L15 : Protein Misfolding and Quality Control Flashcards
In what ways can translation be discontinuous?
Results in pausing and sometimes stalling
- Truncation
- Obstruction or damage within mRNA sequence
- AA-tRNA insufficiency (eg. starvation)
- PolyA in ORF causes non-stop ribosome
- Peptide induced (Gly-Arg, Pro-Arg dipeptide repeats) can stall ribosome
What can stalling ribosomes trigger?
mRNA degradation
Ribosome recycling
Protein degradation
Stress responses
What are collided ribosomes?
Leading ribosome has stalled and formed disome with collided ribosome
How is a collided ribosome recognised and split?
Asc1/RACK (yeast/humans) binds to uS3 and acts to detect collisions
Once bound, Hel2/ZNF598 recruited to small subunit interfaces near es10/uS10 (major sensor)
Triggers K63 ubiquitination of ribosome at uS10 (Hel2) and eS10 (ZNF598)
mRNA cleavage and ribosome splitting by Dom34/Pelota
What happens to nascent chain that was mid-translation?
Listen/Lnt1 (E3 Ub ligase) binds, curving around 60S ribosome towards exit tunnel
Enables ubiquination of stalld NC
Rqc2p/NEMF binds to P-site tRNA 60S subunit (nucleotide binding protein)
Recruits specifically tRNA Ala and Thr to larger 60S peptidyl-tRNA complex
Protein translation is restarted with the addition of CAT tails
Rqc2 C-terminally tags NC with CAT-tail (C-terminal Ala/Thr seq)
Listern/Ltn1 ubiquitinates NC
Why is RQC important?
Deletion/removal/depletion of RQC components = global increase in truncated or premature products misfolding and aggregating within cell
Important links between biosynthesis, RQC, and origins of protein misfolding
Discuss the importance of kinetic control in protein folding?
Protein folding and misfolding during biosynthesis are inherently competing evens
Protein folding is assisted by folding intermediates to overcome kinetic control
Co-translational folding is a kinetically driven process that begins on the ribosome
Chaperones also guide folding and attempt to correct
Explain and give an example of link between protein misfolding and disease?
Proteinaceous inclusions can be amyloid or non-amyloid
Examples of neurological diseases linked to misfolding and aggregation include:
Alzheimer’s (Abeta) plaques, Parkinson’s Lewy bodies, Huntington’s (huntintin) intranuclear inclusions
Diseases such as A + H show severe atrophy of the brain due to accumulation of toxic protein deposits
What are structural characteristics of amyloids?
Amyloid forms a fibrillar structure
Typically 10-100 nm length and unbranched
Representative protein folds (SH3, acylphosphatase, myoglobin) can all be made to form fibrils
Suggests ability of polypeptide chain to form amyloid structure is generic though propensity varies with sequence
How can amyloids be visualised?
Fibrils of different proteins can be distinguished by birefringence using polarised light with Congo red staining (binds between fibrils)
Made up of different numbers of protofilaments (assemblies of component beta strands) twisted around each other in compact or ribbon like arrangements
Where does protein misfolding originate?
- Misfold from other states (eg. intermediates)
- Unfold from native state and then misfold
- Misfolding during synthesis (up to 15% new proteins are ubq/degraded)
Give an example of protein misfolding in disease?
a1-antitrypsin (AAT)
Forms in liver, released into plasma, functions in lungs as inhibitor of elastase
Z-mutation traps protein in intermediate state leading to non-native folding pathway and causes formation of inclusions, eventually cirrhosis of liver
Loss of protection of elasticity in lungs, leading to early onset emphysema
How was AAT biosynthesis monitored?
In vitro cell-free environment using lysate (mammalian environment)
Replace methionine with 35S methionine allowing radiolabelling of newly synthesised proteins
Can analyse products using polyacrylamide gel electrophoresis (PAGE)
What were the differences in wild type and Z-mutant AAT NCs?
Native and denaturing PAGE curves shows AAT completes folding once it finishes synthesis and leaves ribosome (can start coT)
Plots show that folding is affected but generally not biosynthesis
Z-mutation shows smeared band as NCs become trapped and cannot form native structures easily, forming variety of non-native species over time
What experiment showed AAT pausing as RNC?
Performed synchronised reaction showing AAT naturally pauses as full length NC on ribosome
After treating with RNase, additional band disappears so can conclude band corresponds to full length RNC
Difference of 20kDa corresponds to weight of tRNA
Unsure why as 25 AA seq in tunnel has no signature residues (eg. no polyprolines, polybasic)
What experiment showed co-translational misfolding on the ribosome?
PAGE showed smears at different timepoints
Ran in a sucrose gradient to isolate RNCs and run on gel
Laddering effect seen and disappeared after treatment with urea
Suggests formation of higher molecular weight species (polymers)
Translating ribosomes can form higher order assemblies via recruiting released polypeptides
What experiment showed translating ribosomes collide and initiate AAT misassembly (co-co or co-post)
Run in sucrose gradient
Changing conc of starting template can change ribosome behaviour
At low mRNA conc, more polysomes form
At high mRNA conc, more likely to form monosomes
Polysomes may contribute to ribosome collisions
Nuclease treatment to separate difference between polysome and set of collided ribosomes
Can cleave mRNA from between the ribosomes if they are distant
If the ribosomes are collided, nuclease will be ineffective
Proteinase K gives info about arrangement of ribosomes via NCs
How was PEGylation used in the experiment?
PEGylation as a site specific reporter of protein structure
Solvent exposed cys can react with PEG-maleimide moiety
Upshift seen for native C232, but engineered A183C not as reactive with PEG
Can use relative differences in solvent exposure to map the type of solvent accessibility on AAT and find PEGylation rate
PEGylation was used to map AAT proteins co-translational folding pathway
What experiment observed fate of the nascent chain after it leaves the ribosome?
A183C can be used as folding probe - exposed in unfolded state and buried in folded state
Running of PAGE to see how quickly folding occurs
80% wild type AAT folds natively
45% Z mutant AAT persists in long-lived partially folded intermediate (‘trapped’ and cannot fold to native state)
What is the new proposed model of protein misfolding and aggregation?
NC that is partially structured with some accessible conformational information may interact with a released polypeptide that does have complete structure
These can join and form long chain polymers that were observed
Co-post misassembly
