Lecture 15 - Co-translational protein misfolding Flashcards
In what ways can ribosome activity be discontinued?
Normal translational cycle via stop codon
Truncation, where there is no additional mRNA for the ribosome to translate
Obstruction or damage within the mRNA
aa-tRNA insufficiency
stalls at Poly(A) in ORF in the absence of stop codon
What can these stalling pathways trigger?
mRNA degradation
Ribosome recycling
Protein degradation
Stress responses
Describe the structure of collided ribosomes (disomes).
Disomes consist of the leading ribosome and the collided ribosome.
From EM data, we can see that the leading and collided ribosome have a tRNA missing in their A-site and E-site respectively.
30bp of mRNA connects the two ribsomes.
What is the reaction cascade that occurs when collided ribosomes are detected?
Asc1/RACK (yeast/humans) binds to uS3. Asc1/RACK acts as a sensor for ribosome collisions.
Then Hel2/ZNF598 is recruited to the small subunit interfaces.
This triggers K63 ubiquitination at uS10/eS10.
mRNA cleavage and ribosome splitting then occurs via Dom34/Pelota
After ribosome splitting what happens to the NC?
Rqc2/NEMF (yeast/humans) is a nucleotide-binding protein that binds to the P-site tRNA region.
It recruits tRNAs, specifically Ala and Thr to peptidyl-tRNA complex.
Protein translation restarts, and Rqc2/NEMF terminally tags the NC with CAT tails.
Ltn1/Listerin (yeast/humans) binds, curving around the 60s subunit towards the exit tunnel.
This enables the ubiquitination of the stalled NC at lysine residues.
Explain the structural characteristics of amyloid fibrils.
Amyloid forms a fibrilar structure, typically 10-100nm in length.
Fibrils of different proteins can be distinguished by specific immunohistochemical staining
Fibrils are made up of different numbers of component beta strands (protofilaments).
Protofilaments are twisted around each other in either compact or ribbon-like arrangements.
Where does protein misfolding originate?
Misfold from other states
e.g intermediates during folding
Unfold from a native state
and then misfold
Misfolding during synthesis. Up to 15% of new proteins
ubiquitinated/degraded.
Misfolding can also occur co-translationally on the ribosome.
Give an example of misfolding in disease.
a1-antitrypsin is a protein formed in the liver, released into plasma, and transported to the lungs, where it acts as an elastase inhibitor.
A Z-mutation causes the formation of inclusions. These inclusions look like beads on a string under EM.
These inclusions build up in the liver, leading to cirrhosis of the liver.
How was a1-antitrypsin (AAT) folding monitored?
Study the protein biosynthesis in a cell-free scenario, through the use of a lysate.
Replace methionine with 35-S methionine, to allow radiolabeling of polypeptide chain.
Products can then be analysed via polyacrylamide gel electrophoresis (PAGE).
Formation of native isolated protein possible, along with ribosome nascent chain complex.
What were the differences seen in wild type AAT NCs compared to the Z AAT NCs?
PAGE of the NCs shows that the biosynthesis of the proteins compared to one another is unaffected.
The folding over time PAGE shows a variety of species formed with the Z-mutation NC.
This suggests that Z NCs become ‘trapped’ &
cannot form natively-folded structures easily.
What observations were seen in AAT NCs in terms of paused NCs on the ribosome?
After an hour of translation, there were two bands seen on a gel, and after treatment with RNase, there was a downshift seen in the gel.
This downshift corresponds with the weight of tRNA.
So, we can conclude AAT naturally pauses as full-length nascent chains on the ribosome.
What experimental evidence is there that misfolding occurs co-translationally on the ribosome?
Smears seen in the biosynthetic PAGE, so sample was purified using a sucrose cushion.
Laddering effect was seen on gel, reinforced by the fact that treatment with urea removes the laddering effect, leaving a band for the complex.
NC complex sitting in buffer over time shows the NC falling of the ribosome and formation of high molecular weight species.
Looking at high molecular weight species, how were polysomes distinguished from a group of translating ribosomes?
Nuclease treatment can be used, which cleaves exposed mRNA.
If ribosomes are far apart, then nucleases can cleave.
If too close together (polysome), cleavage is not possible.
What is the relationship between the concentration of mRNA and polysome/monosome formation?
Low concentrations of mRNA lead to more polysome formation.
High concentrations of mRNA lead to more monosome formation.
Polysomes may contribute to ribosome collisions.
How is the fate of the NC chain be studied?
PEGylation was used, a PEG-maleimide moiety can react with the solvent-exposed C232 on the a1-antitrypsin (AAT) protein.
On a cystiene-free AAT, an A183C residue is engineered onto the protein.
The A183C is more available than the C232.
