MCB 252 Exam 1

GAPs

GTPase activating proteins: increase intrinsic GDP active RAS --> causes more inactive RAS

What do G alpha(s)s do?

Lead to more cAMP being made -from hormones such as epinephrine, glucagon, ACTH

What do G alpha(i)s do?

Leads to no cAMP being made -from hormones such as PGE and adenosine

Cultured cells

-Provide a homogeneous population of cells -Media and experimental conditions can be precisely controlled -To be grown in culture, animal cells must be provided a liquid mixture that includes nutrients, vitamins, and growth factors (GFs)

RAS pathway inactivation

-RAS interacts with a GAP -phosphatases deactivate kinases

cell surface receptors

-Receptors: receive and transmit signals -2 types of receptors: cell surface (hydrophilic signals) & intracellular receptors (hydrophobic signals) -Cell surface receptors are usually transmembrane proteins -Binding of the ligand causes a conformational change which allows the receptor to interact with downstream proteins -activate one or more effector proteins: can change the function of the cell -Long term effects occur when the product of the genes (from the pathway) has an impact on the cell

Signaling Pathways

-Signaling molecules (ligands) can be proteins, peptides, amino acids, nucleotides, steroids, fatty acid derivatives, and even gases --> these are released by other cells through exocytosis, diffusion, or are present in their cell membrane -Is a series of switches--> there are many ways to turn proteins on and off, one pathway can elicit a variety of responses, geneticists call it a switch pathway

How is a protein polyubiquitinated?

-The conjugation of ubiquitin requires ATP and a three enzyme system: E1--> ubiquitin-activating enzyme (1 type), E2--> ubiquitin-conjugating enzyme (15-20 types), E3--> ubiquitin protein ligase (800+ types) Step 1) ubiquitin is activated at its C-terminal end by E1--> forms a high energy thioester between the C-terminal carboxylate of ubiquitin and a specific cytosine residue of E1 Step 2) ubiquitin is transferred as a thioester to E2 enzyme Step 3) substrate recognition involves an E3 ligase (like a mediator) -There are many types of E3--> specificity for substrate recognition -E3 recognizes certain types of amino acid stretches -sequence examples: D box, KEN box, PEST sequence, hydrophobic patch

Apoptosis

-When a cell needs to die -phosphatidylserine moves to the exoplasmic side--> the implication of this is that cells in our body have receptors that bind to phosphatidylserine and then activate mechanisms

model organism

-Yeast, roundworm, planarian, mouse, alga, fruit fly, zebrafish, plant -Scientist pick certain organisms based on their goal -Planarian --> good for studying stem cells -Mouse--> genetically close to humans -Fruit fly--> strong genetics

Ran

-a GTPase and monomeric like RAS--> controls directionality of molecules -Ran-GTP is active --> has high concentration in the nucleus Ran-GDP is inactive --> has high concentration in the cytoplasm

NFKB

-a master regulator of toxin in cells of the immune system -A two protein complex: p50 & p65 (heterodimer complex) -come into the nucleus and binds to the promoters of 150-200 genes and activate the transcription of them --> kill a virus and other pathogens -don't always want NFKB to be activated

Ubiquitin protein

-a protein of 76AA that is highly conserved and compacts into a globular structure -Ubiquitin attaches to a target protein through a covalent bond between the glycine of the C-terminal ubiquitin and the side chain of a lysine(K) on the target protein - Ubiquitin-protein is a target protein for further ubiquitination, the additional ubiquitin units add to the previous ubiquitin to form a poly-ubiquitin chain (always a lysine & glycine bond)(always linear) -polyubiquitination regulates protein turnover in a cell by closely regulating protein degradation --> need at least 4-6 ubiquitin molecules to be linearly connected and will degrade the target protein that the linear chain is connected to

Nucleoporins

-are estimated to be present in either 16 or 32 copies per NPC (x30 for whole complex) -the relative distribution of nucleoporins within a NPC is defined -contain regions of repeated AA sequences that end in FG (phenylalanine-glycine) or XFXFG. This is the most highly conserved feature between yeast and mammalian nucleoporins --> FG repeats create a hydrophobic environment which prevents the entry of larger molecules -some are glycosylated

Effector proteins

-can change a cells metabolism -can alter gene expression -alter cell morphology or shape

19S proteasome

-cap that controls what goes into the 20S core -multi-subunit protein (19) -has a molecular mass of 700KDa -also called PA700 -recognized proteins to be degraded, hydrolysis ATP, and controls access to the core particle -has proteins called ubiquitin receptors -ubiquitin binds and the DUBs (part of the cap) can remove the ubiquitin part of the molecule connected to the protein being degraded -AAA (6 protein complex) on the cap (with ATP) can unfold the protein of interest --> protein is unfolded to a primary structure in order to expose the peptides for degradation. Linear primary protein can now fit into the 20S core promoter

Fluidity of membrane

-depends on both its composition and temperature -phospholipids can easily move laterally, can flex, and can rotate -proteins can move laterally

peripheral proteins

-do not cross the membrane -Interact with the headgroups of different molecules or just the exoplasmic or cytosolic side -loosely attached to the membrane

GRB2

-has two domains -is an adaptor protein -SH2 domain interacts with the receptor (the tyrosine phosph. receptor, EGFR) -SH3 domain interacts with SOS

Acetylcholine ligand

-heart muscle cells: decrease rate and force of contraction -skeletal muscle cells: causes contraction -salivary gland cell: causes secretion This is because the downstream receptors are different

FRAP (Fluorescence Recovery After Photobleaching) Curve

-how we know something is moving Step 1) label molecule to tag phospholipid (with fluorescence) Step 2) Use laser to bleach a certain region Step 3) Allow time to recover --> over time fluorescence will come back into the bleached area because of lateral shift -if fluorescence doesn't come back --> that type of molecule doesn't move through the membrane -Mobile fraction: how many molecules are coming back

Phospholipids

-hydrophobic fatty acid tails (can be saturated with all single carbon bonds; can be unsaturated with cis alkenes; 12-18 C in chain) -hydrophilic (polar) head groups -name based on headgroup - amphipathic -difficult to switch leaflets because a charged molecule cannot pass through the middle of the hydrophobic region--> flippases (&ATP) can achieve this

Nuclear Pore Complex (NPC)

-involved in the regulation of nucleo-cytoplamic trafficking -125 MDa -has cytoplasmic filaments on the cytoplasmic side -has a nuclear basket on the nucleoplasmic side -there are 30 different types of proteins in the NPC called nucleoporins -Have multiple ring like structures --> rings make sure the NPC is connected to the membrane in a specific way -about 3000-5000 per cell -10^6 molecules are exchanged every second

RAS

-is a lipid anchored protein on the cytoplasmic leaflet that can move laterally -is a GTPase (hydrolyzes GTP into GDP + P) --> alone it is a weak GTPase -functions on transducing signals from many membrane receptors -alternates between an active state (RAS-GTP) and an inactive state (RAS-GDP)

20S core structure

-made up of 4 rings which are each made up of 7 proteins--> beta rings are the inner/middle rings, alpha rings are the outer rings -the four rings form 3 interior chambers--> the middle chamber is the catalytic chamber where actual protein degradation occurs -there is a very small "hole" in the middle (1.5-1.7nm)

How do we know what is upstream/downstream of something within a signaling pathway?

-make a LOF mutation of the receptor & a GOF mutation of RAS --> the cell will grow because what is upstream of RAS no longer matters Examples: -wild type Sev RTK and RAS --> R7 neuron -single mutant Sev --> no R7 neuron -double mutant Sev and RAS^D --> R7 neuron

Catalytic chamber composition

-only 3 of the 7 beta subunits have catalytic activity -Beta 1: caspase-like--> cleaves after acidic residues -Beta 2: trypsin-like--> cleaves after basic residues -Beta 5: chymotrypsin-like--> cleaves after hydrophobic residues -any protein that goes through will be degraded in some way

GCPR

-part of the largest family of receptors--> involved in many different pathways -about 30% of drugs target GCPR receptors -have 7 domains -act through trimeric G proteins --> have 3 subunits: alpha, beta, gamma

Lipid anchored proteins

-present on either side of the membrane -Always has a covalently attached lipid molecule -lipid is part of the membrane -if lipid and protein strongly interact, it may interfere with the fluidity of the membrane

Ways to activate a signal pathway

-protein synthesis -ligand binding -unmasking -release from the membrane -covalent modification -addition of a second subunit -stimulation of nuclear entry

Plasma membrane properties

-receive signals -made from phospholipids (heads face outward, tails face inward) -has two leaflets -asymmetric, semi-permeable, fluid in nature -exoplasmic leaflet: outside of cell -cytoplasmic leaflet: faces inside of cell -sides have different properties because different headgroups act as signalling molecules for different proteins

The 26S proteasome

-the machinery that specifically degrades poly-ubiquitinated proteins -has 60 components -it is very abundant (about 1% of cellular proteins) -present in the cytoplasm and the nucleus -is actively ATP dependent -Has a 20S core structure -Has one or two 19S caps on either one side or both side of the core structure -protein is degraded into 2-20 AA long peptides *not the only thing in the cell that can degrade proteins-->lysosomes*

RAS/MAP kinase pathway

1) EGF interacts with EGFR which is a transmembrane protein -->the cytosolic side of EGFR has the kinase activity 2) Once bound, two EGFRs will dimerize --> dimerization activates the kinase activity of the EGFRs cytoplasmic domains--> tyrosines on either EGFR are phosphorylated 3) Phosphorylated tyrosines act as binding sites for GRB2--> this binding activates GRB2 4) Once GBR2 is activated it recruits SOS (binds to GRB2 and is now activated) 5) Activated SOS binds to inactive RAS and activates RAS (because SOS is a GEF) 6) Once RAS is active, it binds to RAF 7) RAF is usually inhibited by a bound 14-3-3 protein, the binding of RAS causes RAF to release the inhibitor and become active 8) RAF is a kinase. Once activated it will interact with/phosphorylate MEK 9) MEK then interacts with the MAPK kinase

GPCR desensitization depends on receptor phosphorylation

1) Receptor inactivation and can not interact with G proteins 2) Receptor sequestration and are temporarily moved to the interior of the cell 3) Receptor down regulation; destroy in lysosomes after internalization

How many different types of G alpha proteins are there?

16-20 types -two types are G alpha(s) and G alpha(i)

What percent of genes in the genome code for membrane associated proteins?

20-30%

How many protein coding genes do cells have?

25,000 protein coding genes -only a subset are active in each cell -house keeping genes are active in all cells

Stem cells

Have the potential to form all types of cells in the body (it depends on signals)

Cholesterol in plasma membranes

Equal on each leaflet

Which lysine leads to degradation?

lysine 48 degradation is targeted by proteases

Signal pathway

reception & amplification --> transmission (fast response) --> change of gene expression (if a signal goes into nucleus; slow)

RAF and MEK are what type of kinases

serine kinases

Amino acids that can be phosphorylated

serine, threonine, tyrosine, histidine -Proteins are phosphorylated by kinases -Proteins are de-phosphorylated by phosphatases

What acts as a GEF in the GPCR pathway?

the GCPR

What acts as a GAP in the GPCR pathway?

the effector molecule

Acylation

Protein has Glycerine on the C terminus (3' end). There's an addition of a fatty acyl group such as palmitate or myristate

HER2 protein

RTK receptor that is overexpressed in breast cancer -activity can be repressed when herceptin is used

Diffusion through the NPC

The passive diffusion channel of the NPC has an effective diameter of 9nm -Proteins <40KDa: diffuse through relatively fast -Proteins >40KDa: diffuse through very slow, almost negligible -Proteins >60KDa: diffuse through exceedingly slow

First evidence of active transport comes from...

The study of a nuclear protein named Nucleoplasm Nucleoplasm: -a 5 subunit molecule (LARGE) -scientist purified the protein and injected into cytoplasm -over time they saw that they entered the nucleus -broke the protein into fragments, injected this into the cytoplasm, and watched what part of the protein would go into the nucleus -indicated that the tail had something that helped it go into the nucleus Conclusion: the tail had a nuclear localization signal (NLS)

Synapic signal

There is a debate about whether it is paracrine or contact dependent

Specific signals activate certain processes inside the cell

This results in different cell fates

Definition of a gene

a gene: all sequence elements that control the expression of a product --> differential expression of genes is dependent on activation or inhibition of the sequence elements

Location of C-terminus of GPCR

always in the cytosolic side

Location of N-terminus of GPCR

always in the exoplasmic side --> ligands always interact on this side

Receptor binding

can only bind to one type of ligand typically

Oncogenes

cellular genes in which gain-of-function alleles contribute to cancer

tumor suppressor gene

cellular genes in which loss-of-function mutant alleles contribute to cancer

Proteins on the membrane act as:

channels, receptors, transporters, enzymes, cell adhesion molecules

Phosphatidylserine

found on cytosolic leaflet, flips to outside during apoptosis

Phosphatidylcholine

found on the exoplasmic leaflet

How were other players in the RAS/MAPK kinase pathway identified?

genetic screens in drosophila fruit flies -mutagenize an organism and look among a collection of mutagenized organisms for those that display a particular phenotype of interest -this screen was designed to understand development and differentiation -Scientist use the mutant fly "sevenless"

GEFs

guanine exchange factors: facilitate the release of GDP --> RAS is free--> GTP now has a high chance of binding to RAS (causes more active RAS)

TFs cannot enter the nucleus alone, they need what

importin

Several receptors impinge on I-KB by different paths

*the paths are the same from activation of I-KB forward The receptors in the NFKB pathway: IL-1 receptor -the receptor is a dimer -when the dimer is formed it acts as a substrate for several proteins in the cytoplasm -one of these proteins, TRAF6, is an E3 ligase that adds an ubiquitin chain to itself (this doesn't involve degradation) -ubiquitin acts as a binding platform for multiple proteins--> TAK1 is recruited (a kinase) -two I-KB kinases bind (one on each ubiquitin chain, there are two TRAF6s involve that each add a chain to itself) -TAKI phosphorylates the I-KB subunit. I-KB becomes active and phosphorylates the I-KB alpha molecule

MAPK

- is a dual kinase -can phosphorylate 100+ substrates in the cell --> these can be within the cytoplasm or nucleus -can activate the expression of genes that are important for cell proliferation --> uncontrollably proliferation causes cancer -to become active it must dimerize--> phosphorylates another kinase called p90^RSK) -can phosphorylate specific TFs

GPCR desensitization

-Activated GPCR stimulates GRK to phosphorylate the GPCR on multiple sites -phosphorylated receptor has a low binding affinity for the G alpha molecule -phosphorylated site recruits beta-arrestin complex which facilitates the degradation of the receptor

Ligands

-Are very specific--> bind to specific receptors -Do not have covalent attractions--> weak but stable reactions -Can bind to similar types of receptors in different cell types and elicit different reactions

Activation of Kinase EGFR domain by dimerization

-C terminal tail of EGFR has a C lobe and a N lobe -When dimerization occurs, one C terminal tail becomes the donor kinase and one becomes the acceptor domain (conformation is changed) --> all phosphorylation is now done by the acceptor domains

Prenylation

-C terminus (3' end) of protein has a CaaX motif (C=cystine, a=aliphatic residue like Ala, Val, Leu, X=any AA) -Farnesyl group is covalently attached to the cystine -CaaX protease will remove the aaX -Methyl transferase adds a methyl group -hydrocarbon chain (the farnesyl group) will go into the membrane--> now a lipid anchored protein

Cells cannot function independently

-Cells communicate with each other -Book example: Skin is made of layers of cells. These cells interact with each other. Skin cells cannot survive within the body; they must be in the right place. If a cell cannot communicate it will die.

Components of the RAS/MAPK kinase pathway

-EGF: signaling molecule -EGFR: EGF receptor; a receptor tyrosine kinase -GRB2: an adaptor protein (with SH2 and SH3 domains) -SOS: RAS GEF (son of sevenless) -RAS: monomeric G-Protein (GTPase) -RAF, MEK, MAPK: kinases in cascade (ser/thr) -MAPK: the kinase that enters the nucleus -14-3-3 protein: RAF inhibitor

Trimeric G-protein effector proteins are often enzymes that generate second messengers

-Effector molecule: Adenylyl cyclase--> membrane bound protein that produces the small secondary messenger called cAMP (produces it in millions of copies; "amplification") -cAMP can bind to a protein kinase A (PKA) which is made of 4 subunits (2R, 2C) --> two cAMP molecules bind to each R subunit and change the confirmation. The R subunits are then released from the C subunits. This activates the PKA and PKA can go on to activate and deactivate different proteins

How did scientist figure out RAS was part of a growth factor signaling pathway?

-Fibroblast tissue culture cells require EGF and PDGF to grow in culture -inject cell with RAS blocking antibody= cell stop dividing. Ras is required for response to GFs -Transform with RAS^D allele and now divide in the absence of GFs--> RAS acts downstream of GFs *these type of experiments help scientist see what molecules and what response are connected*

Mutant fly "sevenless"

-Fly eyes are compound eyes made up of 800 single small eyes -each small eye is made up of 22 cells. 8 of these cells have neurons (nerve cells) (R) -Scientist looked for mutants that lacked R7 photoreceptor cells--> these mutants cannot see UV light -identified a LOF mutation that didn't make R7 --> sevenless gene --> RTK -Lack of R7 relates to the RAS pathway--> BOSS receptor binds to Sev RTK, this binds to Drk adaptor protein, this binds to SOS (the same RAS-GEF), this binds to RAS

Integral proteins

-Go through the membrane (transmembrane) -have a domain on both sides -amino acids that pass through the membrane are hydrophobic in nature

Sometimes importin doesn't recognize TFs and an external signal must come and modify the TF so importin can now recognize it

1) TFs have a stretch of AAs called a nuclear localization signal (NLS)--> the signal that comes in allows for the NLS to be exposed 2) If a TF is bound to an inhibitor, a signal can come in and cause the inhibitor to be released--> a kinase does this--> TF can now enter the nucleus with importin's help 3) Phosphatase can dephosphorylate a kinase --> inhibitor can bind to the TF and it'll remain in the cytoplasm 4) an inhibitor molecule can be modified by another protein: UBIQUITIN *other signals could impact the TF instead of the inhibitor *one of the genes the TF transcribes is the inhibitor gene (feedback) *biochemical reactions can almost always go both ways

GPCR pathway

1) inactive G protein is sitting on the membrane not interacting with the receptor (alpha and gamma subunits have lipid anchor) 2) Once the GPCR is activated by a ligand, it can specifically bind to the G alpha subunit (G alpha is in a GDP bound form at this point) 3) Once the GPCR and G alpha interaction occurs, a conformational change occurs--> G alpha will release it's GDP molecule and can now bind to a GTP molecule and become active (in this case GPCR acts as a GEF) 4) Activation of G alpha results in a conformational change --> G beta and G gamma are released and G alpha is released from GPCR 5) The free G alpha can now bind to and activate an effector molecule --> this effector molecule acts as a GAP--> G alpha becomes GDP bound-->becomes inactive-->is released from the effector molecule

NFkB pathway

1) there are many different receptors on the immune cell that can interact with different ligands--> all of these activate the kinase I-KB 2) I-KB kinase phosphorylates the protein I-KB alpha--> when non-stimulated, I-KB alpha is already bound to the NFKB complex and acts as an inhibitor 3) When I-KB alpha is activated it phosphorylates and becomes a good substrate for E3 ligase 4) E3 ligase adds the poly-K48-linked ubiquitin to the I-KB alpha --> I-KB alpha is now degraded in the cytoplasm 5) There is now a free NFKB molecule that can enter the nucleus and the result is the transcription of: adhesive proteins, I-KB alpha (feedback), inflammatory kinases, chemokines, enzymes

How many cells are there in an adult?

About 100 trillion

How many cell types are in humans?

About 210 different cell types that all have different functions.

How is the function of polyubiquitin determined?

By the lysine used for inter-ubiquitin isopeptide bond -different E3 ligase used different # of lysine to link between ubiquitin (lysines are linked at different places)

Mutation that causes a white patch on forehead

C-kik (receptor gene) mutation causes this in both mice and humans

What does hyperactive RAS cause?

Cancer

Autocrine signaling

Cell produces the signal and has the target site for the ligand

Four global responses to ligands

Cells need a signal for everything 1) Survive 2) Grow and divide 3) Differentiate 4) Die Cells signal each other--> most signaling is from cells to each other

The first NLS

Characterized on the simian virus 40 (sv40) large T antigen as a short basic peptide -NLS: PKKKRKV -To qualify as a NLS a sequence should be necessary and sufficient for nuclear localization of a protein--> scientist concluded this by studying the cytoplasmic protein pyruvate kinase (PK). They added a PK-SV40 NLS to PK and saw that it was then transported into the nucleus

How is ubiquitination regulated?

Exposure/masking of a signal recognized by an E3 ligase -if the signal on a target protein is blocked, E3 ligase cannot bind. Once another signal comes and causes the signal sequence to be exposed, E3 can bind and the process can occur --> singal: phosphorylation, ligand binding, ect. -maybe an inhibitor protein needs to be removed - a well folded protein can be denatured to expose a signal for E3 (like a hydrophobic region) Many co-chaperons proteins are E3 ligases because they will degrade misfolded proteins Activation/Inhibition of an E3 ligase -something has to happen to the E3 ligase to "activate" it and allow it to interact with the target proteins -an inhibitory protein can bind to an E3 ligase and keep in inactive

Pertussis toxin effects...

G alpha(i)s in their GDP bound form -whooping cough -continuous activation of cAMP

Cholera toxin effects...

G alpha(s)s in their GTP bound form -continuous activation of cAMP

Four key players in the NLS-dependent nuclear pathway

Importin alpha, importin beta, Ran, nuclear transport factor 2 (NTF2)

Homologous genes regulate development in diverse animals

It's important to study organisms like flies because things are evolutionary conserved -Location of gene expression (homeotic genes)--> expression pattern conserved

Do cells that aren't highly metabolic active have more or less NPC per nucleuses?

Less

Mutation in RAS in which it cannot hydrolyze GTP

Mutant RAS will remain RAS-GTP -RAF and downstream receptors will remain active -To stop the pathway, scientist want to remove the location of RAS (from the plasma membrane)--> worked a little, but even non-bound RAS can bind within the cytoplasm

Does lysine 64 binding lead to degradation?

No

Mutation in eyeless in flies results in no eye. What is the similar human mutation?

Pax6 mutation results in no iris

How was the distribution of nucleoporins discovered?

Relative distribution was defined by immuno-EM 1) purified NPC were fixed into a grid 2) an antibody against the nucleoporin of interest was applied to the same (the antibody was conjugated to gold particles) 3) after incubation and extensive washes of the unbound antibody, the sample was analyzed by EM

Determining if anything more than an NLS is required for nuclear import

Scientist took a cell and altered it -permeabilization of the plasma membrane with digitonin (the nuclear envelope remains in tact) -all soluble cytoplasmic material is lost, nucleus is unaffected -added a protein in the culture medium (that as a NLS) -this cell will enter the cell but will not enter the nucleus Conclusion: soluble cytoplasmic factors are required for the nuclear import of NLS-containing proteins

Paracrine signaling

Signal producing cell and target cell are in close proximity -ligands are unstable

Endocrine signaling

Signal producing cell is located in one part of your body. Ligand will transfer via the bloodstream and come in contact with the target cell elsewhere in the body -distant--> ligand is stable

Contact dependent signaling

Signaling cell physically interacts with the target cell