Post Translation Flashcards
where does most protein synthesis start?
on free cytosolic ribosomes not on ER
apart from mitochondria and plastid (chloroplast is one) translation
polysomes
multiple ribosomes can bind on same mRNA and make diff length proteins
macromolecular crowding
crowded state of cytoplasm in eukaryotic
conc. of substrates is high so fast reactions and drives cellular biochemistry, aggregation of proteins
nascent proteins
not fully born
non-native aggregation prone conformation (aggregation because crowded env.)
extruded in close proximity
newly synthesised proteins are…
non-functional
unfolded/misfolded proteins have exposed hydrophobic residues so protease-sensitive and prone to aggregation
when folded, the sites are tucked away so stable and protease resistant (with help of chaperones)
chaperones
favour correct folding for cytosolic proteins
chaperone mechanisms
- hydrophobic patches on unfolded proteins are recognised by Hsp40 co-chaperone which shields them so keeps protein soluble
- Hsp40 transfers protein to Hsc70 chaperone which is ATP-bound in open conformation, but stimulates ATPas activity so ATP to ADP
- ADP-bound Hsc70 is closed and shields hydrophobic patches so prevent aggregation
solubility allows hydrophilic parts to fold and find final conformation - release by NEF binding Hsc70 forming client complex so nucleotide exchange (removes ADP) so ATP to nucleotide binding site of Hsc70 and Hsc70 opens so releases substrate in partly folded shape
(diff co-chaperones release at diff locations (BAG1/2, HASPBP1, CHIP, chips come in bags remember) - multiple fates can occur now: released and find final conformation, or pass to other chaperone, or transport to organelle, or to proteasome for degradation
Hsp40
heat shock protein 40
co-chaperone
Hsc70
heat shock cognate protein 70
chaperone
study to see what is needed for solubility of protein
heat protein and separate aggregated (P) and soluble (S) by centrifugation
most protein is insoluble without chaperones
adding Hsp40 increases solubility and Hsc70 greatly increases and together even more - recover 50% activity
adding ATP is max solubility because system can regenerate
NEF
nucleotide exchange factor
Hsc70 co-chaperones pass protein onto other chaperones like…. or they…..
Hsp90 accepts partly folded and assembles multimeric complexes
pass onto chaperonins
chaperonins (definition and mechanism)
a class of chaperones that assist in folding of (largely) newly synthesized proteins with the help of ATP, i.e. all chaperonins can be referred to as chaperones, however, all chaperones need not be chaperonins
2 cages of 7, protein enters upper cage and finished in lower cage, shuts and lower released, forces conformation
very ATP expensive
how is a protein’s fate decided (role of each co-chaperone)
there is no absolute control over fate but is competition of co-chaperones for protein which depends on conc. of Hsc70 and Hsp90
if HOP get there first then transfer protein from Hsc70 to Hsp90
BAG-1 sends to proteasome
BAG-2 favours folding
HIP maintains Hsc70:client complex so competes with NEF
proteasome structure
central core of 4 stacked rings (7a top and bottom and 2 7b in the middle) forms 20S core
3 proteolytic activities (lecture 8 page 2) inside barrel encoded by b
19S regulatory particle cap on 1 or both ends
so together is 26S or 30S
proteasome targetting
polyubiquitylation - ubiquitin (Ub) covalent addition to protein, chain of 4 Ub means degradation signal
E1 (9 of it): ubiquitin-activating enzyme activates Ub because H removed from SH when Ub added covalently
E2 (30): ubiquitin-conjugating enzyme chosen by E1 transfers Ub from E1 to E2 (on SH)
E3 (100s): ubiquitin ligase, E2-Ub associates with E3 and binds target protein to transfer Ub to it
each E is specific to the next E and to a protein
19S cap of proteasome recognises Ub
monoubiquitylation vs poly
mono sends to lysosome while poly to proteasome
proteasome destruction
protein binds 19SRP (19S regulatory particle cap)
RP uses ATP to unfold protein and feeds through 20S core where it’s degraded and comes out other end as small peptides
deubiquitylases (DUBs) recycles Ub
other function of proteasome
fail-safe mechanism and can re-fold proteins
RPT5 subunit in cap checks if protein worth saving and acts as chaperone so refold and recover activity
UPS fail?
ubiquitin-proteasome system
proteins that need to be destroyed accumulate and aggregate
cell cycle proteins not degraded so cell proliferation and cancer
on the other hand, overactive proteasome causes autoimmune diseases
other protein modification (other than Ub)
proteolytic cleavage lipids for membrane targeting phosphorylation ADP ribosylation methylation
proteolytic cleavage
inactive protein form is cleaved and subunits are rearranged so activated
e.g. so digestive enzymes don’t eat us
lipids for membrane targeting (Rab)
Rabs regulate membrane trafficking
Rab-GDP is cytosolic and nucleotide exchange causes GDI (GDP dissociation inhibitor) to dissociate so no longer mask prenyl group so Rab-GTP enters membrane
phosphorylation
add phosphates by CAK and Wee1 and remove by Cdc25 so alter activity of cell cycle
activated by single phosphate but inactive with more
ADP ribosylation
add ADP-ribose residues for cell signalling/DNA repair/apoptosis
Cholera toxin is an ADP-ribosylase so interferes with signalling
methylation
on arginine or lysine residues
e.g. histones epigenetics to repress/activate gene expression
co-translational targeting
target while translated
nascent protein has N-terminal signal which directs to ER then other secretory pathways
N-terminal signal
5’ first few AAs (15-30) fused to RNA encoding protein
no ER signal peptide is the same but there are 3 key similarities: +ve N-terminus (Arg or Lys), long hydrophobic stretch of AAs, then cleavage site after small AA (serine/cysteine/glycine)
entry into the ER
- binds SRP (signal recognition particle) receptors in cytosol
- SRP/SP to alpha subunit of SRP receptor in ER membrane
- SRP receptor recruits closed translocon and forms channel
- translocon opens so SP enters as loop/nook shape, and SRP recycled
- signal peptidase removes SP so released to ER membrane and now mature protein without SP
cleaves SP so broken and extracted from membrane so SPs don’t build up - translation terminates and protein is released to ER lumen to fold
what happens in the ER lumen?
membrane-bound polysomes are formed during secretion so proteins in ER are formed in crowded conditions and need chaperones
proteins may become N-glycosylated or disulfide-bonded
if not folded properly then fail check by chaperones and ejected from ER for degradation (retro-translocation)
ER chaperones equivalent to cytosol chaperones?
but also ER specific chaperones?
BiP in ER does same thing as Hsp/Hsc70
GRP94 in ER does further folding like Hsp90
PDI and ERp57 do disulfide bonding
CRT and CNX do N-glycan
N-glycosylation definition
covalent addition of oligosaccharide tree of sugars (core N-glycan) from lipid carrier to target protein by OST
OST
oligosaccharide transferase
in membrane with active site in ER lumen
recognise protein motifs and add N-glycan
core n-glycans
2 N-acetyl glucosamines with branch tree of 9 mannose and on largest branch are terminal 3 glucose residues
is soluble
more detail in lecture 9 page 2
N-glycosylation functions
flags for folding and ER quality control
increase protein solubility
influence folding rates and final protein conformation
how does n-glycosylation flag protein for folding and quality control?
removes 2 glucose so allows interaction with chaperones for folding in ER
final glucose and 1 mannose removed so ready to pass to Golgi
so can see how far down folding pathway by how much glucose and mannose it has
how does n-glycosylation increase protein solubility
it adds large and hydrophilic sugars so prevents aggregation
how does n-glycosylation affect folding rates and final conformation
N-glycan is bulky so contrains alpha-C backbone so it’s no longer flexible so affects shape and folding
e.g. determines Ab structure or enzyme rates
disulfide bonding (definition, affect, conditions)
2 cysteines brought close during folding by PDI (protein disulfide isomerase) so covalent bond which contributes to stability or tertiary structure
only under oxidising conditions so in ER and not in reducing conditions of cytosol
PDI
binds to unstable proteins
2 SH of cysteines interact to form a mixed disulfide heterodimer which breaks and shuffles (isomerisation) till stable conformation
breaks bond if in wrong place
coordination of ER protein folding with other things
lecture 9 page 2 bottom
MHC Class 1 assembly
BiP maintains HC solubility till beta2 microglobulin binds and n-glycosylated
HC enter folding env. provided by calreticulin and ERP57 w/ chaperone shuffle and stabilise disulfide bonds
ERP57 bond to tapasin so folding env around HC and folds so groove for peptide fed through TAP transporter recruited by tapasin
other secretory system modifications
O-glycosylation - sugars to oxygen of target protein
proteolytic cleavage to activate
addition of lipids for membrane targeting
Alzheimer’s link to protein modifications
alpha or beta cut first then gamma
if beta cuts first then fragment aggregates and plaques
if alpha cuts first then don’t aggregate
is mitochondrial targeting co-translational?
no
origin of mitochondria
prokaryote Rickettsia obligate intracellular parasite
alpha-proteobacterium
problems with import into mitochondria
2 membranes and crowded env. so aggregation
leader peptide (LP) (definition and types for mitochondria)
N-terminal LPs for targeting from free ribosomes in cytosol to secretory pathway
longer than signal peptides
to the matrix: matrix-targeting sequence, matrix protease, alcohol dehydrogenase III
to inner membrane: matrix protease, IMS targeting, IMS protease, cytochrome b2
to outer membrane: matrix protease, OM localisation, P70, no cleavage so remain as membrane anchors
LPs (leader peptides) for mitochondria
18-80 AAs long
no 2 the same
form amphipathic helices (hydrophobic and +ve part)
+ve N-terminus on 1 face and -ve on another
mitochondrial (mt) targeting process
- precursors of mt proteins made in cytosol are kept soluble with Hsc70 and delivers to mt
- LP is recognised by import receptors of TOM complexes
Hsc70 interact with receptors associated with TOM40 so it opens and proteins enter - channel formed by Tim23 and Tim17 at rare contact sites between OM and IM (membranes)
which lines up with TOM and channel forms to matrix - Tim-mediated transport requires proton-motive force across IM so proteins move through channel, requires ATP
Hsp70 bound to Tim44 uses ATP and drags 1 residue at a time - protein released from mortalin (Hsp70) and matrix signal removed by matrix protease if in matrix
TOM
translocase outer membrane
targeting to other mitochondrial locations (not matrix)
p70 exits laterally from TOM so stays in membrane and matrix signal not cleaved
cytochrome oxidase exits laterally from Tim in IM, no cleavage of IM signal
cytochrome c1 enter matrix into IMS - IMS signal cleaved (have to go to matrix and cross IM to IM space)
differences between cytosolic vs ER vs mt targeting
lecture 10 page 2
