Lecture 25 - PTM, protein localisation and degradation Flashcards
What must proteins do in the cytoplasm to function
Fold
What can cause protein missfolding and aggregation?
stress
How efficient is folding in vitro and in vivo, and why is this?
20-3-%
100% (Vivo)
Because in vivo chaperone proteins are present
Give an example of a molecular chaperone, and where they are found and why it’s important
hsp90 (heat shock protein - origionally identified as proteins upregulated by heat shock, which causes protein denaturation and surgfaces become exposed and proteins aggregate) Found ubiquitiously (although hsp70 is most ubiquitous) Upregulated in response to stress - important for cell health and organismal longevity
What are the primary roles of chaperone proteins
assist nacent protein folding and prevent aggregation
stabilise polypep surfaces in unfolded state
How do chaperones achieve their function - generally
- Bind transiently to newly synthesised protein
- binding during translation too to prevent it
- bind stably to the surface of to misfolded proteins
- Have affinity for exposed hydrophobic peptides rather than specific sequences
Molecularly how do chaperone proteins work
undergo cycles of binding and release, powered by ATP hydrolysis
Have 2 domains joined by tenuous link - the domains can move large distances and rotate (up to 50aa and 100 degrees relative to each other)
binding and release from the polypeptide substrate is what assists the conformation rearrangement
How do the 2 domains of chaperone proteins attach to the substrate polypeptide?
‘lid’ folds over the polypeptide substrate
ATP binds
refolding occurs,
lid opens, allows access of another substrate to the substrate binding domain
it’s the cycle that facilitates the conformational change
How does hsp90 specifically bind to a substrate? (same as a normal chap but just more detail)
It has 3 different structures: open, ATP bound, ADP bound
Open - ATP binds - cofactor assist - substrate attached - conformational changes occur (it’s closed and twisted) - now ADP is bound - ADP released-Open
Other than in nascent protein folding, what roles in homeostasis do chaps have?
-Aid interaction between cellular components for transport and degradation
androgen and oestrogen receptors require hsp90 for efficient binding of steroid hormones
Describe how hsp90 is present in steroid binding to androgen
- steroid receptors are not present on cell surface because steroids diffuse through the membrane
- the steroid receptor (inside the cell) is unstable until hsp90 bound
- Now the androgen (receptor) can bind to the steroid, hsp90 displaced
- receptor ligand complex now stable so can enter nucleus , attach to DNA and act as TF
What are many hsp90 ‘client proteins’ implicated in
cancer. surprise.
Give an example of a hsp90 client prominent in breast cancer, and what the effects would be if drugs could successfully target hsp90
HER2 tyrosine kinase (an estrogen receptor)
If hsp90 inhibited, the oncogenic client proteins such as HER2 are compromised and cancers are less able to withstand the stress of accumulating unfolded proteins - proteolytic stress is induced
how much normal protein is degraded per hour in mammals
1-2% i.e. a significant amount of cellular protein is degraded normally
What are the 3 main types of proteins that are degraded and why have these arisen
INCOMPLETE OR MISSENSE PROTEINS - errors in lation
-cellular errors, disruptive mutations, prem termination, accidental proteolytic cleavage
POST SYNTHETIC DAMAGED PROTEINS - environmental toxins e.g. UV damage, ROS
-misfolded, protein aging, denatured
UNWANTED (SIMPLY NOT NEEDED) PROTEINS
-inactive/used proteins, free subunits of multimeric complexes, proteins made in excess
BASICALLY ANY PROTEIN IN WRONG PLACE AT WRONG TIME
What are the options for where proteins can be degraded in eukaryotic cells
Proteosome
Lysosome
Caspase enzymes (that are not organelle associated)
What are the features the proteins it degraded by the proteosome
cytoplasmic and nuclear proteins
Its is a targeted, selective, highly regulated process
What are the features of the proteins degraded in the lysosome and what is it’s internal environment like
mainly defecitve organelles and extracellular proteins taken up by cell
It’s an acidic environment with hydrolytic enzymes
What are the features of proteins degraded by the caspase enzymes
(This is a proteolytic enzyme)
Degrades cystolic and nuclear proteins
activated by signals that promote programmed cell death
What are the structural features of the proteosome and features of how it process proteins
complex molecular machine
There are about 30000 per cell
made of 50 protein subunits
assembled as a 26s compex - 20s core containing proteolytic enzymes and 2x19s regulatory caps
Caps control entry of ‘marked’ (with ubq) proteins - it denatures them to allow them inside
6 proteolytic sites are inside, accessed only through the cap
produces fragments of viral proteins to be presented on cell surface
proteins broken down to fragments 7-9aa long, then further degraded in cytoplasm by peptidases
What are used to tree multiple melonomas and explain why
proteosome inhibitors
In mmplasma cells produce lots of defective immunoglobin, so high proteolytic stress if proteasomes can’t degrade the extra protein
Give an example of a proteosome inhibiting drug and how it does this
What can it work in comination with?
Bartzomib - targets 1 core enzyme within it. Doubles life expectancy but lots of nausea as side effect, often people become resistant and relapse
hsp90 inhibitors - has synergenic effect because both cause proteotoxic effects fro cancer cells
