Lecture 10. Cytosolic Events

Question 1

Where does most protein synthesis in a eukaryotic cell start/take place?

Answer 1

Free cytosolic ribosomes (Not an ER event)
Exceptions though (mitochondrial and plasmid translation)

Question 2

How does protein synthesis occur in the cytosol?

Answer 2

1. 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
2. Multiple ribosomes assemble, producing free cytosolic polyribosomes (polysomes)
3. Newly made proteins are released to the cytosol and the ribosomes are dismantled, returning to the original common pool

Question 3

What does macromolecular crowding favour?

Answer 3

Aggregation of proteins (when hydrophobic parts are not touching)

Question 4

What is the effect of crowding in the cytoplasm of the eukaryotic cell?

Answer 4

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

Question 5

Because nascent proteins are being made in close proximity to each other and there are exposed hydrophobic residues/patches

Answer 5

Hydrophobic patches will interact and cause the nascent proteins to aggregate
Nascent proteins are in a non-native, aggregation-prone conformation

Question 6

Because newly synthesises proteins are non-functional and prone to misfolding, how do nascent proteins ind their stable conformations?

Answer 6

There is a class of cellular proteins (molecular chaperones) that ensures that the folding of other polypeptides occurs correctly

Question 7

How are molecular chaperones involved in the correct folding of cytosolic proteins?

Answer 7

1. Hydrophobic patches on nascent/unfolded proteins are recognised by Heat shock protein 40 family members (Hsp40 co-chaperone)
2. 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
3. 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

Question 8

How do we study chaperone interactions?

Answer 8

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

Question 9

What is the straightforward principle of how chaperones function?

Answer 9

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)

Question 10

What can the fates of client proteins be?

Answer 10

Both productive (folding, activation) and non-productive (destruction, inactivation) fates can proceed from an Hsc70-bound state

Question 11

How are protein clients released from Hsc70?

Answer 11

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

Question 12

What are examples of Hsc70 co-chaperones?

Answer 12

Multiple NEFs for Hsc70 (e.g. BAG-1, BAG-2, HSPBP1). Hsp40 family members CHIP

Question 13

What is the Hsp90 dimer?

Answer 13

Can provide a platform for further protein folding and also assembly of multimeric complexes (can take two partially folded substrates and combine them)

Question 14

What is the chaperonin?

Answer 14

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

Question 15

What is the role of the chaperonin?

Answer 15

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

Question 16

What is the fate of the chaperon client proteins?

Answer 16

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

Question 17

How does Hsc70 disaggregate material?

Answer 17

1. DNAJB1 (Hsp40) binds as dimers to αSyn fibres and recruits Hsc70
2. Apg2 triggers more Hsc70 binding (persuades bound Hsc70 to let go and try and squeeze more Hsc7 into that space, entropic pulling)
3. Disaggregation (rips apart due to too much entropic pulling)

Question 18

What is the architecture of the proteasome?

Answer 18

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)

Question 19

How is a protein targeted to the proteasome?

Answer 19

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

Question 20

What is the mechanism that allows a protein to be targeted to the proteasome?

Answer 20

1. Ub is activated by an E1 ubiquitin activating enzyme (~9 E1 enzymes in mammalian cells - vital enzymes for life)
2. 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)
3. 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)
4. The E3-E2-Ub conjugate binds the target protein and transfers Ub to the target

Question 21

How can E3s recognise the specific feature of the target protein?

Answer 21

Extended residence in a chaperone system
Their N-terminus
Misfolded regions
Exposure of a degradation signal

Question 22

What proteins involved in key cellular processes do E3s control the stability of?

Answer 22

Timing the key transitions in the cell cycle
Circadian rhythms
Development
Signalling immunity

Question 23

Where on the protein Ub normally added covalently and where do the polyubiquitylated proteins bind to the proteasome?

Answer 23

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

Question 24

How is the polyubiquitylated protein degraded by the proteasome?

Answer 24

1. Polyubiquitylated proteins bind the 19S regulatory particle of the proteasome
2. 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
3. Three proteolytic activities are encoded by the β subunits of the 20S core
4. 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

Question 25

Summary of the ubiquitin proteasome system (UPS)

Answer 25

1. Ub is activated by an E1 ubiquitin-activating enzyme
2. Ub is transferred to an E2 ubiquitin-cojugating enzyme
3. Ub-conjugated E2 selects and E3 ubiquitin ligase
4. The E3 transfers Ub to a lysyl on the target protein (T)
5. The process is repeated until T is tetra-ubiquitylated
6. The Ub-Target protein binds the proteasome RP and is degraded in the core: deubiquitylases (DUBs) recycle Ub

Question 26

Besides from the destructive property of the proteasome, what else does it do?

Answer 26

There is a fail-safe mechanism. It can also bind proteins.
One of the RP subunits acts as a chaperone that directs some clients for destruction and allows re-folding of others back to their native conformation
The proteasome is judge, jury and executioner

Question 27

What happens when the proteasomes or E3s fail?

Answer 27

Proteins that would normally be destroyed accumulate instead. This can lead to the formation of aggregates e.g. in neurons of people with Parkinson’s and
Alzheimer’s
If cell cycle proteins are not degraded properly, it can lead to cell proliferation (as in cancer)

Question 28

What happens when the proteasomes are overactive?

Answer 28

Have been implicated in autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis

Question 29

What is targeted specific proteolytic cleavage (e.g to activate a protein)?

Answer 29

The effector proteases of apoptosis are stored in an inactive condition. Activation is by proteolytic cleavage and subunit rearrangement
Cleavage allows molecular rearrangement of the subunits

Question 30

What is addition of lipids to permit membrane targeting?

Answer 30

Many regulatory proteins (e.g Rabs that regulate membrane traffic) are modified by the addition of lipids. Rabs are doubly prenylated (prenylated lipids are either at 15C farsenyl or a 20C geranylgeranyl)
When the prenyl groups are masked by GDI (GDP dissociation inhibitor), Rab-GDP is cytosolic
Following nucleotide exchange, GDI dissociates and the prenyl groups of Rab-GTP enter the target membrane

Question 31

What is phosphorylation in the context of post-translational modifications?

Answer 31

Addition/removal of phosphates can alter the activity of a protein: phosphates can activate or inactivate e.g control of CDK activation

Question 32

How do we study phosphorylation?

Answer 32

In vitro recapitulation
U0126 is an inhibitor of MEK, a MAP kinase kinase
SCH772984 is an inhibitor of ERK
Nuclear entry of cyclin B1 requires phosphorylation by ERK

Question 33

What is ADP ribosylation?

Answer 33

Addition of one or more ADP-ribose residues to a protein (can occur in prokaryotes as well as eukaryotes). ADP-ribosylated proteins have roles in cell signalling, DNA repair and apoptosis

Question 34

What is methylation in terms of post-translational modifications?

Answer 34

(Can occur in all organisms): Protein methylation typically takes place on arginine (R) or lysine (K) residues in the protein sequence