Lecture 15 case study Flashcards
What is autophagy dysfunction a feature of?
Normal aging and many neurodegenerative disorders.
What is MND?
Encompasses a group of diseases which involve upper and lower motor neurons. Progressive life limiting condition.
What genes are highly relevant in autophagy that are mutated in ALS?
OPTN, SQSTM1(p62), UBQLN2, TBK1.
Which ALS-linked mutations in p62 map to the UBA domain or LIR motif?
LC3 region- D335N, D337E, L341V.
UBA region- T430P, P438L, P439L.
Why focus on the L341V mutant to identify ALS in patients?
Conservative mutation. Of interest due to the WxxL motif which is the core motif of an LCR interacting region. Switches the L for a V. Do the mutations affect the ability of the autophagy to bind to LCR? Same idea with the UBD.
What is the protein pull down?
Affinity chromatography where beads have either glutathione attached to them purified LC3 B protein or purified ubq protein. Try to capture the GSP62 protein.
What did the protein pull down show?
L341V: The weaker band in the LC3 shows capture of less GSP 62 in the LC3 protein mutation. The ubiquitin beads still capture the normal amount of GSP62 because the UBA domain is intact.
G425R: Vice versa
How is artificial avidity generated in this experiment?
The ubiquitin is immobilised on the surface of the beads at high concentration in different conformations. This mimics a poly ubiquitin chain. Have sufficient oligomers and sufficient concentration of the ubiquitin to recreate,. They aren’t linked by peptides but they are close in space due to being on the beads.
What is the ITC experiment?
ITC measures heat that is released or absorbed during a biomolecular binding event. Allows estimation of Kd values. The WT LIR shows weak binding. Tighter binding from p62 oligomerisation (avidity) and LIR phosphorylation (affinity).
Can we show this loss of LC3B binding by p62 is relevant in a cellular context?
NSC34 cells (motor neurone-like cells) used. Difficult to show due to the tagging of proteins in the autophagy system being degraded so not measurable by the tag.
What was the solution to measuring autophagy?
Could exploit the trafficking of the p62 into the autophagosome to get a measure of the rate of autophagy in a living cell. Engineer a plasmid that allows p62 to be expressed in the NSC34 cells with two fluorescent tags covalently attached to this N terminus. A red protein and a GFP. The autophagosome has the same pH as the cytoplasm. mCherry is an acidic stable fluorophore. Still fluoresces in acidic conditions. GFP is acid sensitive so fluoresces less. After fusion with lysosome the GFP will be quenched by acid and mCherry will continue. Change from yellow to red would show rate of autophagy. Could do this with WT and mutations.
What was done with the results?
Quantified using PCC values. Pearson correlation coefficient. ALS mutant shows defective autophagy which can be rescued by rapamycin (autophagy initiator). Important to define autophagy as a therapeutic target. Drug repurposing.
What else does p62 bind to?
Has a preference for LC3B but also binds to GABARAPL2. A neomorphic mutation happened where L341V mutation caused loss of LC3B binding but optimised the LIR to bind to GABARAPL2, the wrong ATG8 protein.
