LEC32: Protein Quality Control in the Cell Flashcards
(46 cards)
how is aggregation of newly-made proteins prevented in a crowded cellular environment?
chaperones prevent protein aggregation
how do molecular chaperones promote polypeptide folding?
Bind to, stabilize otherwise unstable conformers of a protein and facilitate its folding
Hsp70, Hsp60, Hsp90 families bind/relase polypeptides
e.g. = Hsp60, chaperonin- barrel-shaped, takes protein into its inner core, allows it to fold there where it is sequestered and protected
what is mechanism by which misfolded proteins are targeted for degredation?
Proteasome/ubiquitin pathway
Ubiquitin tags proteins for degredation; works via E1, E2, E3; brings protein to proteasome for degredation
what are processes of protein quality control?
1) protein folding
2) protein degredation
how do proteins fold? how do we know this?
_by a self assembly process _
Anfinsen took RNase, treated w/ reducing agent that unfolded it via breaking disulfide bondes and w/ a **chaotrope **
saw that purified protein in dialysis bag w/o reagents of reducing agent and chaotrope refolded and activity was restored
indicates that proteins self assemble
what is a chaotrope?
agent that disrupts hydrophobic interactions
what are phases of polypeptide chain folding?
1) burst phase: 0-5 ms; hydrophobic collapse, formation of secondary structures
2) intermediate phase: 5-100 ms; secondary structures form molten globule intermediate; loose; high state of equilibirum
3) rate-limiting step: 100 ms-several minutes; global repacking of hydrophobic side chains and association of domains that were folded independently in intermediate stages; lowest free energy state so stops trying to fold itself
why doesn’t protein folding happen spontaneously in cells?
b/c conditions are not ideal:
1) we do not exist at 4 degrees celsius, we are 37 degrees
2) concentration of proteins he used were on order of ng/ml, and protein concentration in humans is 100s of mg/ml
what would happen if did Anfinsen’s experiment in human cell conditions? why?
aggregation would occur: when proteins **unspecifically interacts w/ its neighbor(s) **
occurs via **hydrophobic a.a. of other proteins that it normally isn’t interacting with **
occurs b/c hydrophobc Van Der Waals interactions get stronger when heated; so leads to aggregation
what are molecular chaperones? what do they do?
proteins that promote protein folding and prevent aggregation in vivo by stabilizing an otherwise unstable conformer of another protein
may help with:
1) folding
2) oligomeric assembly
3) transport to a particular subcellular compartment
4) controlled switching between active, inactive conformations
what do chaperones do to the folding reaction?
they **increase the yield, but not the rate **
thus a thermodynamic, not kinetic, effect
what are the major families of molecular chaperones?
how do they work?
Hsp70
Hsp60 (chaperonin)
Hsp90
bind got and release polypeptides in a manner dependent on ATP binding, hydrolysis and ntd exchange
do molecular chaperones work alone?
no; function in association w/ many co-chaperone proteins that regulate reaction cycle of polypeptide binding and release by chapersone
regulated cycles promote polypeptide folding
what is the structure of bacterial chaperonin?
homo-oligomer of 14 subunits, each 60 kDa,
arranged in **2 stacked rings each of 7 subunits **
each ring = like a donut w/ a 7-fold axis and a chamber
smaller lid structure of 7 subunits on top of the barrels
how does chaperonin act on misfolded proteins?
1) misfolded proteins bind to rim of barrel
2) proteins displaced into cavity of barrel by lid structure
3) protein folds in sequestered, protected environment of barrel chamber, which is lined w/ hydrophilic a.a. that promote folding
4) ATP hydrolysis **causes lid to dissociate, due to conformational changes in large subunits **
5) folded protein emerges into cytosol
what controls chaperone gene txn?
Hsf, heat shock factor
txn factor
Hsf responds to presence of unfolded protein or heat shock or other type sof proteotoxic stress
stressed cells’ Hsf activates upon stress signal, binds to promoter element of Hsp70 gene, stimulates expression, sometimes hundred fold, and quickly
what does Hsf respond to?
1) environmental stress: heat shock, a.a. analogues, heavy metals, inhibitors of energy metabolism
2) pathophysiological state: fever/inflammation, hypertrophy, oxidative injury, ischemia, infection, xenobiotics
3) non-stress: cell cycle, growth factors, development, differentiation
what does this demonstrate?
Hsf expression is increased under conditions of stress, i.e. high temperature
what are methods of protein degredation?
1) proteasome
2) target system that targets proteins in the proteasome, via covalent attachment of ubiquitin
what is the proteasome, what is its structure?
large gated protease
central catalytic core (20S proteasome) and regulatory cap (19S) come together = 26S proteasome
what are the functions of the 20S subunit of the proteasome?
catalytic particle
outer subunits = alpha, inner subunits = beta
access to inside is via tunnel formed by alpha subunit rings
catalytic activity occurs in beta subunits
what is the structure of the 19S regulatory cap?
what are the functions of the 19S regulatory cap of the proteasome?
comprised of at least 15 subunits, 6 of which are AAA-ATPases that perform unfolding
1) binds ubiquitinated proteins
2) de-ubiquitinates proteins
3) unfolds substrates
4) presents substrate to catalytic particle (CP) of the proteasome
what are the activities of eukaryotic proteasomes?
1) chymotrypsin-like: cleaves after hydrophobic amino acid chains
2) trypsin-like: cleaves after basic amino acids
3) peptidyl-glutamyl peptide hydrolyzing activity: cleaves after a.as
what special functions have been described in mammalian proteasomes?
1) cleaving after branched chain amino acids
2) cleaving between small neutral amino acids
