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?
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:
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?
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
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?
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?
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
size of peptides resulting from proteasome activity?
7-9 amino acids
when are proteasomes therapeutic targets?
eg: velcade is v. useful for multiple myeloma
what is ubiquitin? its use?
76 a.a. protein that becomes covalently attached to polypeptides that're substrates for degredation by the proteasome
has a G at the end
has a K at position 48 which conjugates via an isopeptide bond of its epsilon-amino group to the glycine of the next ubiquitin
how do successive ubiquitins become attached to each other?
via action of enzymes E1, E2, E3:
E1: activates ubiqutin via ATP-dependent activation of C-terminal glycine
E2: ubiquitin-conjugating enzymes, accept ubiquitin from E1 & transfer it to protein substrate
E3: ubiquitin ligase, specifies substrate selection, brings it in close proximity to ubiquitin, catalyzes transfer to substrate
describe process of E1, E2, E3 w/ ubiquitin --> protein substrate
1) E1 carries out ATP-dependent activation of C-temrinal glycine by first forming a ubiquitin-adenylate, then transfers activated ubiquitin to thiol site in E1
high energy thiol important for Ub tarnsfer to E2
2) E2 enzymes accept ubiquitin from E1, transfer it to protein substrate w/ help of E3 enzyme
what is CHIP? what does it do?
C-terminal Hsp interacting protein
protein w/ an E3 ligase domain & a chaperone binding domain
misfolded proteins bind chaperone binding domain of CHIP; this stimulates ubiquitin ligation
CHIP binds directly to Hsp70 and catalyzes ubiquitinylation of misfolded proteins
how does Ub chain grow?
as several ubiquitins tagged on to substrate, higher number of ubiquitins tagged = growing affinity = chain grows, too
what is this?
ubiquitylation visualized by gel electrophoresis
multiple ubiquitins added to chain successively
what happens to misfolded proteins that cannot be destroyed quickly enough?
form aggresomes, aggregates of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibers
occurs if overwhelm the ubiquitin/proteasome pathway
how are aggresomes cleared?
what is healthy/normal function of aggregates in cells?
large collections of misfodled aggregated proteins
are central meeting point for molecular chaperones & proteasome- so although aggregated, it's ok
form in micro aggregate that are then transported along cytoskeleton to form a central protective aggresome near center of cell- way to path misfolded proteins and make sure they don't do harm to the cell
usually get cleared by autophagy
Model for aggresome formation
Small aggregates formed at the cell periphery are delivered along microtubule tracks (green lines) on retrograde motors (grey) to a juxtanuclear, pericentriolar location
A pair of centrioles is shown in blue next to the nuclear indentation
what are prions?
proteins that form toxic amyloids that're transmissible
what are amyloids?
extremely stable aggregates of proteins that're formed by stacked beta sheets
build up of amyloids in brain is toxic, causes death
leads to diseases like alzheimer's
what is genetic characterization of Alzheimer's disease?
extracellular amyloids of a peptide called AB, A-beta40 and A-beta42
are 40 and 42 a.a. long peptides which are formed by clipping a protein called APP, alzheimer's precursor protein
A.D. also caused by intracellular deposits, or neurofibrillary tangles, of microtubule binding protein, Tau
aggregating form of Tau is hyperphosphorylated
what is genetic association of Parkinson's disease?
formation of aggregates called Lewy Bodies
= aggregates enriched in alpha-synuclein
also involves loss of dopaminergic neurons in substantia nigra
what causes poly-glutamine?
what does this cause?
some cases, the triplet encoding Q (CAG) expands during replication
if expands beyond ~36 Q residues then get diseases state
currently, know of 9 proteins w/ polyQ resulting in neurotoxicity
what diseases are known to be caused by poly-Q strectches?
must be beyond ~36 Q residues to cause disease state
DRPLA (Atrophin 1)
SCA 1, 2, 3, 6, 7, 17 (spinocerebellar ataxias: Ataxin 1,2, 3,7,17 & TATA box binding protein)
what is it about poly-Q that causes disease?
when >36 bp of polyQ, toxicity results b/c glutamines in nucleus of these neurons aggregate, this aggregation causes neurons to degenerate
what are prions?
how are they transmitted?
what do they cause?
proteins that form transmissible amyloids that cause disease in humans & other mammals
transmission occurs by eating contaminated food, so they're infectious
cause scrapie, CJD, KURU, BSE, fatal fetal insomnia
all known prion diseases attack the brain
what is the purpose of protein quality control?
cellular process that integrates folding, degredation of proteins
why are so many late-onset diseases assocaite w/ protein misfolding?
b/c quality control apparatus becomes less robust w/ age