein synthesis bear complimentary ribonucleotides to the transcribed mRNA codons thereby ensuring that the correct protein sequence is translated. DNA is Transcribed into mRNA and that is Translated into Proteins. DNA stores the genetic information its sequence of nucleotides from which all other cellular components are generated.

DNA base paring forms the genetic basis of replication

-DNA stores the genetic information in the sequence of nucleotides from which all other cellular components are generated The complementary anti-parallel strands of DNA follow base pairing rules; the base- paired anti-parallel strands differ in base composition and also differ in in sequence when each chain is read 5' to 3'. The linear sequence of deoxynucleotides arranged in a precise sequence encodes the genetic information. Two of the polymeric stands form the DNA double helix in which each deoxynucleotide in one strand pairs specifically with a complimentary deoxynucleotide in the opposite strand. Before the cell divides, the two strands separate and each serves as a template for the synthesis of a new, complementary strand, generating two identical double-helixal molecules, one for each daughter cell.

Fluoxetine Hydrochloride (Prozac)

-Early member new class of antidepressants -SSRI -1st publication 1974 (more than 16 years to develop) -US FDA approval in 1987 -Sustained effective treatment of depression --low side-effect profile -2002, 1 year after patent expiration --prescribed to >40 M patients --total sales US $22 billion --Peak annual sales US $2.3 billion in 1998 a) Tryptophan hydroxyls (TH) catalyses the conversion of tryptophan (TRYP) to 5-hydroxytryptophan (5-HTP) b) aromatic aminoacid decarbocylase (AADC) catalyses the conversion of 5-HTP to 5-hydroxytrptamine (5-HT, serotonin) c) 5-HT is taken up into storage vesicles d)5-HT is released from storage vesicles into the synaptic space e) 5-HT can activate subtypes of 5-HT receptor families (1-7), which couple with their respective system of signal transduction inside the postsynaptic neuron f) 5-HT is taken up into the presynaptic 5-HT terminals by the 5-HT transporter g,h) Within the presynaptic 5-HT terminals, 5-HT would either be taken up by the storage vesicles or degraded by monoamine oxidase (MAO) i) 5-HT activates the presynaptic somatodendriatic 5-HT1A auto receptor, which can be blocked by selective 5-HT1A antagonists j) selective serotonin-reuptake inhibitors (SSRIs) including fluoxetine inhibit the 5-HT transporter. 5-HIAA, 5 hydroxyindolacetic acid; AC, adenylate cyclase; DAG, diacylglycerol; IP3, inositol- 1,4,5-triphosphate; PIP2, phosphatidylinositol-4,5-bisphosphate

tumor supressor phosphatase and tennis homolog (PTEN)

-PTEn is a non-redundant phosphatase that regulates one of the most critical cancer-promoting pathways: the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway --PTEN dephosphorylates phosphatidylinositol- 3,4,5- trisphospate (PIP3) -PTEN has additional roles --regulating genomic instability, DNA repair, stem cell self-renewal, cellular senescence, and cell migration and/or metastasis -clinically, PTEN mutations and deficiencies are prevalent in many types of human cancers -severe PTEN deficiency is also 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

STAT phosphorylation and activation

-STAT tyrosine phosphorylation RTKs, JAKs, Src and Tec tyrosine kinases --phosphorylation of single carboxyl-terminus tyrosine (Y701/Y705/Y695) --STATs form homo- or hetero- dimers via reciprocal SH2 domain interacts --dimerization uncovers nuclear localization signal and dimers translocate to nucleus --STAT dimers bind specific 9 base pair DNA sequences (5'TTCCNGGAA3') in target gen regulatory regions- promoters and enhancers -STAT serine phosphorylation JNK and p38 MAPKs, PKC, PKA --phosphorylation of single carboxy-terminus serine residue (S727) --enhances STAT transcriptional activation post tyrosine phosphorylation -STAT activation is transient- shut down by multiple regulatory mechanisms --protein inhibitors of activated STATs (PIAS) --supressor of cytokine signaling (SOCS) --genes associated with retinoid IFN induced morality (GRIM) --dephorphorylation by membrane and cytoplasmic and nuclear phosphatases

Ion Channels

-maintain membrane potential -critical for neuronal and cardiac action potentials

V-type ATPases

-V0V1 H+ ATPase uses ATP to pump protons into vacuoles and lysosomes lowering their pH

Biosignaling

-ability to receive and act on signals in the environment beyond the plasma membrane -information detected by a specific receptor and converted into a cellular responses

selective androgen receptor modulators (SARMs)

-androgen receptor (AR) plays a critical role in the function of several organs --primary and accessory sexual organs, skeletal muscle, and bon -SARMs bind to the AR and demonstrate osteo- and myo- anabolic activity -unlike testosterone and other anabolic steroids --SARMs produce less of a growth effect on prostate and other secondary sexual organs -SARMs provide therapeutic opportunities in a variety of diseases --muscle wasting associated with burns, cancer, end-stage renal disease, osteoporosis, frailty and hypogonadism

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

-cGMP ubiquitous intracellular second-messenger that mediates many physiologic processes -generated by soluble and receptor guanylyl cyclase isoforms -cGMP experts its actions through --cGMP-dependent protein kinases (PKGs) --cGMP-gated cation channels --cGMP-regulated phosphodiesterase (PDEs) that hydrolyze cyclic nucleotides

senescence

-cellular senescence is a physiological program of terminal growth arrest which can be triggered by various endogenous or exogenous stress signals -cellular senescence can be induced in response to ontogenetic activation, acting as a barrier to tumorignensis - tumor cells undergo senescence when exposed to chemotherapeutic agents -in addition to suppressing tumorigenesis however, senescent cells remain metabolically active and may contribute to tumor formation and to therapy resistance In response to therapeutic intervention, cancer cells can rapidly undergo apoptosis or enter therapy induced senescence (TIS). These senescent cells can be cleared by the immune system, which is beneficial for the host. In contrast, some cancer cells in a senescence-like state might remain as 'dormant' or possess a secretory phenotype, therefore represent a dangerous potential for tumor relapse, which is considered a detrimental outcome for the host.

signal transducers and activators of transcription

-common STAT domain structure --NH2-terminus dimerization domain (SDD) --coiled-con protein-protein interaction domain (CC) --central DNA-binding domain (DBD) --src-homology 2 domain (SH2) --conserved tyrosine residue- Y701, Y705, Y695 --COOH-terminus transcription activation domain (TAD) -STAT family- STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 -cytoplasmic transcription factors regulating cytokine and growth factor gene expression -interferons, IFN alpha/beta and IFN gamma (prototypic activators of STAT1 and STAT2) -activated by receptor tyrosine kinases, Janus Src, and Tec family kinases -STATs 1, 3, and 5 activated by a large number of cytokines, STATs 2, 4, and 6 by relatively few

Type 3 kinase inhibitors

-compounds binding exclusively to less conserved allosteric sites outside the ATP pocket beyond the gatekeeper residue -allosteric 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

autophagy

-conserved pathway that maintains homeostasis by degrading proteins and cytosolic content of cells -clears miss-folded and long-lived proteins, damaged organelles and invading microorganisms --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

biological membranes

-define the external boundaries of the cell -control molecular traffic across those boundaries -Divide internal space into discreet compartments --segregate organelles, processes and components -organize complex reaction sequences -central to biological energy conservation -central to cell-to-cell communication -flexible- shape changes- growth/movement -self sealing- fission and/or fusion --endocytosis, exocytosis, mitosis -selectively permeable to polar/charged solutes

cellular senescence

-defined as an irreversible growth arrest --characterized morphology, gene expression pattern and chromatin structure with an activated DNA damage response -has dual role for 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 --oncogene induced senescent cells found early, pre-malignant tumor stages suggests that senescence has to be overcome during tumorigenesis in order to progress to malignancy

Cancer drug discovery and development

-despite significant investments in cancer research, drug discovery and drug development --rate of new cancer drug approval is less than 5% -most cases of metastatic cancer remain incurable -95% of new cancer drugs fail in clinical development --lack therapeutic efficacy and/or have unacceptable toxicity -average clinical development success rate for new drugs --11% in other therapeutic areas and 20% for CV diseases -several factors contribute to low success rate of cancer drug therapy development --preclinical tumor models fail to adequately recapitulate the complexity and heterogeneity of cancer

Natriuretic Peptides and Heart Disease

-heart muscle contracts cyclically and continuously to provide the mechanical force needed to move blood about the circulation and help 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 generation of myocardial cAMP stimulates function and/or growth -natriuretic peptides of the atrial, brain, and C-type are endocrine/autocrine regulatory factors --ANP, BNP, &CNP activate NPR-A and NPR-B receptors-- membrane-bound guanylyl cyclases --cGMP activates PKG and interacts with PDE-5 serves as a counter-brake to a variety of myocardial stressors -Organo-nitrates and NPs that stimulate cGMP synthesis- used to treat clinical heart failure --cGMP-selective PDE5 inhibitors such as sifenafil and tadalafil ameliorate cardiac pressure, volume overload, ischemic injury, and cardiotoxicity -clinical trials initiated to explore PDE-5 inhibitors for dilated cardiomyopathy and heart failure with a preserved ejection fraction

Intergrins

-heterodimers of non-covalently associates alpha and beta subunits each with 1 TM helix -extracellular domains bind to extracellular matrix (ECM) proteins or counter receptors on adjacent cells -crucial for embryonic development, tissue maintenance and repair, host defense, and hemostasis -cytoplasmic tails are linked to cytoskeleton and permit the bidirectional transmission of force across the plasma membrane -have ligand binding region -heterodimers, heterophilic bind to ECM, Ig-CAMs, cadherins adhesion, polarity, migration -cytoplasmic tails are linked to cytoskeleton -- permit the bidirectional transmission of force across the plasma membrane -outside-in signaling- transmit chemical signals into the cell --provide information on location, local environment, adhesive state and surrounding matrix --determine cellular responses such as migration, survival, differentiation and motility --provide a context for responding to other inputs, including those transmitted by growth factor or GPCRs -inside-out signaling/activation- regulate their affinity for extracellular ligands --undergo conformational changes in their extracellular domains in response to signals that impinge upon their cytoplasmic tails -integrins have key roles in a diverse range of diseases --cancer, infection, thrombosis and autoimmune disorders

Adhesion molecules

-hold neighboring cells together -cell-cell and cell-extracellular matrix (ECM) interactions

mechanisms to evade apoptosis

-impaired receptor signaling pathway -disrupted balance of Bcl-2 family of proteins (over or under expressed) -increased expression of IAPs -reduced expression of caspases -defects/ mutations in p53

combination therapy potential

-improved efficacy -decreased dosage --equivalent/increased efficacy -control/delay resistance -enhanced therapeutic benefit with reduced side effects -synergistic drug combinations --improved efficacy --lower does required --les toxicity --improved quality of life

vasculogenesis, angiogenesis and vascular remodeling

-in development, a vascular network is formed throughout the body to meet the tissue requirements for oxygen and nutrients --three major processes are involves -vasculogeneis- de novo blood vessel formation --vascular precursor cells (angioblasts) migrate to sites of vascularization differentiate into endothelial cells and coalesce form the initial vascular plexus -angiogensis --the budding of new capillary branches from existing blood vessels -vascular remodeling --a later phase when a newly formed vessel increases its luminal diameter in response to increased blood flow and acquired identity as an artery, vein or capillary -once these processes are completed during postnatal development, adult vasculature is stable and rarely proliferates under physiological conditions -in cancer, existing vessels again start to grow to meet the abnormal requirements for oxygen and nutrients of the expanding tumor.

traditional oncogenesis

-initiation and progression of cancer --initiating genetic alteration takes place and is required for the immortalization of a committed/differentiated target cell --cell acquires additional genetic hits over time that aggravates the deregulation of the differentiated target cell leading to the clinically recognized features of cancers --phenotype of the tumor cell reflects that of the normal cll that gave rise to it Human cancer genetic defects act on cells already committed to a differentiation program, in such a way that the tumoral phenotype is derived from that of the initial differentiated target cell.

Integrin Signaling

-integrin activation -integrin engagement and initial signaling -integrin clustering and focal adhesion assembly -integrin inactivation -steps are reversible- depend on the specific interns involves; the nature, organization and mechanical properties of the ECM; the cell type and its contractility; the presence of co-signaling receptors, and even the sub cellular localization of the integrin -considerable diversity in integrin-based adhesions and signaling -integrins regulate a wide range of cellular processes Integrin signaling depicted as an ordered series of events from integrin activation, to integrin engagement and initial signalling, to integrin clustering and focal adhesion assembly and, subsequently, to integrin inactivation. These steps are reversible and depend on the specific integrins that are involved, the nature, organization and mechanical properties of the ECM, the cell type and its contractility, the presence of co-signaling receptors, and even the subcellular localization of the integrin. These variables contribute both to the considerable diversity in integrin-based adhesions and to the flexibility in signaling that enables integrins to regulate a wide range of cellular processes.

FDA approved drugs for melanoma

-interleukin-2 -intron a -sylatron -imlygic -yervoy -keytruda -opdivo -pembrolizumab -DTIC-Dome -tafinlar -mekinst -zelborad -cotellic

cancer

-involves dynamic changes to the human genome -tumorigenesis is a multistep process of genetic alterations that drives progressive transformation from normal cells to highly malignant derivatives -many cancers exhibit an age-dependent incidence -cells evolve progressively from normalcy via pre-malignant states into invasive cancers -molecular machinery that regulates proliferation, differentiation and cell death is highly conserved and cancer cells have defects in regulatory circuits that govern cell proliferation and homeostasis -tumor cells invariably bear multiple mutations -mutations to tumor supressor genes- recessive loss of function -tumor development involves a succession of genetic changes, each conferring some form of growth advantage that leads to the progressive conversion of normal cells to cancer cells

Membranes organize cellular processes

-lipid synthesis and energy transduction

G-Protein Coupled Receptors (GPCRs)

-membrane receptors with 7 transmembrane helices --serpentine receptors, 7TM receptors, GPCRs -Guanosine Nucleotide-binding Protein (G-protein) -- cycles between GTP-bout active <--> GDP-bound inactive -Effector enzyme or ion channel modulated by the G-protein that generates a second messenger 3 common features - 7TM domains, an associated GTP-binding protein that activates an enzyme/ion channel to generate second messengers transduce a large number of signals.

Transporters

-move specific organic solutes and inorganic ions -high specificity substrate binding -transport rate < free diffusion -saturable (like enzymes and receptors)

tumor cells acquire a self sufficiency fro growth signals

-normal cells require mitogenic signals to move from quiescence to active proliferation --diffusible growth factors and their cognate plasma membrane receptors --instructed to grow by parching/ endocrine signals, extracellular matrix components and cell- cell interactions via interns and/or adhesion molecules -tumor cells exhibit a reduced dependence for growth stimulation --exposed to elevate concentrations of extracellular growth signals --synthesize and secrete own growth factors to create an autocrine positive feedback signaling loop --oncogenes may mimic growth signaling pathways --over expression of growth factor receptors and or mutations that confer constitutive activity --changes in integrin expression to ones that transmit growth signals --alterations to intracellular signaling pathways that transmit growth signals

backbone and base paring interactions of DNA and RNA

-phosphodiester linkages form the coolant backbone of DNA and RNA -hydrogen bonding patterns between base pairs -specific pairing od bases permits the duplication of genetic information and transcription of mRNA and DNA Phosphodiester linkages form the covalent backbone of DNA & RNA that by convention is written in the 5' end to 3' end. Hydrogen bonds form the most important interactions between the base pairs of the complementary strands of nucleic acid and the specific pairing of bases permits the duplication of genetic information, transcription of mRNA from DNA, and translation of mRNA into proteins.

role of telomeres and telomerase in cancer

-normal cels progressively lose telomeres with each cell division until a few short telomeres become uncapped leading to a growth arrest known as senescence -senescent cells without genomic alterations do not die but remain quiescent producing a different constellation of proteins compare to young quiescent cells -upon specific genetic and epigenetic alterations do not die but remain quiescent producing a different constellation of proteins compared to young quiescent cells -upon specific genetic and epigenetic alterations, normal human cell bypass replicative senescence and continue to proliferate until many telomere ends become uncapped leading to a phenomenon known as crisis -in crisis cells have critically shortened telomeres but continue to attempt to divide leading to significant cell death (apoptosis) and progressive genomic instability -very rarely cells escape crisis and theres cells almost universally express the ribonucleoprotein, telomerase, and maintain stable but short telomeres -activation of telomerase may be thought of as a mechanism to slow down the rate of genomic instability due to dysfunctional telomeres -telomerase does not drive the oncogenetic process, it is permissive and required for the sustained growth of most advanced cancers. -since telomerase is not expressed in most normal human cells, targeted telomerase cancer therapeutic approaches that are in development.

tumor cells acquire insensitivity to anti-growth signals

-numerous anti-proliferated signals keep normal cells quiescent and maintain tissue homeostasis --soluble growth inhibitors, cell-ECM and cell-cell contacts --signals transducer by cell surface receptors --primarily cell cycle associated signaling pathways --cells are forced to exit active proliferation via the cell cycle into G0 quiescent phase --terminally differentiated cells relinquish their proliferation potential and enter post-mitotic states -tumor cells evade these anti-proliferative signals -anti-proliferative receptor down regulation and/or disfunction -disruptive of Rb and/or Rb signaling pathways -over expression of the cMyc oncogene impairs differentiation and promotes growth In G1, in the presence of low level of CKIs, active CDK/cyclin complexes trigger the hyperphosphorylation of Rb, releasing the dimeric transcription factor E2F/DP and thereby inducing the transactivation of genes with functional E2F-binding sites including growth and cell cycle regulators and genes encoding proteins required for nucleotide and DNA biosynthesis. Disruption of Rb or the Rb pathway liberates E2Fs and allows cell proliferation rendering the cell insensitive to anti-growth factors that normally use this strategy to block advance through the G1 phase of the cell cycle. Two broad classes of CKIs regulate cylin-dependent kinase (CDK) driven progression through the cell cycle; the INK4 family (p15/p16/p19) specific for CDK4/Cyclin D and the CIP/KIP family (p21, p27) can regulate all CDK-Cyclin complexes. Interestingly, transforming growth factor beta (TGFb) functions as an anti-proliferative signal in some cells because it induces expression of p15 and p21 which block the CDK4/6- Cyclin D complexes that phosphorylate Rb and release EF2 to activate the genes required for DNA synthesis in S phase.

ion channels

-orders of magnitude higher transmembrane rate -exhibit some degree of ion specificity -not saturable -direction of transport dictated by ion's charge and/or the electrochemical gradient

F-type ATPases

-oxidative phosphoryltion -energy coupling factors -F0F1 ATPase drives H+ up gradient into *mitochondria* -large proton gradient can drive pump in reverse ~ ATP synthase

cancer mono therapy issues

-partial patient responses -emergence of resistance -narrow therapeutic index --dose limiting toxicities --reduced quality of life --poor patient adherence

three types of senescence

-replicative senescence (RS) -oncogene-induced senescence (OIS) -accelerated cellular senescence (ACS) Human cells may experience diverse forms of stress in their lifetimes. If left unchecked, it could be detrimental to the cells and the organism. One of the mechanisms that cells may use to keep the damage to the cellular DNA in-check is by inducing cellular senescence. Depending on the stress stimuli, cells may activate pathways to undergo replicative senescence (RS), oncogene-induced senescence (OIS) or accelerated cellular senescence (ACS).

Receptors

-sense extracellular environment and receive signals -downstream signaling induces molecular charges in the cell

Common Features of Signal Transduction

-signal interact with a receptor -activated receptor interacts with cellular machinery -produces a second signal or alters activity of cellular protein -metabolic activity and/or gene expression profile of target cell changes -signal transduction event is terminated

Types of membrane transport

-simple diffusion (nonpolar compounds only, down concentration gradient) -facilitated diffusion (down electrochemical gradient) -primary active transport (against electrochemical gradient) -secondary active transport ( against electrochemical gradient, driven by ion moving down its gradient) -ion channel (down electrochemical gradient; may be gated by ligand or ion) -ionophore-mediated ion transport (down electrochemical gradient) -active transport (expends energy of ATP to move solutes against a gradient primary or secondary to ATP hydrolysis) -carrier/facilitated diffusion (speed transmembrane movement/ diffusion of solutes down a concentration &/or electrochemical gradient) -Mediated transport across membranes is either passive or active. Passive transport goes down a concentration or electrochemical gradient. Active transport utilizes the energy from the hydrolysis of ATP to move solutes against a concentration/electrochemical gradient.

SNARE

1) Neurotransmitter-filled vesicle approaches plasma membrane 2) v-SNARE and t-SNARE bind to each, zipping up from the amino termini and drawing the two membranes together 3) Zipping causes curvature and lateral tension on bilayers favoring hemifusion between outer leaflets and causing formation of an energetically unfavorable void space 4) Inner leaflets of both membranes come into contact 5) complete fusion creates a fusion pore 6) pore widens; vesicle contents are released outside cell. Synaptic vesicles contain the v-SNARE synaptobrevin and the pre-synaptic membrane contains the t-SNAREs syntaxin and SNAP 25. Arriving action potentials activate voltage gated Ca2+ channels which raises intracellular [Ca2+] and the v-SNARE, t- SNARE and SNAP 25 proteins form a coiled bundle of 4 alpha helices that pull the synaptic vesicle and pre-synaptic membranes together to disrupt the bilayer, form a fusion pore and release neurotransmitters into the synapse.

beta-adrenergic signaling pathway

1) epinephrine binds to its specific receptor 2) the occupied receptor causes replacement of the GDP bout to Gs by GTP, activating Gs 3) Gs (alpha subunit) moves to adenylyl cyclase and activates it 4) Adenylyl cyclase catalyzes the formation of cAMP 5) cAMP activates PKA 6) Phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine 7) cAMP is degraded reversing the activation of PKA.

Beta Adrenergic signaling Pathway: Integration, Cascases and Amplification

1. 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. 2. Epinephrine activation of the GPCR expressed cells initiates a cascade of signaling events involving activation of adenylyl cyclase, increased cAMP levels, activation of PKA and downstream kinases that mediate a number of functional responses.

Integrin family

24 alpha-beta chain integrin heterodimers comprised of 18 alpha subunits and 10 beta-subunits. AlphaV subunit can associate with multiple beta-subunits, others alpha-subunits tend to bind to singular beta-subunits. Alpha-subunits define the ligand binding properties of the integrin. The extracellular regions of both the alpha and beta subunits are comprised of multiple domains. The resting state is represented by the bent, closed conformation, which has a low affinity for the ligand. The intermediate state is extended; however, the headpiece is still closed, and therefore this state also has a low affinity for the ligand. In the fully activated state, the hybrid domain of the thigh domain swings out opening up the head domain into a high-affinity state. Following separation of the two integrin tails by activation events that direct talin binding to the carboxy-terminal NPXY phosphotyrosine domain of the β-subunit tail, the membrane proximal regions of both α and β subunits become available to bind a range of proteins including; tensin, filamin, paxillin and, either directly or indirectly, focal-adhesion kinase (FAK).

cell differentiation

3 types of cells: -germ cells -somatic cells -stem cells In a fertilized egg, the zygote has the ability to divide and differentiate into all cell types in the body and create a new organism. Such cells are omnipotent or totipotent - they have the potential to become any type of cell. 1st three divisions of the zygote give birth to eight totipotent cells, each of which also has the ability to become an entire organism. As the embryonic cells divide and the daughter cells differentiate, they become increasingly specific. The early mammalian embryo consists of the extra-embryonic cell layers—the trophoblast and a body of cells called the inner cell mass (ICM), which eventually become the embryo proper. The cells of the ICM are no longer omnipotent, because they no longer share the fate of the trophoblast, and they have committed themselves to an embryonic fate with the ability to become any cell in the body (but not the trophoblast). These cells are considered pluripotent. The ICM continues to differentiate into three germ layers—ectoderm, mesoderm and endoderm, each of which follows a specific developmental destiny that takes them along an ever-specifying path at which end the daughter cells will make up the different organs of the human body. Each specialized cell type in an organism expresses a subset of all the genes that constitute the genome of that species. Each cell type is defined by its particular pattern of regulated gene expression. Cell differentiation is thus a transition of a cell from one cell type to another and it involves a switch from one pattern of gene expression to another. Cellular differentiation during development can be understood as the result of a gene regulatory network.

cellular cues

3D cellular micro environments alter cellular cues a) chondrogenesis matrix synthesis and remodeling, proliferation -soluble cues, adhesive cues, mechanical cues, interstitial flow b) angiogenesis matrix remodeling, multicellular reorganization, depolarization, migration, proliferation -mechanical cues, soluble and ECM bound cues, topographical cues, cell-cell adhesion c) metastasis polarization, matrix remodeling, migration -adhesive cues, mechanical cues, topographical cues, soluble and EMC-bound cues

PTEN pathway and regulation

A, PTEN protein structure. PTEN protein contains: an N-terminal PIP2-binding motif, a phosphatase domain, a C2 domain, a C-terminal tail containing PEST sequences, and a PDZ interaction motif at the end. Two naturally occurring mutations on the phosphatase domain disrupt PTEN's phosphatase activity: C124S mutation, which abrogates both lipid and protein phosphatase actively, and G129E mutation, which abrogates only lipid phosphatase but not protein phosphatase activity. B, PTEN is regulated at different levels. 1, PTEN mRNA transcription is activated by EGR1, IGF2, PPARγ, p53, etc., and inhibited by MEK-mediated NF-κB activation. 2, PTEN mRNA is also post-transcriptionally regulated by PTEN targeting miRs, including miR21, miR221/222, and miR25. PTEN protein is extensively regulated by post-translational modifications. 3, Protein stability is primarily regulated by phosphorylation of C- terminal tail domains (Thr366, Ser370, Ser380, Thr382, Thr383, and Ser385). The phosphorylation leads to a "closed" state of PTEN and maintains PTEN stability. Dephosphorylation of the C-terminal tail opens the PTEN phosphatase domain, thereby increasing PTEN activity. 4, NEDD4-1 is an E3 ligase of PTEN, which mediates PTEN poly- and mono-ubiquitination. Polyubiquitin leads to proteasomal degradation of PTEN. Mono-ubiquitination of PTEN promotes its nuclear translocation. 5, Ubiquitin-specific protease HAUSP deubiquitinates PTEN in the nucleus, and leads to PTEN nuclear exclusion. PTEN biological function includes membrane function and nuclear function. 6, On the cell membrane, PTEN dephosphorylates PIP3 and consequently suppresses the PI3K pathway. 7, PTEN also regulates the cell cycle through Akt-mediated cytoplasmic sequestration of cell-cycle regulator CHK1. 8, PTEN physically associates with centromeres in the nucleus and maintains chromosome stability.

p53 a tumor supressor

Activated by a wide variety of different stresses, including oncogene activation, telomere dysfunction, and DNA damage, p53 transcriptionally induces a broad spectrum of genes implicated in apoptosis, cell cycle progression, DNA repair, autophagy, metabolism, antioxidant signaling, angiogenesis, and fertility. In addition, p53 interacts with Bcl-2 family proteins, and mitochondrial membranes to induce mitochondrial outer membrane permeabilization (MOMP), to trigger cytochrome c release and subsequent caspase activation. Importantly, p53 is regulated by the E3 ubiquitin ligases Mdm2, and Mdm4. Cytoplasmic and nuclear functions of p53 are intertwined, as Mdm2, a p53 transcriptional target, promotes mitochondrial localization of p53 through mono-ubiquitination , and p53-induced PUMA can release Bax and/or p53 from inhibitory Bax/Bcl-xL and p53/Bcl-xL complexes, to trigger MOMP.

Pregnant X receptor (PXR) and constitutive androstane receptor (CAR)- xenobiotic receptors

Activation of CAR and PXR and their target genes. CAR can be activated by either direct (ligand binding) or indirect mechanisms, while activation of PXR is purely ligand dependent. CAR and PXR shared target genes are grouped in a red box, CAR-specific targets in a blue box, and PXR-specific targets in a purple box. Target genes include Phase I and II drug metabolizing enzymes and drug transporters that affect drug clearance. Potential sites of drug-drug interactions. affect on drug metabolism and energy metabolism: Transcriptional circuits and metabolic relevance controlled by PXR. Major interactions between drug metabolism and energy metabolism and the central roles of PXR and CAR in these cross-talks.

GPCR genes

Almost 800 GPCR genes; Class A/Family1 Rhodopsin-like GPCR largest family, class B/Family 2 secretin-like, and class C/Family 3 metabatropic glutamate (mGluR) family. 271 Orphan GPCRs - endogenous ligand as yet not identified

Monomeric building blocks

Amino acids, nitrogenous bases, sugars and lipids are the building blocks of macromolecules, supramolecular structures and cells.

signaling networks regulate the operations of the cancer cell

An elaborate integrated circuit operates within normal cells and is reprogrammed to regulate hallmark capabilities within cancer cells. Separate sub-circuits, depicted here in differently colored fields, are specialized to orchestrate the various capabilities. At one level, this depiction is simplistic, as there is considerable crosstalk between such sub-circuits. In addition, because each cancer cell is exposed to a complex mixture of signals from its microenvironment, each of these sub-circuits is connected with signals originating from other cells in the tumor microenvironment.

emerging hallmarks and enabling characteristics of cancer

An increasing body of research suggests that in addition to the original six acquired capabilities of cancer two additional hallmarks of cancer are involved in the pathogenesis of some and perhaps all cancers. One involves the capability to modify, or reprogram, cellular metabolism in order to most effectively support neoplastic proliferation. The second allows cancer cells to evade immunological destruction, in particular by T and B lymphocytes, macrophages, and natural killer cells. Because neither capability is yet generalized and fully validated, they are labeled as emerging hallmarks. Additionally, two consequential characteristics of neoplasia facilitate acquisition of both core and emerging hallmarks. Genomic instability and thus mutability endow cancer cells with genetic alterations that drive tumor progression. Inflammation by innate immune cells designed to fight infections and heal wounds can instead result in their inadvertent support of multiple hallmark capabilities, thereby manifesting the now widely appreciated tumor-promoting consequences of inflammatory responses.

caspases

Caspases are a family of proteins that are one of the main effectors of apoptosis - caspase activation is a hallmark of apoptosis. 14 mammalian caspases have been identified that are synthesized as inactive zymogens that are cleaved to form active enzymes after the induction of apoptosis. All caspases can cleave substrates at Asp-Xxx bond. Caspases can be classified into three groups; inflammatory caspases, apoptotic initiator caspases, and apoptotic effector caspases. Caspases serve as signaling mediators that orchestrate apoptotic execution pathways by cleaving a subset of cellular proteins. More than 100 substrates have been identified thus far that can be subdivided into four major categories: (1) mediators and regulators of apoptosis, (2) structural proteins, (3) cellular DNA repair proteins, (4) cell cycle- related proteins. Apoptosis can be initiated by the death-receptor (extrinsic) pathway that acts through caspase 8 or mitochondrial (intrinsic) pathway that acts through caspase 9, but both pathways converge to activate the effector caspases (caspase 3), which act on the death substrates. Extrinsic pathway - the ligand-induced activation of death receptors induces the assembly of the death-inducing signaling complex (DISC) on the cytoplasm side of the plasma membrane. This promotes the activation of caspase-8 (and possibly of caspase-10), which in turn is able to cleave effector caspase-3, -6, and -7. Caspase-8 can also proteolytically activate Bid, which promotes mitochondrial membrane permeabilization (MMP) and represents the main link between the extrinsic and intrinsic apoptotic pathways. The extrinsic pathway includes also the dependency receptors, which deliver a death signal in the absence of their ligands, through yet unidentified mediators. Intrinsic pathway - several intracellular signals, including DNA damage and endoplasmic reticulum (ER) stress, converge on mitochondria to induce MMP, which causes the release of proapoptotic factors from the intermembrane space (IMS). Among these, cytochrome c (Cytc) induces the apoptosis protease-activating factor 1 (APAF-1) and ATP/dATP to assemble the apoptosome, a molecular platform which promotes the proteolytic maturation of caspase-9. Active caspase- 9, in turn, cleaves and activates the effector caspases, which finally lead to the apoptotic phenotype. DNA damage may signal also through the activation of caspase-2, which acts upstream mitochondria to favor MMP.

Steady-State in Cells

Cells are open systems that exchange energy and matter with their surroundings. Extract energy/matter from their environment and metabolize/convert them to waste products (catabolic) or use the energy to move and perform functions (anabolic) - replicate DNA, transcribe mRNA and translate proteins.

Integration

Cells often require multiple signal proteins coincidentally to trigger a response. Multiple signals may use integrator proteins which require > one signal input to generate a downstream output or signaling cascade. A. Coincidence detectors - a single protein requires phosphorylation on two different residues by two independent signaling pathways to become activated. B. Two proteins, after phosphorylation by two different signaling pathways associate together to form an active signaling complex.

IL-6 cytosine family receptor complexes

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

(5-HT) Seratonin and Depression

Criteria for Neurotransmitters: -synthesis -storage -release -breakdown -uptake -Serum factor --vasoconstriction smooth muscle -distinct and separate 5-HT neurons --catecholamines DA and NE -antidepressants --Monoamine oxidase inhibitors (MAO) --tricyclic antidepressants (TCA) --- imipramine and desipramine~ inhibited uptake of monoamines --- Carlsson et al, proposed inhibitie of 5-HT uptake basis for mood elevation -lower 5-HT in autopsy samples of brains from depressed patients

stages of mitosis

During interphase the chromosomes, in the form of chromatin, will duplicate their chromatids (DNA replication) in S-phase and the cell then grows in G2. The cell then enters mitosis. In prophase, the chromatin condenses into well defined chromosomes, each chromosome is made up of 2 chromatids joined by a centromere, the centrosomes duplicate and transform into asters, the nuclear membrane disappears and a spindle of tubulin fibers forms (achromatic spindle). In metaphase, the chromosomes move to the center of the cell and line up along the equator of the cell forming the "Equatorial Plate". In anaphase, the centromeres split and the sister chromatids separate. Each chromosome is now formed of one chromatid with a centromere. Sister chromatids move towards opposite poles of the cell . This is called "Polar Ascension". In telophase, The chromosomes de-condense and go back to the chromatin state (tall and thin). The asters become centrosomes. A new nuclear membrane forms and the spindle fibers break down and disappear. In cytokinesis, the cytoplasm of the mother cell will be cleaved in half, and the cell membrane will finish its constriction at the middle of the cell to divide the mother cell into two separate daughter cells that enter into a new interphase.

oncogene activation of the ERK MAPK cascade

EGFR overexpression: -colorectal cancer -pancreatic cancer -lung cancer -non-small cell lung cancer EGFR mutation: -NSCLC -glioblastoma Ras mutation: -pancreatic cancer -papillary thyroid cancer -colon cancer -non-small cell lung cancer B-Rad mutation: -Melanoma -papillary thyroid cancer -colon cancer Mutationally activated B-Raf, Ras and mutationally activated (by missense mutations in the cytoplasmic kinase domain in NSCLC or by extracellular domain truncations (e.g., VIII) in glioblastomas) and/or overexpressed EGFR causes persistent activation of the ERK MAPK cascade in human cancers. Activated ERKs translocate to the nucleus, where they phosphorylate and regulate various transcription factors leading to changes in gene expression. In particular, 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.

effects of oncogenic RAS on energy metabolism in caner cells

ERK and PI3K signalling downstream of oncogenic RAS converge to activate mTOR by inhibiting its negative regulators TSC2, LKB1, & AMPK. TSC2 can be directly phosphorylated by both ERK and RSK, as well as by AKT, and, likewise, RAF-ERK1 or RAF-ERK2 signaling disrupts the LKB1-AMPK checkpoint. This leads to mTOR-eIF4- dependent translation of hypoxia-inducible factor 1α (HIF1α). Activated RAS can also result in the transcriptional up-regulation of HIF1A. Increased levels of HIF1α augment multiple steps in glycolytic metabolism (shown in blue). The upregulation of hexokinase (HK) facilitates the conversion of glucose to glucose-6- phosphate, a glycolytic intermediate that is used in pentose phosphate pathway-dependent nucleotide synthesis. Higher levels of phosphofructokinase (PFK) lead to an enhanced glycolytic flux and the production of pyruvate, which, in conjunction with the oncogenic RAS-dependent increase in lactose dehydrogenase (LDH) levels, can allow glycolysis to persist by regenerating NAD+, a necessary cofactor for glycolytic reactions. In addition, some of the pyruvate can enter the tricarboxylic acid (TCA) cycle where its conversion to citrate generates intermediates that are necessary for the synthesis of fatty acids and non-essential amino acids.

adaption of metastatic cells to a foreign environment

Homing and colonization of a cancer cell to a distant organ are complex processes with many questions still unanswered. CTCs transiting from the primary tumor to a metastatic site can arrive at their destination via a variety of mechanisms: (A) CTCs may become lodged in the capillary beds of specific organs due to size. (B) CTCs may display specific adhesion molecules that enable them to adhere to microvessels in specific organs, or they may respond to a chemoattractive gradient arising from a particular tissue. (C) CTCs may preferentially home to organs where a premetastatic niche has prepared a microenvironment conducive to their survival. (D) Once cancer cells have exited the blood stream (extravasated) they may first experience a period of quiescence (dormancy) while they adapt to their newfound microenvironment. (E) Dormant cells may progress to micrometastatic deposits (perhaps in response to the recruitment of an appropriate stroma or an enhanced ability to respond to proliferative signals present in the host microenvironment) where their size is kept in check because of a balance in proliferation, apoptosis, and phagocytosis by the host- tissue immune system. (F) To develop into a macrometastasis, cancer cells must recruit an adequate blood supply (necessary for growth beyond 1 to 2 mm). The signals or mechanisms responsible for the transition from dormancy to micrometastasis to macrometastasis remain largely unknown.

senescence in the development and treatment of cancer

Human chromosomes have small repetitive structures known as telomeres that prevent genomic instability and tumorigenesis. Human telomerase is the enzyme that controls the telomere length and is composed of a protein (hTERT) and an RNA component (hTR). Replicative senescence (RS) mainly occurs in response to dysfunctional telomeres, such as telomere erosion or telomere shortening. In cancer cells, telomerase or alternative lengthening of telomeres (ALT) can prevent telomere erosion or telomere shortening, and subsequent cellular senescence. (C) Telomere shortening and uncapping activates DNA-damage response (DDR) pathways and, as depicted, either induces apoptosis or senescence. (D) The fact that only immortalized and cancer cells have elevated levels of telomerase (hTERT) allows specific targeting of these cells with anti-cancer drugs. Oncogene-induced senescence (OIS) acts as a barrier to neoplastic transformation and is regulated by various pathways. Primary cells, upon acquiring activating mutations in oncogenes, undergo OIS. Thus, for a cell to be immortalized and eventually transformed, it must bypass oncogene induced senescence. When tumor suppressor genes are lost or oncogenes are over- expressed, cells will bypass OIS and eventually be transformed. The most common pathways that OIS uses are p53 and Rb pathways. OIS may also be induced by other mechanisms, such as secretion of senescence inducing proteins, autophagy and miRNAs. Oncogenic stimuli that induce a senescence response may also have the potential to initiate tumor promotion. Accelerated cellular senescence 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 thus are important for cancer chemotherapy. Bypass of ACS may contribute to cancer recurrence and drug resistance.

cell cycle phases

In S phase the DNA is replicated to produce copies for both daughter cells. In the G2 phase (G = gap between divisions) new protein and RNA synthesis occurs and the cell doubles in size. In Mitosis, M phase, the maternal nuclear envelope breaks down, the mitotic spindle is formed and the paired chromosomes are pulled to opposite poles of the cell and the two daughter cells are formed by cytokinesis. After mitosis, cells pass into G1, another growth phase where new protein and RNA synthesis occurs. In rapidly proliferating cells the cells pass through G1 into S and the cell division cycle begins again. Cells that cease to divide, such as terminally differentiated cells, may enter a quiescent phase G0. Cells in G0 that re-enter the cell cycle do so in G1.

serpentine receptor

external ligand binding to receptor (R) activates an intracellular GTP-binding proteins (G), which regulates an enzyme (Enzyme-Substrate) that generates an intracellular second messenger, X.

intrinsic and extrinsic apoptosis pathways, pro- and anti- apoptotic Bcl-2 proteins, and the inhibitors of apoptosis proteins (XIAP and cIAPs)

Intrinsic and extrinsic apoptotic pathways of Apoptosis. In the intrinsic (mitochondrial) pathway, mitochondrial outer membrane permeabilization (MOMP) results in the release of cytochrome c and other apoptogenic factors from mitochondria into the cytosol and the ensuing formation of the apoptosome, which triggers activation of the apoptosis-inducing caspase cascade via activation of caspase-9. Interactions among BCL-2 family proteins play a critical role in the mediating MOMP induction and consequent apoptosis. BH3-only proteins (activators such as BIM and tBID and sensitizers like BAD) can relay apoptotic signals to the mitochondria through activation of BAX or BAK, the principal effectors of the intrinsic apoptotic pathway. In contrast, anti-apoptotic BCL-2 proteins (represented by BCL-2) serve to inhibit apoptosis by blocking BAX/BAK activation. In the extrinsic (death receptor) pathway, binding of death receptors such as FAS or TRAIL receptors (TRAILR1, TRAILR2) by their cognate ligands triggers the recruitment of death domain (DD)-containing adaptor proteins (represented by FADD) and procaspases with a death effector domain (DED), specifically procaspase-8 and procaspase-10. The resulting complex is known as the death inducing signaling complex (DISC). High levels of active caspase-8 generated by large amounts of procaspase-8 processing at the DISC lead to the activation of executioner caspases, including caspase-3, and the induction of apoptosis. Activation of caspase-8 can also result in the cleavage of the BH3-only protein BID to generate the activated BID fragment tBID, which serves to transmit the death signal from the extrinsic to the intrinsic signaling pathway. BCL-2 proteins play a key role in mediating the delicate balance between cell survival and cell 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 can enable the evasion of apoptosis, which tips the balance to favor cell survival and thus promotes the development and progression of cancer. The inhibitors of apoptosis (IAP) proteins, XIAP and cIAPs inhibit apoptosis and promote cell survival. The BIR2 domain of XIAP, along with residues in its N- terminal flanking linker region, mediates the binding and inhibition of caspase-3 and caspase-7. Inactivation of caspase-9 by XIAP involves the BIR3 domain of XIAP binding to caspase-9. In addition to blocking caspase activity, XIAP can also promote cell survival through regulation of important cellular signaling pathways, including signaling mechanisms of NF-κB activation. IAP-binding motif (IBM)- containing proteins, such as SMAC, interact with the BIR2 and BIR3 domains of XIAP to neutralize its anti-apoptotic activity.

tumors acquire sustained angiogenesis

Many extracellular, cell surface and intracellular molecules modulate angiogenesis. They include; growth factors and their receptors, adhesion molecules, extracellular matrix (ECM) proteins, remodeling and guidance molecules, matrix-degrading proteinases, signaling molecules, and transcription factors and inhibitors, and homeobox gene products. Adult vasculature is stable and rarely proliferates under physiological conditions. However, in cancer, existing vessels again start to grow (neoangiogenesis) in response to hypoxia inducible factor (HIF)-driven VEGF expression in tumors. Newly formed vessels provide oxygen and nutrients to rapidly expanding tumors. The stroma of solid cancers often show typical signs of inflammation and is infiltrated by many leukocyte populations, i.e. neutrophils, eosinophils, basophils, monocytes/macrophages, dendritic cells, natural killer cells and lymphocytes. Although many of these leukocytes are potentially capable of killing tumor cells, experimental and clinical evidence suggest that in most cases they actually contribute to tumor progression.

differentiation of mesoderm cells

Mesenchymal stem cells (MSCs) exhibit extensive diversity in differentiation, production of trophic mediators, and interaction with the host environment. Adult human MSCs are capable of differentiating into a number of phenotypes, which include cells capable of fabricating bone, cartilage, muscle, marrow, tendon/ligament, adipocytes, and connective tissue . Furthermore, hMSCs have been shown to produce large quantities of bioactive factors which provide molecular cueing for regenerative pathways as well as affecting the status of responding cells intrinsic in the tissue. Although MSCs can differentiate into various phenotypes of mature cells, their intrinsic capacity to secrete cytokines and growth factors (trophic mediators) is defined by their in vivo location, niche, and microenvironment. MSCs are reservoirs for the production of cytokines, chemokines, and extracellular matrix which have the ability to support stem cell survival and proliferation

estrogen receptor signaling

Molecular networks potentially influence the expression of SERM action in a target tissue. The structure of the ligands that bind to the estrogen receptors (ERs) alpha or beta direct the complex to become an estrogenic or anti-estrogenic signal. The tissue context of the ER complex (ERC) can influence the expression of the response through the numbers of co- repressors (CoR) or coactivators (CoA). The expression of estrogenic action is not simply the binding of the receptor complex to the promoter of the estrogen-responsive gene, but a dynamic process of CoA complex assembly and destruction A core CoA, such as the steroid receptor coactivator protein 3 (SRC3), and the ERC are influenced by phosphorylation cascades that phosphorylate target sites on both complexes. The core CoA then assembles an activated multiprotein complex containing specific co-co-activators (CoCo) that might include p300, each of which has a specific enzymatic activity to be activated later. The CoA complex (CoAc) binds to the ERC at the estrogen-responsive gene promoter to switch on transcription. The CoCo proteins then perform methylation (Me) or acetylation (Ac) to activate dissociation of the complex. Simultaneously, ubiquitiylation by the bound ubiquitin-conjugating enzyme (Ubc) targets ubiquitin ligase (UbL) destruction of protein members of the complex through the 26S proteasome. The ERs are also ubiquitylated and destroyed in the 26S proteasome. Therefore, a regimented cycle of assembly, activation and destruction occurs on the basis of the preprogrammed ER complex . However, the co-activator, specifically SRC3, has ubiquitous action and can further modulate or amplify the ligand-activated trigger through many modulating genes that can consolidate and increase the stimulatory response of the ERC in a tissue. Therefore, the target tissue is programmed to express a spectrum of responses between full estrogen action and anti-estrogen action on the basis of the shape of the ligand and the sophistication of the tissue-modulating network.

Nuclear receptor super family

NRs broadly classified into three sub-groups based on their physiologic ligands and potential functions. 1st class is endocrine receptors which all act as high affinity (Kd = nM range) receptors for fat-soluble hormones and vitamins; includes receptors for the steroid hormones, thyroid hormone (thyroid hormone receptor; TR), and vitamins A (retinoic acid receptors; RAR) and D (vitamin D receptor; VDR), which are all essential for homeostatic control of endocrine system. Mechanistically, the steroid receptors function as homodimers and TR, VDR, and RAR form heterodimers with the Retinoid X Receptor (RXR). The endocrine receptors are very successful drug targets, and ligands for each of these receptors are in use clinically. 2nd class is the adopted orphans, which were identified originally based on sequence homology to endocrine receptors, called orphan receptors, but "de-orphanized" by the identification of a naturally occurring ligand. Identification of a vitamin A derivative, 9-cis retinoic acid, as an endogenous high-affinity ligand for RXR represented the first true adoption of an orphan NR. This class now includes low affinity (Kd = μM range) receptors for dietary lipids and xenobiotics, which all function as heterodimers with RXR. These NRs regulate lipid and/or glucose homeostasis by controlling uptake, synthesis, storage or clearance. The "enigmatic" adopted orphans, for which a ligand has been identified, at least for one of the subtypes, but the nature of ligand-dependent regulation in physiology has not been established. The 3rd class is comprised of true orphans whose (natural or synthetic) ligand has not been identified.

Voltage gated neuronal Na+ channels

Na channels have multiple subunits but only the alpha subunit is essential. Alpha subunit has 4 homologous domains each containing 6 TM helices; helix 4 is the voltage sensor, 6 is the activation gate, connector between 5 & 6 is the selectivity filter/pore region, & connector between domains III & IV is the inactivation gate. Four domains wrapped around a central channel lined with polar amino acids: the 4 pore regions come together near the extracellular surface to form the selectivity filter that distinguishes between ions of similar charge; the inactivation gate closes soon after the activation gate opens. The voltage sensing helix 4 moves perpendicular to the plane of the membrane in response to a change in transmembrane potential. Positively charged helix 4 pulled to the negatively charged inner membrane surface of polarized membrane closing the activation gate. Upon depolarization of the membrane the pull on helix 4 relaxes and it moves out causing a conformational change in the activation gate and channel opening.

Glucose Transport in Intestinal epithelial cells

Na/K ATPase, Na+-glucose symporter, GLUT2 passive diffusion uniporter Na+ K+ ATPase uses energy from ATP hydrolysis to pump Na+ out of epithelial cells establishing & maintaining a Na+ gradient. Glucose and Na+ in lumen of the gut are transported across the apical plasma membrane by the Na+ -glucose symporter down the Na+ gradient. Glucose moves through the epithelial cell to the basal membrane and passes into the blood via GLUT 2 a passive glucose uniporter.

Nicotinic acetyl choline ligrand gated ion channel

Neurotransmitters like acetylcholine released into the synaptic gap bind to GPCRs and ligand-gated ion channels in the membrane 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 pass the signal on. The ligand-gated acetylcholine ion channel has 5 homologous subunits (α2, β,γ, δ) each with 4 TM domains (M1, M2 M3 & M4). M2 helices are amphipathic while the others have mainly hydrophobic residues. The five subunits are arranged around a central transmembrane channel lined with the polar sides of the M2 helices. When both actylcholine sites are occupied a conformational change occurs twisting the bulky hydrophobic Leu residues of the M2 helices away from the channel and presenting smaller polar residues than open the channel to the passage of Na+, Ca2+ and K+ ions.

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 the transcriptional up-regulation 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, as well as the nuclear accumulation of SMAD2 and SMAD3. RAS also upregulates the levels of cyclin D1 and suppresses the cyclin- dependent kinase inhibitor (CDKI) p27. Newly synthesized cyclin D1 associates with and activates CDK4 and CDK6, leading to the phosphorylation of RB and the subsequent dissolution of the RB-E2F transcription factor 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 activation of the RAS oncogene 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 repair of DNA damage can lead to mutations and chromosome aberrations, thereby contributing to tumorigenesis.

RAS Oncogene effects on apoptosis

Oncogenic RAS may have both pro-apoptotic and anti-apoptotic functions depending on the status of RAS effector pathways and the apoptotic machinery. Oncogenic RAS signaling through the RAF pathway engages an apoptotic response that is mediated by p53 & other effectors including RASSF1, NORE1, & JNK can lead to apoptotic death via the activation of caspase 3 and the pro-apoptotic proteins BAX & BAK1. Acquisition of a tumorigenic phenotype is marked by the suppression of such mediators of RAS-induced apoptosis. In this context, the anti-apoptotic activity of RAS prevails. The anti-apoptotic function of oncogenic RAS is 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 BAD. How RAS induces the epigenetic silencing of the pro-apoptotic CD95 gene remains undetermined.

Membrane Proteins facilite transport

Passive transport goes down a concentration or electrochemical gradient until equilibrium is achieved. Primary active transport utilizes the energy from the hydrolysis of ATP to move solutes against a concentration/electrochemical gradient directly. Secondary active transport uses the energy from the hydrolysis of ATP to create a concentration or electrochemical gradient and then harnesses these to drive the co-transport of a solute transport via a symporter or antiporter carrier. Active transport-expends energy of ATP to move solutes against an electrochemical gradient - passive transport: simple diffusion (high to low), passive diffusion (channel mediated high to low) facilitated diffusion (uniport, carrier mediated, high to low) -active transport: secondary: cotransport (symport, carrier mediated, solutes being pumped in same directions) exchange (antiport, carrier mediated, solutes being pumped in different directions) primary: pump-mediated requires ATP

Cooperativity

Positive cooperativity occurs when a substrate binds to an enzyme with multiple binding sites the other binding sites are affected by this change. An example is the binding of oxygen to hemoglobin to form oxyhemoglobin. Hemoglobin has four subunits, two alpha and two beta. They come together to form a tetramer, each subunit having its own active site to bind oxygen to. This active site contains a porphyrin ring structure with an iron atom in the center. When the subunit is not bound to an oxygen the iron is about 0.4 A below the plane of the ring. When the tetramer is in this state, it is considered to be in the T-state or tense state. The R- state, or relaxed state occurs when hemoglobin has bound to oxygen. Deoxyhemoglobin, or the T-state, has a low affinity for oxygen. When one molecule binds to a single heme, though, the oxygen affinity increases, which allows the following molecules to bind more easily in succession. Overall the R-state is more stable than T-state but under certain conditions this can change. The oxygen affinity of the 3-oxyhemoglobin is about 300 times greater than that of its deoxyhemoglobin counterpart. This behavior leads to the affinity curve of hemoglobin to become sigmoidal, not hyperbolic as with the monomeric myoglobin's affinity curve. In the same way, the ability for hemoglobin to lose oxygen is greater as fewer oxygen molecules are bound. This cooperativity can be seen in Hemoglobin when one of the oxygen binds to one of the tetramer's subunits. This will increase the probability that the other three sites will bind to oxygen.

Type V Integral membrane proteins

Proteins covalently linked to lipids

Type VI Integral membrane proteins

Proteins hace transmembrane helices and lipid anchors

G1 phase

RNA and protein synthesis. No DNA synthesis

cell membranes

Signals from the environment must traverse the plasma membrane and sometimes additional intracellular membranes such as the nuclear envelope to trigger a response. Extracellular and intracellular signals must transverse one of more membranes. Functions: Barrier, support, protection, transport of wastes, nutrients, hormones, neurotransmitters Selectively permeable and made of phospholipid bilayer and proteins w/ some carbohydrates.

Bacteriorhodopsin Seven Transmembrane Receptor (7TM)

Single polypeptide chain folds into seven hydrophobic alpha-helices that traverse the membrane roughly perpendicular to the plane of membrane. The 7TM helices cluster together surrounded by the acyl side chains of the lipids. The corresponding hydropathy plot color coded by the 7TM helices shows the hydropathy index - a measure of the free energy change accompanying the movement of an amino acid from a hydrophobic solvent into water.

extrinsic "death receptor" pathway of apoptosis activation

The extrinsic pathway is activated by ligand-bound death receptors mainly including TNF-TNFR1, FasL-Fas and TRAIL-DR4 or -DR5. Members of the TNF ligand family are primarily produced as type II transmembrane proteins arranged in stable homotrimers. They exert their biological functions via interaction with their cognate membrane receptors. Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily and generally have several functions including initiating apoptosis. The ability of preassembled TNF-R1 complexes to signal is masked by binding of the silencer of death domain (SODD) in the un-induced condition. After TNF binding, SODD is released from TNF-R1 complexes and the death domain- containing adaptor protein TRADD is recruited to the death domain of TNF-R1 by homophilic interactions of the death domains. It is generally believed that TNF-R1- bound TRADD then serves as a common assembly platform for binding of TNF receptor-associated factor (TRAF) 2 and the death domain-containing serine- threonine kinase RIP (receptor-interacting kinase). TNFR1 is also able to mediate apoptosis through the recruitment of an adaptor molecule called RAIDD (RIP- associated ICH-1/CED-3 homologous protein with a death domain). RAIDD associates with RIP through interactions between death domains and can recruit caspase 2 through an interaction with a motif, similar to the death effector domain, known as the CARD (caspase recruitment domain). Recruitment of caspase 2 leads to induction of apoptosis. Ligation of death receptors is followed by the formation of the DEATH-INDUCIBLE SIGNALLING COMPLEX (DISC), which results in the activation of pro-caspase-8. In type I cells, caspase-8 activates pro-caspase-3, which cleaves target proteins, leading to apoptosis. In type II cells, caspase-8 cleaves Bid, which, in turn, induces the translocation, oligomerization and insertion of Bax and/or Bak into the mitochondrial outer membrane. This is followed by the release of several proteins from the mitochondrial intermembrane space, including cytochrome c, which forms a cytosolic APOPTOSOME complex with apoptosis activating factor-1 (Apaf-1) and pro-caspase-9 in the presence of dATP. This results in the activation of pro- caspase-9, which leads to the activation of pro-caspase-3. The yellow circle represents a complex of Apaf-1, pro- caspase-9 and dATP (apoptosome), which can not be separated in the scheme.

cell cycle checkpoints

The primary goal of eukaryotic cell division is to successfully pass accurate DNA strands (mutation free) from parental genomes to daughter cells as cells replicate. This will ensure the cycle produces healthy and functional cells. However, DNA does not always exist mutation free and DNA with mutations will likely lead to cancer. To prevent the passing of mutated/damaged DNA which could cause replication of cancerous cells, the cell cycle includes an impressive system of checkpoints that, more or less, "scan" the DNA passing through the cycle for mutations and acts as a natural prevention against the duplication of cancerous cells. The term 'cell-cycle checkpoint' refers to mechanisms by which the cell actively halts progression through the cell cycle until it can ensure that an earlier process, such as DNA replication or mitosis, is complete. Checkpoints along the cycle not only assess the DNA for damage but can actually act upon it in efforts to correct any mutation which is hindering its advancement in the cycle. Mechanisms within the checkpoints can delay the cycle until mutations are corrected. If mutations are irreversible, they can tag a cell for self- destruction (apoptosis) and thereby eliminating the chance that mutated DNA will be replicated.

the tumor microenvironment

The tumor microenvironment (Upper) is an assemblage of distinct cell types that constitutes most solid tumors. Both the parenchyma and stroma of tumors contain distinct cell types and subtypes that collectively enable tumor growth and progression. Notably, the immune inflammatory cells present in tumors can include both tumor-promoting as well as tumor-killing subclasses. (Lower) The distinctive microenvironments of tumors. The multiple stromal cell types create a succession of tumor microenvironments that change as tumors invade normal tissue and thereafter seed and colonize distant tissues. The abundance, histologic organization, and phenotypic characteristics of the stromal cell types, as well as of the extracellular matrix (hatched background), evolve during progression, thereby enabling primary, invasive, and then metastatic growth. The surrounding normal cells of the primary and metastatic sites, shown only schematically, likely also affect the character of the various neoplastic microenvironments. (Not shown are the premalignant stages in tumorigenesis, which also have distinctive microenvironments that are created by the abundance and characteristics of the assembled cells.)

Ion Channels and transporters

There are 443 genes in the human genome (20-30K total) encoding transport proteins and 315 encoding ion channels. The solute carriers (SLC) and ATP binding cassette (ABC) transporters are the major super families of the transporters. Ion channels are split between voltage-gated and ligand-gated super families together with a number of ATPases that pump specific ions to create electrochemical gradients.

genetic basis of evolution

Unrepaired rare mistakes that occur during DNA replication may result in mutations. A mutation is a change of the nucleotide sequence of the genome of an organism. Mutations result from unrepaired damage to DNA or to RNA genomes (typically caused by radiation or chemical mutagens), errors in the process of replication, or from the insertion or deletion of segments of DNA. Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism. Mutations play a part in both normal and abnormal biological processes including: evolution, cancer, and the development of the immune system. A gene may be accidently duplicated and over many generations rare mistakes may occur in one the duplicated genes. In a very few rare cases, the altered protein produced as the result of the mutation alters the function of the duplicated gene - for example the mutant protein may bind a different substrate. A cell containing both the wild type and mutant gene may have acquired a new capability that allows it to survive in an ecological niche.

autophagy supports the growth of aggressive tumors

a) Autophagy is up-regulated in RAS-driven cancers and is also induced in hypoxic tumour regions where it supports tumour cell survival. b Autophagy deficient tumour cells accumulate defective mitochondria and are prone to cell death in hypoxic regions. This can lead to impairment of the growth of RAS-driven cancers and perhaps other cancers.

cyclin dependent kinase inhibitors (CKIs)

act as brakes to stop the cell cycle progression Active forms of the cyclin dependent kinases (CDKs) are a complex of at least two proteins, a kinase and a cyclin. CDK-Cyclin complexes drive the cell from one stage of the cell cycle to another. The cell cycle is determined by the constellation of proteins that are activated or inactivated by phosphorylation, a result of the activity of the CDKs during that stage. In mammalian cells, a succession of kinase subunits (CDK4, CDK6, CDK2, and CDC2) are expressed along with a succession of cyclins (cyclin D, E, A, and B), as the cells progress from G1 to mitosis. CDK4 and CDK6 complexed with one of several D-type cyclins function early in the G1 phase, probably in response to growth factors. CDK2 that complexed with cyclin E, cyclin A, or both is essential for the G1 S transition and DNA replication, respectively. CDC2 that complexed with cyclin A and cyclin B is essential for mitosis. The passage of cells from one stage of the cell cycle to another is tightly regulated by a wealth of controls that act on the transcription of cyclin genes, the degradation of cyclins, and modification of the kinase subunits by phosphorylation. A number of positive or negative feedback loops also contribute to the cell cycle progression. CDKs and their cyclin partners are positive regulators or accelerators that induce cell cycle progression; whereas, important negative regulators, such as cyclin dependent kinase inhibitors (CKIs), act as brakes to stop the cell cycle progression in response to regulatory signals. By direct association with CDK, CKIs can negatively regulate CDK activity. There are two types of CKIs. The four members of the INK family, INK4A (p16), INK4B (p15), INK4C (p18), and INK4D (p19), exert their inhibitory activity by binding to CDK4 and CDK6, and preventing their association with D-type cyclins. The three members of the CIP/KIP family, CIP1 (p21), KIP1 (p27), and KIP2 (p57), form heterotrimeric complexes with the G1/S CDKs. CKIs are induced in response to different cellular processes. The critical point of cell cycle control is the restriction point. After passing this point, the cell is irreversibly committed to the next phase of the cell cycle. The restriction point control is mediated by the cyclin D and cyclin E-dependent kinases. The primary substrates of CDK4/6 and CDK2 in G1 progression are the members of the retinoblastoma protein family pRB, p107, and p130. These molecules function as negative regulators at the restriction point. One important target of pRB for regulating the early G1 cell cycle progression is the E2F family of transcription factors. E2Fs regulate the expression of a host of genes that mediates both restriction point transversals, such as cyclin E, and the S phase progression, such as dihydrofolate reductase and thymidylate synthase.

Synaptic vesical fusion

action potential triggers synaptic vesicle fusion with the pre-synaptic membrane and neurotransmitter release Arriving action potentials depolarize the membrane at the pre-synaptic terminal activating voltage gated Ca2+ channels which raises intracellular [Ca2+] and trigger synaptic vesicles containing neurotransmitters to fuse with the membrane and release their contents into the synaptic gap.

Phospholipase C

activated by Gq-coupled GPCRs to generate Diacylglycerol and Inositol 1,4,5-triphosphate (IP3) that mobilized Ca+2 1) Hormone (H) binds to a specific receptor 2) The occupied receptor causes GDP-GTP exchange on Gq 3) Gq with bound GTP, moves to PLC and activates it 4) Activate PLC cleaves phosphatidyl-inositol 4,5-bisphosphate to inositol triphosphate (IP3) and diacylglycerol 5) IP3 binds to a specific receptor on the endoplasmic reticulum, releasing sequestered Ca+2 6) Diacylglycerol and Ca+2 activate protein kinase V at the surface of the plasma membrane 7) Phosphorylation of cellular responses to the hormone

Heterotrimeric G-proteins

alpha, beta, and gamma subunits distinct Alpha subunits activate different second messengers alpha i--> adenylyl cyclase, inhibition of cAMP production, ion channels, phosphiodiesterases, phospholipases alpha q--> PLC beta, DAG, Ca+2, PKC alpha s--> adenylyl cyclase, increase cAMP concentration alpha 12--> RhoGEFs, Rho G beta gamma subunits also produce a variety responses like dec Ca+, and produce cAMP G alpha responses mostly involve cAMP (alpha i, t, 12/14) both G alpha and beta gamma subunits have a variety of secondary messengers

tumor cell microenvironment

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

GR agonists glucocorticoids

amount the most effective and prescribed anti-inflammatory therapies therapy issues and future: -patients develop resistance -adverse side effects -SERGAs selective GR agonists

patch clamp

an electrode is placed on a cell membrane to alter the voltage of the membrane

peripheral membrane proteins

associated through electrostatic interactions and H-bonds with integral proteins and polar head groups

PDE-5 Inhibitors for ED

avanafil (standra or spedra) lodenaful (phase III) mirodenaful (mvix) sildenafil (viagra) tadalafil (cialis) vardenafil (levitra) udenafil (zydena)

microautophagy

nonselective process whereby cytosolic proteins are sequestered by imagination of the lysosomal membrane

G1/S checkpoint (restriction) point

end of G1 before S phase. If the environment or DNA unsuitable for DNA synthesis cell can arrest the process or go into G0 The transition from G1 to S phase is initiated by the increasing mitogen-stimulated activity of Cyclin D/Cdk4/6 which phosphorylates pRb resulting in the initial release of the E2F transcription factor. This activates a positive feedback loop rendering cell cycle progression independent of mitogenic stimulation, an event that has been termed restriction point (R-point). The feedback loop involves the up-regulation of CyclinE which, in complex with Cdk2, further stimulates E2F release and the transcription of genes necessary for S phase. After double-strand break (DSB) induction, two parallel checkpoint pathways target the activity of Cyclin/Cdk complexes. The slower pathway involves the stabilization of p53 and transcriptional up-regulation of p21 which binds and inhibits the Cyclin/Cdk complexes. The faster pathway acts via the activation of Chk2 and the inactivation of Cdc25. Thus, inhibitory phosphates of the CyclinE/Cdk2 complex can no longer be removed.

DNA polymerases

ensure fidelity of DNA replication A large number and amazing diversity of transactions are involved in DNA synthesis and are required to faithfully replicate genomes and to stably maintain them in the face of constant challenges from cellular metabolism and the external environment. To perform these tasks, cells harbor multiple DNA polymerases. In addition to DNA extension capabilities, DNA polymerases have proofreading and exonuclease activities

G2-M checkpoint

ensures that DNA has been copied without major errors Upon DNA damage, cell division cycle 25 (CDC25) proteins are inhibited through various mechanisms, including activation of ATM/ATR signaling cascades leading to checkpoint kinase activation and CHK-dependent CDC25 degradation or cytoplasmic sequestration of CDC25 through 14-3-3 binding. The inhibitory kinases WEE1 and MYT1 are activated by checkpoint kinases. CDK-cyclin complexes are in turn maintained in their inactive state and the cell remains arrested in the G2 to M transition phase

spindle checkpoint

ensures that chromosome separation has occurred correctly

EGFR signaling and drug therapy

epidermal growth factors are related to breast, and head and neck cancers small molecules that inhibit tyrosine kinases -> stop cancers

stem cells and differentiation

epigenetic changes sich as DNA methylation and histone modification, alters how genes are expressed without altering the underlying DNA sequence clinical potential: replacement therapy regenerative medicine embryonic stem cells adult stem cells induced pluripotent stem cells (IPSCs) Totipotent cells have unlimited potential to become any type of cell. Pluripotent cells are capable of giving rise to most but not all cell types. Multipotent cells are committed to giving rise to cells from a specific lineage with a specific function. Epigenetic processes play a crucial role in regulating the decision to adopt a stem, progenitor, or mature cell fate. Lysine-specific demethylase 1 (Lsd1), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible 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 required for proper hematopoietic maturation. Stem cells derived from embryos, adult tissues or by reprogramming (iPSCs) are being investigated and developed for their clinical potential in both regenerative medicine and for replacement therapy.

Anion exchanger (AE)- antiporter

example, erythrocyte chloride bicarbonate AE anti porter CO2 converted to bicarbonate (HCO3-) by carbonic anhydrase 1) Essential for CO2 transport between tissues and lungs 2) In tissues [CO2] high, in RBCs CO2 converts to HCO3- by carbonic anhydrase 3) The AE anti porter electroneural exchange 1 HCO3- OUT for 1 Cl- IN to RBC 4) HCO3- is the primary pH buffer in the blood and more soluble than CO2-- raises carrying capacity for CO2 in blood 5) In lungs [CO2] low, AE antiproton exchanges 1 HCO3- IN for 1 Cl- Out of RBC 6) In RBCs in HCO3- converted to CO2 vy carbonic anhydrase 7) CO2 exhaled by lungs

tumor cells

exhibit self- deficiency for growth and insensitivity to anti-growth signals leading to uncontrolled survival and proliferation normal cells receive survival cues and proliferation cures or inhibition and death cues causing survival and proliferation or cell death or ceased division

cGMP

generated by receptor or soluble gyanylyl cyclase isoforms in response to Natriuretic peptides and Nitric Oxide generated by two mechanisms: a nitric oxide synthase (NOS)- soluble guanylate cyclase (sGC) coupled pathway, and membrane natriuretic receptor-guanylate cyclase (NPR-A/B rGC) pathway. These two pools of cGMP interact with different effectors - namely other PDEs that regulate both cGMP and cAMP, and protein kinase G (PKG). PDE5 is a cGMP-selective PDE that is also activated by cGMP binding to the enzyme as well as PKG phosphorylation of the enzyme. sGC-generated cGMP can both augment and reduce cAMP in the cell by inhibiting PDE3 and activating PDE2, respectively. Inhibiting PDE5 also augments cGMP from this pool. Natriuretic peptide receptor (NPR)-coupled cGMP also activates PKG signaling. Cardiac stress regulation by PDE5 inhibition is primarily controlled by PKG targeting, and PDE5 inhibition enhances PKG signaling to stimulate several pathways; sarcoplasmic reticulum (SR) calcium handling proteins (PLB[phospholamban]), mitochondrial signaling involving ATP-sensitive potassium channels (KATP), mitogen activated kinases (MAPK), calcineurin (Cn), Rho-activated kinase (ROCK), protein kinase Cα (PKCα) their regulation of nuclear transcription factors (nuclear factor of activated T-cells [NFAT], myocyte enhancer factor-2 [MEF2], SRF, and GATA4). Upstream, these activators are blunted by PKG due to direct inhibition of transient receptor potential channel 6 (TRPC6), and regulator of G-coupled signaling 2 and −4 (RGS2/4) coupled inhibition of Gq-coupled receptors. At1 angiotensin I; Et1 endothelin 1; a-AR α-adrenergic receptor).

selective estrogen modulators (SERMs)

have different response profiles T tamoxifen (breast cancer) R raloxifene (osteoporosis) Tamoxifen is currently used to treat all stages of breast cancer, chemotherapy in women at high risk for breast cancer- also has beneficial effects on bone mineral density and serum lipids in postmenopausal some. Raloxifene is approved for the prevention and treatment of postmenopausal women. Raxloifene is approved for the prevention and treatment of postmenopausal osteoporosis and vertebral fractures. However SERMs have some potentially serious adverse effects, thromboembolic disorders and, for tamoxifen, uterine cancer.

neural cell adhesion molecule (NCAM, CD56)

hemophiliac binding glycoproteins expressed on the surface of neurons, glia, skeletal muscle and natural killer cells have immunoglobulin-like domains

Cadherins

hemophiliac binding interactions with cadherins on adjacent cells have adhesive domain Ca+2 dep, homophilic adhesion functional unit = dimer

Neuronal transmission

involves both voltage and ligand gated ion channels Initially the plasma membrane of the pre-synaptic neuron is polarized due to the Na+ K+ ATPase. Stimulation of the pre-synaptic neuron induces an action potential to move along the axon away from the cell body towards the synapse causing voltage gated Na+ channels to open and depolarize the membrane. This triggers adjacent voltage gated Na+ channels to open followed by the opening of K+ voltage gated channels that serve to repolarize the membrane. Directionality is ensured by a brief refractory period following the opening of voltage-gated Na+ channels. When the wave of depolarization reaches the synaptic terminal voltage-gated Ca2+ channels raise intracellular [Ca2+] and trigger synaptic vesicles containing neurotransmitters such as acetylcholine to fuse with the membrane and release their contents into the synaptic gap. Neurotransmitters like acetylcholine released into the synaptic gap bind to GPCRs and ligand-gated ion channels in the membrane 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 pass the signal on using the same process.

transcriptional regulation

involves the recruitment and interactions of multiple proteins activation and repression

hERG

inwardly rectifying voltage gated K+ chainels -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 -pharmacological effects of compounds --hERG channel studied by command-voltage protocol --upon depolarization from a holding potential of 80 mV, currents develop and rapidly inactivate (C-type) --when cell is rapidly repolarized to -40mV, channel recovers to open state and tail currents ensue --pharmacological effects on tail currents measured

NADPH

is an electron carrying cofactor that collects electrons from oxidative reactions and then donates them to reducing reactions during biosynthesis. Present at low concentrations these cofactors are essential to anabolic reactions and must be regenerated by catabolic reactions.

ATP

is the shared chemical intermediate linking energy-releasing and energy-consuming cellular processes.

chaperone-mediated autophagy

selective process- proteins with defined consensus sequences recognized by molecular chaperones, including Hsp70, are delivered to the lysosome

repair mechanisms

maintain stability of DNA To maintain genetic stability, cells have developed extensive DNA repair mechanisms to correct any damage that occurs arising from cellular metabolism and/or the external environment. Direct repair results in the reversal of an alkylated base to a normal base without any intervening steps or the generation of DNA strand breaks. The O 6- methylguanine DNA methyltransferase (MGMT) catalyses the transfer of the aberrant methyl group onto a cysteine in the active site of MGMT, thereby removing the lesion from DNA. The AlkB homologue (ALKBH) family of α-ketoglutarate-dependent dioxygenases catalyse the hydroxylation of the aberrant methyl group, which then spontaneously leaves as formaldehyde (HCOH) to regenerate the normal base. b | The base excision repair (BER) pathway removes simple alkyl and oxidative base lesions. BER is initiated by a DNA glycosylase, such as alkyladenine-DNA glycosylase (AAG), which excises the damaged base to generate an abasic site for subsequent processing by either short-patch BER or long-patch BER (not shown). In short-patch BER, the apurinic-apyrimidinic endonuclease (APE) incises the abasic site to yield a 3′ OH adjacent to a 5′-deoxyribose phosphate (5′-dRP) moiety. DNA polymerase β (Pol β) removes the 5′-dRP and replicates DNA from the 3′ OH, generating a nick ready for subsequent ligation by DNA ligase (LIG). Long-patch repair occurs in a similar manner except that a longer piece of DNA is removed. c | Nucleotide excision repair (NER) removes bulky, helix-distorting lesions but it can also serve as a backup repair mechanism for the excision of certain alkylated bases16. NER is initiated by the recognition of a distorted region containing the DNA adducts followed by a 5′ and 3′ incision on either side of the lesion to create a 27-29 nucleotide gap. The excised region is then filled in by Pol δ or Pol ɛ for subsequent ligation. d | The mismatch repair (MMR) pathway mediates the removal of mismatched bases, as well as mispairs that are caused by base alkylation lesions such as O 6meG:T. In mammals, MMR is initiated by the recognition of mismatches by the MUTSα heterodimer (MSH2-MSH6) followed by recruitment of the MUTLα heterodimer (PMS2- MLH1). Diffusion of MUTSα-MUTLα leads to nicking of the DNA either upstream or downstream of the mismatch; this serves as an entry point for exonuclease 1 (EXO1) that removes a segment of DNA. This is subsequently filled in and repaired by a combination of Pol δ, Pol ɛ and LIG to complete repair. TFIIH, transcription factor IIH; XP, xeroderma pigmentosum protein.

Na/K ATPase active transport

maintains intracellular [Na+] and [K+] to generate membrane potential chemical gradient drives Na= and Ca+2 inward and K+ outward electrical gradient drives Cl- outward -for every ATP Na+ K+ ATPase moves 3 Na+ out and 2 K+ in to a cell *-membrane potential across the cell is central to neuronal signaling- action potential* -Na+ gradient used to drive secondary active transport of solutes

beta-adrenergic receptor orthosteric ligands ****

mediates effects effects or epinephrine-- target of beta blockers beta lockers are prescribed for hypertension, cardiac arrhythmia, glaucoma, anxiety, and migraine headache

cell cycle/ DNA damage checkpoints and cancer

mutations in apoptosis pathways, DNA-damage, DNA-repair or mitotic-checkpoint pathways can permit the survival or continued growth cells of genomic abnormalities, thus enhancing the likelihood of malignant transformation A. Replication-fork arrest stimulates the initiation of cellular ATR activity, whereas DNA damage can directly activate ATM and can lead to replication-fork arrest, thereby also activating cellular ATR kinase. Once active, both the ATM and ATR kinases, functioning in combination with other proteins and substrates, help determine the outcome of the cell. If genomic instability ensues, this can contribute to cellular transformation. B. If DNA damage is repaired efficiently, the likelihood of tumour development is low. If cells have mutations in DNA-damage response signaling pathways — either sporadic or inherited — this will lead to enhanced genomic abnormalities. Cells with damaged DNA frequently arrest or do not survive, thus reducing the probability that they will progress to malignancy. Mutations in apoptosis pathways, DNA-damage, DNA-repair or mitotic-checkpoint pathways can permit the survival or continued growth of cells with genomic abnormalities, thus enhancing the likelihood of malignant transformation.

Janus Tyrosine Kinase (JAKs)

named after Janus the roman god of gates, doors, doorways, beginnings and endings FERM (four point one, erin, radian, 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 -constitutively associated with cytokine receptors -ligand binding catalyzes JAK auto-phorphorylation and subsequent cytokine receptor phosphorylation -creates docking sites for STAT-SH2 domain binding

acquisition of the metastatic phenotype

neoplastic cells with tumors are not homogeneous. Heterogeneous populations of cells within a tumor are organized hierarchically with self-renewing stem cells (SCs), partially differentiated trait amplifying (i.e. progenitor) cells, and fully differentiated end-stage cells. epithelial-to-mesenchymal transition (EMT) can induce noon-CSCs to enter into a CSC-like state conferring traits that empower them to disseminate from primary tumors and seed metastases Tumors are heterogeneous populations of cells. Cancer stem cell (CSC) subpopulations are particularly well poised to complete the metastatic cascade. Two alternative means of generating CSCs are depicted here. Intrinsic CSCs are thought to exist in primary tumors from the very early stages of tumorigenesis and may be the oncogenic derivatives of normal-tissue stem or progenitor cells. The epithelial-to- mesenchymal transition (EMT) plays critical roles in early embryonic morphogenesis . This trans differentiation program, driven by EMT-inducing transcription factors (EMT- TFs), is deployed during a number of critical steps of morphogenesis, enabling cells of epithelial phenotype to generate mesenchymal derivatives. Induced CSCs may arise as a consequence of the EMT. In this case, carcinoma cells initially recruit a variety of stromal cells, such as fibroblasts, myofibroblasts, granulocytes, macrophages, mesenchymal stem cells, and lymphocytes. Together these cells create a reactive microenvironment that releases factors (e.g., Wnt, transforming growth factor-b, fibroblast growth factor) that cause the neighboring cancer cells to undergo the EMT and acquire CSC-like characteristics.

G2 phase

no DNA synthesis RNA and protein synthesis continue

Karyorrhexis

nuclear fragmentation, pyknotic nuclear membrane ruptures and fragments

Pyknosis

nuclear shrinkage due to DNA condensation

autophagolysosome formation and regulation of autophagy

on induction of autophagy, the isolation membrane or phagopore elongates and engulfs the cellular contents. Sealing of the tips leads to completion of the double-membrane autophagosome. The outer membrane then fuses with the lysosome resulting in degradation of its contents. Key elements of this process are the autophagy-related (ATG)12-ATG5-ATG16L complex, which recruits light chain (LC) 3 to the membrane, and ATG4, which controls the lipidation and recycling of LC3. The mammalian complex ULK1/2-mAtg13-FIP200 is required for autophagy. mTORC1 acts as a negative regulator of autophagy by phosphorylating Atg13 and ULK1/2. During starvation, mTORC1 is released from this complex resulting in dephosphorylation of the components and activation of ULK1 and ULK2, ubiquitin-like conjugation systems that play roles in the early steps of autophagy. Early steps in autophagosome formation are regulated by two ubiquitin-like conjugation systems. In both cases, a ubiquitin-like protein (Atg8 and Atg12) is conjugated to an E1-like enzyme (Atg7), and then to an E2-like enzyme (Atg3 and Atg10). These are then used to form phosphatidylethanolamine (PE) conjugates of Atg8 (LC3 II) as well as protein conjugates of Atg5/12/16. Upstream inhibitors of autophagy include insulin and amino acids (AAs), whereas glucagon, starvation and reactive oxygen species (ROS) activate autophagy. Additional regulators of autophagy are the mammalian target of rapamycin (mTOR) and Beclin-1. The AMP-activated protein kinase (AMPK) senses energy deprivation and enhances autophagy via mTOR inhibition. The main pharmacological mTOR inhibitor is rapamycin. Beclin-1 and ultaviolet radiation resistance-associated gene protein (UVRAG) activate autophagy, whereas binding of the anti-apoptotic Bcl-2/Bcl-Xl to Beclin-1 leads to inhibition of autophagy. The class III phosphatidylinositol-3-kinase (PI3K) regulates autophagy in concert with Beclin-1 and can be blocked by the pharmacological agent 3-methyladenine (3MA), thereby blocking autophagy. Both the tumour suppressor protein p53 and Bcl-2/Bcl-Xl connect autophagy with apoptosis.

cancer stem cells

oncogenes may reprogram early stem cells of precursor cells towards specific differentiated tumor cell fates Oncogenic lesions act on stem/progenitor cells by imposing a given, oncogene- specific, tumor-differentiated cell fate. Cancer stem cells (CSCs exhibit traits such as motility, invasiveness, and self-renewal, which are central to malignancy, may in fact be the reflection of the actions of the elusive CSC subpopulations within larger populations of neoplastic cells. In many tumors, such cells may represent a tiny fraction of the total cellularity of individual tumors, yet these CSCs may be the critical drivers of their malignant progression.

gated ion channel

opens or closes in response to concentration of signal ligand (S) or membrane potential

CDK activating kinases (CAK) and cell division cycle 25 (CDC25) phosphatases

regulate CDK-cyclin mediated progression through the cell cycle a) CDK activity is positively regulated by the association with the cyclins, and by phosphorylation of the T-loop threonine by the CDK activating kinase (CAK), a serine/threonine kinase. Inhibitory phosphorylation of adjacent threonine and tyrosine residues (T14/Y15 in CDC2) is mediated by dual specific kinases (Wee1 and MYT1). This inhibition is relieved when the cell division cycle 25 (CDC25) phosphatases dephosphorylate these residues, which triggers entry into mitosis. b) CDC25A, B and C are involved in de-phosphorylating CDK-cyclin complexes at all stages of the cell cycle. c) CDC25A, B and C control entry and progression into mitosis. CDC25B is thought to be responsible for the initial activation of CDK1-cyclin B at the centrosome that contributes to microtubule network reorganization and mitotic spindle assembly. Nuclear translocation leads to an auto-amplification process (bold arrows) of CDC25s that then fire the bulk of CDK1-cyclin B complexes and trigger mitosis.

phosphatases

regulate kinase signaling 285 phosphatase genes Classification: PTP- protein-tyrosine phosphatase PPP- serine/threonine-specific protein phosphatase PPM- protein phosphatase 2C-like HAD- halfacid dehalogenase-like LP- phosphatidix acid phosphatase, inositol monophosphatase, and inositol polyphosphate-related phosphatase NUDT- NUDIX hydrolase Protein & Lipid phosphatases regulate kinase signaling - terminate kinase cascade signaling pathways

CDK-cyclin complexes

regulated by phosphorylation, proteolysis, and gene expression a) In the absence of the cyclin partner the cyclin dependent kinase (CDK) is inactive. As cyclin protein levels increase they form active Cyclin-CDK complexes. However, the CDK activated at the time of mitosis has a T-loop that can fold into the substrate binding site. Phopshorylation of Tyr-160 on the T-loop by a CDK activating kinase (CAK) causes a conformational change that opens up the substrate binding site. However phosphorylation of threonine and tyrosine residues (T14/Y15 in CDC2) by dual specific kinases (Wee1 and MYT1) makes the Cyclin-CDK complex inactive. This inhibition is relieved when CDC25 phosphatases dephosphorylate these residues, which triggers entry into mitosis. b) the fully active Cyclin-CDK triggers its own destruction by phosphorylation of destruction box recognizing protein (DRBP) which activates the addition of ubquitin by ubiquitin ligases targeting it for destruction by the 26S proteasome system. The activity of the various Cyclin-CDK complexes varies with the phase of the cell cycle. The retinoblastoma phosphoprotein (Rb) is phosphorylated and dephosphorylated during the cell cycle; the hyperphosphorylated (inactive) form predominates in proliferating cells, whereas the hypophosphorylated (active) form is generally more abundant in quiescent or differentiating cells. Rb function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors. E2F sites are found in the promoters of many genes that are important for cell cycle progression, and Rb appears to repress transcription of these genes through its interaction with E2F .

neuronal action potential

regulated by voltage gated ion channels 1) RMP (cytosolic face negative) 2) goal potential change; graded potential 3) threshold level 4) depolarization: opening of voltage gated Na+ channels 5) depolarization: closure of Na+ and opening of K+ voltage gated channels (cytosolic face positive) 6) hyperpolarization: voltage gated K+ channels reamin open after the potential reaches resting level

hematopoiesis in bone marrow

regulated by: -colony stimulating factors/ cytokines -location/site(s) of differentiation spleen, liver, lymph nodes and bone marrow -other factors in microenvironment cell-cell and cell-ECM interactions Haematopoietic stem cells (HSCs) reside in the medulla of the bone (bone marrow) have the ability to give rise to all of the different mature blood cell types and tissues; lymphocytes (T & B cells), myelocytes (monocytes, neutrophils, eosinophils & basophils) & erythrocytes. In embryos, blood formation occurs in the yolk sac, but as development progresses, blood formation occurs in the spleen, liver and lymph nodes. When bone marrow develops, it eventually assumes the task of forming most of the blood cells, however, maturation, activation, and some proliferation of lymphoid cells occurs in secondary lymphoid organs (spleen, thymus, and lymph nodes). Haematopoietic growth factors initiate signal transduction pathways, altering transcription factors, that, in turn activate genes that determine the differentiation of blood cells. Cell determination appears to be dictated by the location of differentiation. For the stem cells and other undifferentiated blood cells in the bone marrow, the determinism theory of haematopoiesis suggests that colony stimulating factors and other factors of the haematopoietic microenvironment determine the cells to follow a certain path of cell differentiation. The stochastic theory suggests that undifferentiated blood cells are determined to specific cell types by randomness. The haematopoietic microenvironment prevails upon some of the cells to survive and some, on the other hand, to perform apoptosis and die. By regulating this balance between different cell types, the bone marrow can alter the quantity of different cells to ultimately be produced.

Protein Kinase C (PKC) activation

regulates numerous downstream pathways. Cellular effects: inc protein synthesis inc cell surface area in transcription = hypertrophy inc fibroblast proliferation inc fibroblast migration inc collagen deposition = fibrosis inc secretion of cytokines inc migration of inflammatory cells inc transcription = inflammation inc calcium handling inc myofilament sensitivity inc phosphorylation of troponins = contractile dysfunction

increased Ca+2 mobilization

regulates numerous downstream signaling pathways 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.

RTK dimerization mechanisms

required to become active ligand binding to the receptor stabilizes a specific relationship between individual receptor molecules in an active dimer or oligomer TrkA: ligand-mediated dimerization KIT: a ligand-mediated dimer with receptor contacts FGFR: multiple contacts with FGF and heparin molecules The EGFR/ErbB family: the receptor mediated extreme In general, RTKs associate into dimers when ligand (red) binds to their extracellular regions. The bound ligand, which can form all, a portion, or none of the dimer interface, activates the receptors by stabilizing a specific relationship between two individual receptor molecules. A. A nerve growth factor dimer (red) cross-links two TrkA molecules without any direct contact between the two receptors. B. A stem cell factor dimer (red) also cross links two KIT molecules. In addition, two Ig-like domains (D4 and D5), which reorient upon receptor activation, interact across the dimer interface. Thus, KIT combines ligand-mediated and receptor mediated dimerization modes. C. 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. In addition, each fibroblast growth factor molecule (red) contacts Ig-like domains D2 and D3 of both FGFR molecules. D. Dimerization of ErbB receptors is mediated entirely by the receptor. Binding simultaneously to two sites (DI and DIII) within the same receptor molecule, the ligand drives conformational changes in epidermal growth factor receptor (EGFR) that expose a previously-occluded dimerization site in Domain II

cancer cell metastasis

responsible for over 90% of cancer-associated mortality envisioned as a process that occurs in two major phases: (i) physical translocation of cancer cells from the primary tumor to a distant organ (ii) colonization of the translocated cells within that organ In metastatic dissemination, a cancer cell from a primary tumor executes the following sequence of steps:locally invades the surrounding tissue, enters the microvasculature of lymph &/or blood systems (intravasation), survives & translocates largely through the bloodstream to microvessels of distant tissues, exits the bloodstream (extravasation), survives in the microenvironment of distant tissues, and adapts to the foreign microenvironment of these tissues in ways that facilitate cell proliferation and the formation of a macroscopic secondary tumor (colonization).

RTK multi-domain interactions

specify signaling complex formation and branching points -1st and primary substrates that RTKs phosphorylate are the receptors themselves -auto-phosphorylation sites in the TKD play and important regulatory role in most RTKs -phosphorylation of insulin receptor activation loop increases catalytic efficiency 50-200 fold -additional tyrosine are the phosphorylated in other parts of the cytoplasmic region of most RTKs (IRS proteins fulfill this function for the insulin receptor) -P-Tyr's function as specific sites for the assembly of downstream signaling molecules that are recruited to the receptor and activated in response to growth factors stimulation Coordinated assembly of multi-protein complexes in receptor tyrosine kinase (RTK) signaling provides branching points in a signaling network. A. The docking protein FGF receptor substrate-2 (FRS2α forms a complex with activated fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors via its phosphotyrosine-binding domain (PTB). The activated RTK phosphorylates FRS2α on multiple tyrosines, and the resulting phosphotyrosines recruit multiple Grb2 and Shp2 molecules, which bring a second docking protein, Gab1, into the complex. Gab1 is tyrosine phosphorylated and recruits additional signaling proteins, including phosphoinositide 3-kinase (PI3K). PI3K initiates a positive feedback loop in which PtdIns(3,4,5)P3 (PIP3), generated by PI3K, recruits more Gab1, leading to further PI3K activation. B. The multiple domains of phospholipase C-γ (PLCγ) cooperate to integrate multiple signals at the plasma membrane. The N-terminal SH2 domain is responsible for complex formation with activated RTKs. The C2 and PH domains cooperate with the SH2 domain to target PLCγ to the plasma membrane. One or both of the PH domains may also specifically recognize products of RTK-activated PI3K. RTK-mediated tyrosine phosphorylation of PLCγ leads to intra-molecular binding of the C-terminal SH2 domain to phosphotyrosine 783. This stimulates enzymatic activity of PLCγ, leading to hydrolysis of PtdIns(4,5)P2 (PIP2) and consequently leads to the formation of Ins(1,4,5)P3 (IP3) and diacylglycerol (DG).

protein kinase inhibitor challenges

spot for phosphate and ATP, drug prevents ATP binding then there is no phosphorylation of a cancer protein specificity and selectivity -kinase active site highly conserved -ATP competitive inhibitors

steroid receptor

steroid binding to a nuclear receptor protein allows the receptor to regulate the expression of specific genes

Voltage-gated K+ channels

structure: Kv1.2 (KCNA2) potential therapeutic applications decreased frequency of action potential firing --> CNS depression, neuronal conduction disorders (for example, multiple sclerosis) and cognition disorders --> Kv Channel inhibitor--> normal neuronal action potential firing (therapeutic intervention) increased frequency of action potential firing--> CNS hyper excitability, seizures, pain, ADHD, anxiety, bipolar disease and schizophrenia--> Kv channel activator --> normal neuronal action potential firing (therapeutic intervention)