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
What molecule must proteins be labelled with so they are degraded by admission to proteosome and how long is this
ubiquitin - 76aa
Proteins actually polyubiquitinated (multiple chains added to a protein)
Is ubq degraded by the proteosome?
No , cleaved off and recycled
What are the 3 enzymes involved in polyubiquitination?
E1, E2, E3
Describe the process of ubiquitination
E1 binds to and activates ubiquitin in ATP dependant manner
Ubq transfered, in it’s activated state, to E2 conjugating enzyme
E3 ubq ligase recognised the target protein to be destroyed and associates with it
ubq (via E2) is targetted to this protein by E3.
Ubq tramsfered from E2 to a lysine on the target protein
E3 releases the now ubqated target protein
The cycle is repeated until target protein has a short chain of ubq
Proteosome recognises this chain
ubq remove and target protein swallowed by proteosome
Can you have monoubiquitination?
Yes - but regulates protein function rather than triggers degradation
GIve an example of an E3 ligase with a specific substrate (or class of substates)
e3 ligase that regulate cell cycle by degrading cyclins
Entry into mitosis is via cyclins A and B, exit from it requires their destruction therefore a specific E3
diff e3’s are specific to diff substrates
Give another, very specific example of a E3 ligase and it’s substrate
Why is it necessary
e3 = mdm2
substrate = p53 (tumour surpressor)
necessary because p53 triggers apoptosis very easily
What experiement proved mdm2 was crucial to organism survival
KO in mouse - die in utero because p53 induced apoptosis
KO for mdm2 crossed with KO for p53 - survived
HOw do some cancers incorporate mdm2 into their survival
some overexpress it so as to detroy more p53 so it won;t come along and surpress the tumours
What cancer therapy involved mdm2 and p53 and how does it work
nutin - occupies p53 binding site of mdm2 (a hydrophobic pocket pf alpha helices) to prevent p53 binding and being ubiquitinated and then depleted
As well as ubq, what other ptm regulate p53 and where do most modifications occur and why
neddylation, methylation, sumoylation, acetylation, phosphorylation
most occur at either terminus - becasue the middle is the DNA binding domain so less mods needed
What effects do neddylation, methylation and sumoylation have on p53
influence it’s ability to regulate the gene transcription (positvely or negatively) ie. regulates scriptional activity
What phosphorylates p53? GIve and example and the result
many kinases, different ones in different conditions and not always the same effects
E.g. ATM kinase phosphorylates p53 on ser15 in response to DNA damage
Effect of stabilising p53 by inhibiting it’s interaction with mdm2. Therefore p53 can accumulate and direct apoptosis or cell cycle arrest until DNA repaired
What is acetylation of p53 done by? Give and example and general results
done by acetyltransferases e.g. p300 - adds acyl group to lysine residue, triggered by various stresses
Effects: Stimulates p53 ability to bind to DNA and activate scription
also stabilises p53 if residues that would be targeted by mdm2 are acetylated polyubq can’t occur and it won;t be destroyed
Overall how do PTMs have regulatory versatility?
Diff ptms induced in diff conditions
diff ptms can regulate a protein’s function in many diff ways
Give an example of a histone PTM
N-terminal tail is acetylated on lys residues, so the +ve charge is neutralised, therefore accesibility of DNA increases because no longer so tightly wound around histone
Apart from acetylation what other tail ptms occur on histones
phosphorylation of ser or thr residues
methylation of lys or arg residues
What do histone tail modifications states contain info about?
The gene regions where they reside
What do some TFs recruit to stimulate transcription?
acetyl transferases - they take advantage of acetylation to stimulate expression of certain genes
Give an example where acetylation leads to promotion of gene transcription
p53 is acetylated, followed by the nucleosomes around it once the p53 has bound to DNA (post acetylation)
This therefore promotes trascription of genes because they’re now more accessible - - because acetylated regions are more accessible to basal factor and rna polymerases
what is the histone code hypothesis
specific combinations of histone ptm may convey infor about what genes are invovled because some modifications are recognised by particular proteins
Give 3 examples of histone modifications and what they are associated with
histone 3 - lysine 9 trimethylated - silent genes
histone 3 - lysine 4 trimethylated - active promotors
histone 3 - lysine 4 monomethylated - active enhancers
Give an example of a protein modification dictating protein location
the enzyme catalysed fatty acid attachments that anchor protein to the membrane - this protein has been modified so it knows to go here
rubbish example I know but it’s in the notes
What is a lipid anchor?
= a glycophosphatidyl inositol attched to protein c terminus. ie the anchor is the ptm
composed of oligosaccharides and inosotil phospholipids
There’s not more that 1 GPI anchor per protein
added to proteins in the er
What do GPI anchors do?
- anchor cell surface proteins to plasma membrane
- target proteins to outer leaflet
How are proteins targetted to the ER? And on what other proteins is it normally found
via an N terminal signal sequence that contains 6-12 hydrophobic residues
This sequence is found on most proteins destined for secretion, the lysosome, or for integration into the plasma membrane
If we need to know the sequence reqired for different destinations we can look at the glorious table in my notes
I really don;t think we do actually need to know them
What is the signal sequence of a nacent polypeptide recognised by, so it can be targetted to the right place?
Signal recognition particle - a ribonucleoprotein particle found in the cytoplasm
Contains 6 proteins, 7 slRNA and a 300 nucleotide RNA scaffold
Name a subunit of an SRP and it’s role in the SRP’s overall function
-p54
-Binds to the signal peptide of the nascent polypeptide
-SRP docks with SRP receptor (a 2 subunit integral membrane protein on cytolic face of the ER ), docks specifically with the alpha subunit of the receptor
The interaction allows GTP hydrolysis, and the SRP is released and recylced
What is a translocon
protein lined membrane channel
Describe co translational translocation
- SRP has bound to polypep (which is still attached to ribosome)
- this brings ribosome to ER surface
- GTP hydrolysis, release of SRP - this pushes poly pep through the translocon
- more pushing through is driven by the energy of protein synthesis, as growing polypep is fed through to ER
- N terminus emerges 1st -ie signal sequence
- signal sequence cleaved off by siganl peptidase enzyme
- remainer of poly pep pushed through to ER lumen and folds up
What changes are inflicted in the protein once released into the ER lumen?
Glycosylation - covalant additiona nd processing of carbohydrates. O and N linked oligosaccharides added
Disulphide bond formation - catalysed by protein disulphide isomerase
Folding and assembly - of multi subunit proteins
specific proteolytic cleavages - in both ER and Golgi
What is the ultimate goal of proteins secretion through ER and golgi
functional proteins for secretion
Describe vesivular transport from the ER, via the golgi to the plasma membrane
proteins secreted via trafficking vesicles from ER
vesicles bud off
move into golgi
then trafficked to cell surface or lysosome
