M2-1 - M3-2

small GTPases

guanine tri-phosphatases; Ras superfamily of small GTPases; these relay signals from rtks

GEF

guanine nucleotide exchange factor; causes the exchange of GDP for GTP leading to activation of the protein; the specific GEF for the Ras/MAPK pathway is SOS; catalyze nucleotide exchange through their DH and PH domains

GAP

GTPase activating proteins; stimulate the intrinsic ability of GTPases to hydrolyze GTP to GDP causes inactivation of the protein; causes the additional phosphate group to be cleaved off turned GTP to GDP; GTPases can hydrolyze the phosphate group themselves however they just work incredibly slow; GAP activates the GTPase function of the g-proteins and trigger the function to activate the cleaving of the phosphate group; a coenzyme - the GTPase does the catalytic activity

Ras/MAPK signaling pathway

epidermal growth factor (EGF)/mitogen binds to two EGF receptors/RTKs; the EGF receptor kinases transautophosphorylate; the SH2 domain of Grb2 binds to the phosphorylated RTK; the SH3 domain of Grb2 then binds to the PXXP of SOS; the DH domain of SOS kicks out the GDP of Ras; Ras activates and then associates with the GTP binding domain of Raf; Raf autophosphorylates on the kinase and then phosphorylates Mek; Mek phosphorylates the regulatory domain of Erk; Erk starts transcription of the mitotic genes

ways to stop a signal

phosphatases can remove phosphates to stop the signal; internalize the membrane receptor by putting it in a vesicle; GAPs can turn off GTPases - negative feedback; EGF is lost or degraded

Ras/MAPK in T-cell activation

the t-cell receptor complex (TCR/CD3) is a complex of 8 transmembrane receptors; the binding of the t-cell receptors to foreign antigens causes many different intracellular signaling cascades- causes the immune response; the Ras/MAPK signaling is required for t-cell proliferations upon activation of CD3 by an antigen presenting cell; activation of the TCR can also be triggered by an anti-CD3 antibody

studying protein-protein interactions

co-immunoprecipitation, GST pulldowns, immunohistochemistry/immunocytochemistry, FRET

phospho-deficient mutant

cannot be phosphorylated even if the upstream kinase is activated; a mutation removes the hydroxyl group where phosphates are added- therefore phosphorylation cannot occur; tyrorsine becomes phenylalinine, tyrosine become alanine, or serine/threonine becomes alanine; kinases can still bind to this mutant because it has the same surrounding amino acids but cannot phosphorylate it

GST pulldown assays

specifically used to determine binding partners of specific protein domains; exogenously expressed bait protein domain is tagged with glutathione S-transferase (GST) - the DNA construct has the promoter of the protein gene, GST, and SH2; lyse the cells and add glutathione-bound beads; SH2 is used a fusion protein to be a bait and see if the protein is being pulled down or not; GST-tagged protein domain binds with high affinity to glutathione-beads; pulldown or precipitate prey proteins which bind to the bait protein domain; if B has phosphorylated tyrosine it will be pulled down and if not it won't be; for the control just express GST and take a sample of the lysate to make sure that the protein is actually being expressed in the cell

Km

michaelis constant; tells you how well the substrate can bind to the enzyme; a low km shows that you don't need a lot of substrate and there must be a high affinity; high km shows that the substrate can't bind the enzyme very efficiently and must have a low affinity

lineweaver-burke plot

double reciprocal graph of the Michaelis-Menten equation- plot the inverse of both velocity and substrate concentration;

useful in determining what type of inhibition the enzyme is under because Km and V max can be compared without estimation; 1/v = (Km/Vmax)(1/substrate concentration) + (1/Vmax)

cholesterol

inserts itself between the phospholipids; when inserted into the hydrophobic core it acts as a fluidity buffer; can prevent tight packing and increase fluidity when needed or fill gaps and increase cohesiveness which reduces fluidity when needed; also has a polar head group and non-polar hydrocarbon tail`

not permeable molecules

charged ions; H+, Na+, K+, Ca2+, Cl-, Mg2+, HCO3-

single-pass transmembrane protein

one on transmembrane segment; one amino acid chain in the core of the cell membrane; many signaling receptors, cell-cell and cell-matrix adhesion proteins

multi-pass transmembrane protein

more than one transmembrane segment; transporters, carriers, channels, enzymes

lysosome

degrades materials

importin-cargo complex

NLS on the the cargo protein that is bound by importin

proteolytic exclusion assay

used to determine whether proteins localize to membranous compartments; isolate ER membranes by subcellular fractionation; translate proteins in the presence of ER membranes for exogenous proteins; treat samples with protease: cytoplasmsic proteins will be completely degraded, transmembrane proteins are partially degraded, and secreted proteins are not degraded; for a control put in detergent so that you see that transmembrane and secreted are completely degraded

GTP exchange and hydrolysis

occurs on GTPases or G proteins; in monomeric/small GTpases there is one subunit for GTP exchange; to activate a protein the GDP is completely exchanged for a GTP by a GEF; but to deactivate a protein the phosphate group of GTP is removed by a GAP to revert it back to GDP and deactivate the protein

GTPase structure

contain a prenylated tail, switch I and II regions, and a nucleotide binding pocket; in the inactive form the switch regions are pointing out- allowing the nucleotide binding pocket to be accessible; in the active form the switch regions are pulled together through binding the to the additional phosphate group; this also opens up other binding spots for additional proteins

prenylated tail

a chain of lipids which anchors the GTPase in the plasma membrane

switch regions

bind to the terminal phosphate of GTP; in the inactive form the switch regions are pointing out- allowing the nucleotide binding pocket to be accessible; in the active form the switch regions are pulled together through binding the to the additional phosphate group; move through the loaded spring mechanism

nucleotide binding pocket

in the area between the switch regions; binds GTP or GDP

loaded spring mechanism

when the terminal phosphate of GTP associates with the switch regions GTPase can bind to GTPase effectors via their GTP binding domains; hydrolysis of GTP causes conformational changes in the switch regions so GTPase can no longer bind GTPase effectors

PH domain

plekstrin homology domain; part of the GEF that allows the protein to associate with the cell membrane; binds to phospholipids in the cell membrane

DH domain

dbl homology domain; interacts with the switch I and II regions to poke out to the bound nucleotide; does not actually bind to the GDP or GTP; just kicks out the GDP which is automatically replaced with GTP due to the higher concentrations of GTP than GDP in the cell

GTPase effector proteins

these are often kinases; contained GTP binding domains; can only bind to the the GTPase when it is in it's active conformation; this binding causes the second arm to open up more and move into an active conformation to allow the binding of other proteins; association with GTP-bound GTPases relieves inhibitory intermolecular interactions which expose catalyitc kinase domains

PH domain binding partner

phospholipids

GTP-binding domain binding partner

GTP-bound GTPases

DH domain catalytic domain

exchanges GDP for GTP

GAP catalytic domain

GTP hydrolysis

dimerization

the bringing together of two proteins; when two RTKs bind to one growth factor in the Ras/MAPK signaling pathway

SOS

the GEF of the Ras/MAPK signaling pathway; has the PH and DH domains that are connected with a proline-rich motif (PXXP); the PXXP binds the SH3 domain of Grb2 to activate and then the DH domain kicks out the GDP of Ras

Grb2

the SH2 domain binds to the phosphorylated EGF receptor and then the SH3 domain binds to the PXXP of SOS

Ras

the GTPase of the Ras/MAPK signaling pathway; SOS kicks out the GDP to activate it and then Ras binds to the GTP binding domain of Raf

Raf

the effector kinase of the Ras/MAPK signaling pathway; MAPKK kinase/MAP3K; GTP binding domain binds to Raf; the kinase domain autophosphorylates and then Raf phosphorylates the kinase domain of Mek

Mek

MAPK kinase/MAP2K; after it's kinase domain is phosphorylated it phosphorylates the regulatory domain of Erk

Erk

regulatory transcription factor and mitogen-activated protein kinase of the Ras/MAPK signaling pathway - inside the nucleus; the regulatory domain gets phosphorylated causing the DNA-binding domain to bind to the mitotic genes of the cell and the trans-activation domain recruits GTFs

Ras/MAPK mutations

one of the most commonly mutated pathways in cancer; EGFR mutation causing overexpression, Ras mutation causing it to always be active, Raf mutation causing it to always be active

antigen-presenting cells

engulf, break down, and then display part of a foreign pathogen on the surface of the cell

protein activation

constitutively active mutant; dominant negative mutant

phospho-mimetic mutant

behaves as if it is phosphorylated even if the upstream kinase is not activated; serine/threonine becomes aspartic acid/glutamic acid which are negatively charged and mimic the negatively charged phosphate group; more phosphorylation of the traget

mutated protein introduction

can be introduced into a cell via random mutation in one or more copies of a gene; via exogenous expression of mutated genes

analysis of protein mutation

often occurs in the context of the wildtype signaling pathway; in a wild-type/normal pathway there are two function genes making the normal amount of functional proteins; with one nonfunctional gene there is half the normal proteins; in very rare cases there are two mutations causing no functional proteins

constitutively active mutant

a mutation on protein x makes it phosphomimetic- so it is always expressed and always turned on; even if it is not receiving signals from the upstream signal it is sending signals to the downstream protein causing more phosphorylation of the downstream protein

negative mutant

caused by something like the deletion of the consensus sequence making it unable to interact with the upstream signal; the negative mutant combined with the wild-type protein usually won't have much of an effect on the cellular function

dominant negative

mutation still allows for the upstream protein to signal to it; the mutant and wildtype proteins are both getting signals from the upstream protein but only the wildtype is signaling to the downstream protein- halves the amount of signal the downstream protein receives

co-immunoprecipitation

use an antibody to one of two proteins that are suspected of binding to each other and attach the antibody to a bead; whatever is attached to the bead is precipitated out- can evaluate what is in the precipitate

enzymes

catalyze chemical reactions; substrates are converted to products in the active site; enzymes are not altered/modified during chemical reactions

enzyme active site

can bind to substrates and bring them together, weaken bonds that have to be broken (terminal phosphate of GTP/ATP), transfer/accept chemical groups to facilitate chemical reactions, provide the proper chemical microenvironment in the cell

targeting enzymatic activity with drugs

many drugs, including chemotherapeutics, are designed to either block protein binding or modify the active site of enzymes

naproxen

aleve; cox-2

acetylsalicyclic acid

aspirin; cox-2

pseudoephedrine

pseudofed; alpha/beta adrenergic receptors

diphenhydramine

benedryl; histamine receptors

emtricitabine

HIV medication; HIV-1 reverse transcriptase

irreversible inhibitors

covalent bond between inhibitor and enzyme; not easily reversible; heavy metals, insecticides, nerve gases

reversible inhibitors

dissociable non-covalent binding between inhibitor and enzyme

competitive inhibitors

bind to the enzyme at the active site and block substrate binding

non-competitive inhibitor

inhibitor binds to the allosteric site not the active site; this binding causes a change in the conformation of the active site so the substrate can't bind anymore

enzyme kinetics

the rate of an enzymatic reaction is controlled by enzyme availability and substrate concentration; graph plateaus because there is a finite amount of enzyme which doesn't change regardless of substrate concentration and there is only a certain speed at which the reaction can be completed

Vmax

maximum velocity of the enzymatic reaction; when it hits Vmax the amount of available active sites have been maxed out

1/2 Vmax

the half-maximal velocity of a reaction; the substrate concentration at this point is the Km

competitive inhibition on kinetics graph

the Km increases making it so that more substrate is needed to get to 1/2 Vmax; but Vmax won't change because the substrate can outcompete the inhibitor

non-competitive inhibitor on kinetics graph

no change in the Km but a decreased Vmax; this is because you can take the enzymes with the non-competitive inhibitor out of the equation as they can't contribute to enzymatic activity and can't be outcompeted

competitive inhibitor on lineweaver-burke plot

the 1/Vmax is the same but the -1/Km increases; the Vmax is the same but Km increases

non-competitive inhibitor on lineweaver-burke plot

the -1/Km stays the same but the 1/Vmax increases;Vmax decreases but the Km stays the same

diabetes mellitus

characterized by abnormally elevated plasma glucose concentrations; hyperglycemia; in type 1 there is an insulin deficiency and in type 2 there is an insulin resistance

cell membrane functions

maintain a specific internal environment, organize functions of the cell, control signaling and transport, production of energy

cell membrane structure

8-10 nm bilayers with inner and outer leaflets; all cell membranes are made up of glycerophospholipds, sterols/cholesterol, and glycoproteins

glycoproteins

only on the the external plasma because the attachment is very specific

fluid-mosaic model

the lipid bilayer permits movement of both lipids and proteins; as the fluidity of the membrane increases the permeability of the membrane increases

lateral diffusion/rotation

movement of a glycophospholipid in the same leaflet; occurs frequently

transverse diffusion

movement from one leaflet to another; highly restricted due to the fact that the hydrophilic head group would have to move into the hydrophobic tail region

fry and edidin's experiment

fluorescently tag membrane proteins of different cells and then fuse the cells; see how the proteins mix and move together

highly permeable molecules

small and hydrophobic/non-polar molecules; O2, CO2, N2, steroids, hormones

somewhat permeable molecules

small and hydrophilic/polar; H2O, ethanol, and glycerol

barely permeable molecules

large and hydrophilic/polar; amino acids, glucose, nucleotides

integral proteins

embedded in the bilayer; can be fully embedded in one or two leaflets; if it's embedded so that a portion sticks out on their side of the membrane it's transmembrane; integral proteins can only be associated with only one leaflet; RTKs

peripheral proteins

associated with the surface of the inner or outer leaflet; associate with the phospholipids; SOS

lipid-anchored proteins

covalently linked to lipids tails with anchor into a leaflet; Ras and Sarc

glycerophospholipids

have two fatty acid/hydrocarbon tails attached to a hydrophilic head; the head is made up of a phosphate group and alcohol head group; glycerol connects the tails and the head; there are multiple alcohol groups that they are named after- phosphatidylserine

amphiphilic

has hydrophilic and hydrophobic parts

glycerophospholipid structure and membrane fluidity

the longer the hydrocarbon tail the less fluid it is, the shorter the hydrocarbon tail the more fluid it is; tails that are saturated with hydrogen and have no double bonds are straight, packed tightly, and less fluid; tails that are unsaturated and have double bonds are kinked, packed loosely, and more fluid

FRAP

fluorescence recovery after photobleaching; measures the mobility of proteins and fluidity of membranes; label a membrane protein or phospholipid with a fluorescence tag; use a laster with a high intensity to bleach the fluorescence in a certain area; can see the rate at which adjacent membrane proteins move into a bleached area; if recovery is slow the membrane is not very fluid but if recovery is fast the membrane is very fluid

lipid bilayer asymmetry

glycerophospholipids are not randomly distributed in the inner and outer leaflets; phosphatidylserine is only in the inner leaflet- if it were to flip to the outer leaflet this would be a signal for apoptosis

flippases

maintain membrane asymmetry; can flip phospholipids from one leaflet to another; atp-dependent enzymes

lipid rafts

ordered membrane microdomains that contain phospholipids with longer, saturated tails, excess cholesterol, membrane proteins with longer transmembrane regions, and clusters of proteins which function/signal together; highly ordered and cohesive; enhance signaling activity by bringing signaling proteins together so that they interact

endoplasmic reticulum

protein synthesis; has the largest endomembrane system; the er membrane is contiguous with the outer nuclear membrane

glycosylation

tagging of molecules; done by the er and golgi apparatus

vesicles

transport and sort materials in a cell

nuclear membrane

transport in and out of the nucleus occurs via the nuclear pore complex that is made up of nucleoporins which provide passage through the nuclear envelope; mRNA is transported out of the nucleus for translation; specific proteins like transcription factors and histomes are imported back in; some proteins shuttle in and out of the nucleus

rough er

stacked cisternae- layers and layers of membranes; involved in co-translational import of a third of all proteins; studded with ribosomes which synthesize the proteins

smooth er

tubules; has an important role in lipid synthesis- glycerophospholipids and cholesterol; the fragmented tubules send lipids to membranes around the cell

golgi apparatus

consists of the flattened membrane disc cisternae; the cis-gogli network faces the nucleus/er and is completely biochemically distinct from the trans-gogli network which faces the plasma membrane; protein stracels from the cis-golgi to the trans-golgi

insulin in glucose metabolsim

glucose is not able to cross the cell membrane by itself- therefore insulin is needed; insulin binds to the RTK insulin receptor; this causes the translocation and fusion of Glucose transporter 4 storage vesicles (GSV) with the plasma membrane; glut4 is kept inside these vesicles when blood glucose is low; when glut4 is inserted into the membrane cellular uptake of glucose occurs and glucose metabolism/atp production can occur; some is converted to glycogen for longer term storage

GSV

glucose transporter 4 storage vesicles; necessary because you don't want glut4 to always be present in the membrane; glucose levels would be depleted in the blood if glut4 was always active and there would be no glucose available to the cells that need it

protein sorting

all protein synthesis begins in the cytosol but then either split into post-translational import or co-translational import

post-translational import

cytoplasmic proteins are transported to their required location in the cytosol; nuclear proteins are imported into the nucleus via nuclear pore complexes

co-translational import

transmembrane or secreted proteins are translocated into the er during translation; translation starts in the cytoplasm, and then a certain sequence signals the whole ribosomal complex to move to the surface of the er; the amino acids are threaded into the er lumen and then a vacuole will transport it either out of the cell or into the cell membrane