Lecture 10. Cytosolic Events Flashcards
Where does most protein synthesis in a eukaryotic cell start/take place?
Free cytosolic ribosomes (Not an ER event)
Exceptions though (mitochondrial and plasmid translation)
How does protein synthesis occur in the cytosol?
- A common pool of ribosomal subunits in the cytosol is used to assemble ribosomes on mRNAs encoding cytosolic proteins: these remain free in the cytosol
- Multiple ribosomes assemble, producing free cytosolic polyribosomes (polysomes)
- Newly made proteins are released to the cytosol and the ribosomes are dismantled, returning to the original common pool
What does macromolecular crowding favour?
Aggregation of proteins (when hydrophobic parts are not touching)
What is the effect of crowding in the cytoplasm of the eukaryotic cell?
The effects of crowding are very large: estimates of reaction rates and equilibria made in test tubes may differ by orders of magnitude from those of the same reactions operating within cells
Promotes rapid fire biochemistry, but many proteins making inappropriate contact with other molecules
Because nascent proteins are being made in close proximity to each other and there are exposed hydrophobic residues/patches
Hydrophobic patches will interact and cause the nascent proteins to aggregate
Nascent proteins are in a non-native, aggregation-prone conformation
Because newly synthesises proteins are non-functional and prone to misfolding, how do nascent proteins ind their stable conformations?
There is a class of cellular proteins (molecular chaperones) that ensures that the folding of other polypeptides occurs correctly
How are molecular chaperones involved in the correct folding of cytosolic proteins?
- Hydrophobic patches on nascent/unfolded proteins are recognised by Heat shock protein 40 family members (Hsp40 co-chaperone)
- Hsp40 co-chaperones deliver the substrate to ATP bound (open conformation) Heat shock cognate protein 70 (Hsc70 chaperone) and stimulates its ATPase activity
3.This results in ADP-bound (closed conformation) Hsc70 shielding the hydrophobic patches of the substrate, preventing aggregation, and allowing time for the hydrophilic parts of the substrate to fold - Upon nucleotide exchange, Hsc70 adopts its open conformation, releasing the substrate, with folded soluble structure: this partly folded protein may now snap into its final conformation
How do we study chaperone interactions?
Invigilate all of the reactions in vitro and then follow the process
1. Heat targeted protein (PrT) at e.g 45ºC for 15 mins and sperate aggregated (P, pellet) and soluble (S) fractions by centrifugation. SDS-PAGE, silver stain and quantify. Most is insoluble
2.Heating in the presence of Hsp40 increases the S fraction
3.Heating in the presence of Hsc70 has a larger effect
4. Hsp40 and Hsc70 together have even more effect
5. Maximal solubilisation requires Hsp40, Hsc70 and AT
What is the straightforward principle of how chaperones function?
Hsc70 shields the hydrophobic regions of its clients and so it reduces aggregation of nascent or unfolded proteins (acts as a holdase)
A partially-folded Hsc70 client protein may be productive (released, and find its stable conformation - passed on to other chaperones for further folding and/or assembly into multimeric complexes)
Or destructive (transported to a lysosome - or passed to proteasomes for degradation)
What can the fates of client proteins be?
Both productive (folding, activation) and non-productive (destruction, inactivation) fates can proceed from an Hsc70-bound state
How are protein clients released from Hsc70?
A nucleotide exchange factor (NEF) binds the Hsc70:client complex and removes ADP from the nucleotide-binding site of Hsc70.This promotes nucleotide exchange, allowing entry of ATP into the nucleotide binding site of Hsc70
Hsc70:ATP adopts an OPEN conformation, releasing the client protein
What are examples of Hsc70 co-chaperones?
Multiple NEFs for Hsc70 (e.g. BAG-1, BAG-2, HSPBP1). Hsp40 family members CHIP
What is the Hsp90 dimer?
Can provide a platform for further protein folding and also assembly of multimeric complexes (can take two partially folded substrates and combine them)
What is the chaperonin?
14, 16 or 18 x Hsc60 depending on the species arranged in two, stacked rings
Can isolate the client from the rest of the cell and allow the specific client to fold correctly
What is the role of the chaperonin?
Chaperonins provide a cage that isolates small (<70 kDa) folding proteins (e.g tubulin, actin) from the cytosol. Residence time ~10s
Depending on the species utilises 7-9 ADP per cycle
What is the fate of the chaperon client proteins?
Not pre-determined
Co-chaperones make decisions by competing to release chaperone clients
There is an interacting, competing network of co-chaperones that determines the fate of a chaperone client
How does Hsc70 disaggregate material?
- DNAJB1 (Hsp40) binds as dimers to αSyn fibres and recruits Hsc70
- Apg2 triggers more Hsc70 binding (persuades bound Hsc70 to let go and try and squeeze more Hsc7 into that space, entropic pulling)
- Disaggregation (rips apart due to too much entropic pulling)
What is the architecture of the proteasome?
Proteasomes are abundant in the cytosol and nucleoplasm (~30,000: 1-2% of total cellular protein)
The 20S core particles are cylinders with three proteolytic activities: chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolysing (caspase-like)
The active sites are inside the barrel, encoded by the β subunits (20S core particle made of two 7β rings on top of each other, flanked by two rings of 7α subunits)
Each 20S core has 19S caps, regulatory particles (RP) at one or both ends (makes decisions about what proteins can enter, and what rate they can enter at)
How is a protein targeted to the proteasome?
Addition of ubiquitin (Ub)
Ubiquitin (Ub) is a conserved 76 amino acid protein found in all eukaryotic cells
Need a chain of four or more Ub additions to generate a degradation signal
What is the mechanism that allows a protein to be targeted to the proteasome?
- Ub is activated by an E1 ubiquitin activating enzyme (~9 E1 enzymes in mammalian cells - vital enzymes for life)
- Activated Ub is transferred to an E2 ubiquitin-conjugating enzyme (>30 E2 enzymes in mammalian cells - each can select their owen E3s, so they ultimately provide some specificity)
- The E2-Ub conjugate associates with an E3 ubiquitin ligase (100s of E3 ligases in mammalian cells - each type selects its target proteins by recognising some specific feature)
- The E3-E2-Ub conjugate binds the target protein and transfers Ub to the target
How can E3s recognise the specific feature of the target protein?
Extended residence in a chaperone system
Their N-terminus
Misfolded regions
Exposure of a degradation signal
What proteins involved in key cellular processes do E3s control the stability of?
Timing the key transitions in the cell cycle
Circadian rhythms
Development
Signalling immunity
Where on the protein Ub normally added covalently and where do the polyubiquitylated proteins bind to the proteasome?
Ubiquitin is normally added covalently to the side chain of an available lysine residue on the target molecule until 4 Ub are in a chain
Polyubiquitylated proteins can be bound by the proteasomal 19S RP
How is the polyubiquitylated protein degraded by the proteasome?
- Polyubiquitylated proteins bind the 19S regulatory particle of the proteasome
- The RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core. Deubiquitylases (DUBs) remove Ub molecules and 7α return them to a common pool for recyclin
- Three proteolytic activities are encoded by the β subunits of the 20S core
- The target protein is degraded into small peptides (typically 7–9 amino-acid residues long, though they can range from 4 to 25 residues), which are ejected from the proteasome
