Lecture 10: Cytosolic events Flashcards
1
Q
Where does protein synthesis occur in eukaryotic cells?
A
- Cytosol starting on free cytosolic ribosomes
- Except tin amounts that occur in mitochondria and plastids
2
Q
What are the stages of protein synthesis in a eukaryotic cell?
A
- Common pool of ribosomal subunits in the cytosol is used to assemble ribosomes on mRNAs encoding cytosolic proteins
○ These remain free in the ribosomes- Multiple ribosomes assembly -> produces free cytosolic polyribosomes
- Starts to produce a protein
○ Room for docking of another ribosome
○ Gangs of ribosome move along mRNA -> polysome, continue making protein until end of translation - Newly made proteins are released to cytosol, ribosomes are dismantled n re-enters pool into cytosol
- Recycling of the ribosomes, engaging n disengaging w the mRNA
- Eukaryotic genes are pseudo circularized thus this process is v efficient
3
Q
What are the characteristics that distinguish unfolded or misfolded proteins from folded proteins?
A
- Unfolded proteins and misfolded proteins
○ Protease-sensitive
○ Non-functional
○ Prone to aggression- Folded proteins
○ Stable
○ Resistant to proteases
○ Functional
- Folded proteins
4
Q
What is the effect does overcrowding of macromolecules in cells hv?
A
- Promotes rapid biochemistry: overrides diffusion and affinity
- Favours aggregation of proteins: many proteins making inappropriate contacts
5
Q
How do nascent proteins find their stable conformations?
A
- Molecular chaperones: class of cellular proteins that ensures polypeptide folding occurs correctly
- Hydrophobic patches on nascent/unfolded proteins are recognized by Hsp40 (heat shock protein)
- Hsp40 then delivers the substrate to ATP-bound (OPEN conformation)
- Hsc70 stimulate its ATPase activity -> ADP-bound (CLOSED conformation)
- Hsc70 shields the hydrophobic patches of the substrate -> prevents aggregation -> allows time for the hydrophilic parts of the substrate to fold
- Hsc70 adopts open conformation upon nucleotide exchange -> releases substrate w folded soluble structure
- Partly folded protein may now snap into final conformation
6
Q
Describe an experiment that allows us to study chaperones
A
- Heat target protein at e.g. 45C for 15 min
- Separate aggregated (P, pellet) n soluble fractions by centrifugation
- SDS-PAGE, silver stain n quantify. Most is insoluble.
- Heating in the presence of Hsp40 -> increases S fraction
- Heating in the presence of Hsc70 has a larger effect
- Hsp40 + Hsc70 = even more effect
- Maximal solubilization requires Hsp40, Hsc70 n ATP
- Target protein is chosen
- Large proportion of target protein becomes insoluble
7
Q
Explain how Hsc70 reduces the aggregation of nascent/unfolded proteins
A
- Hsc70 shields the hydrophobic regions of its clients
8
Q
What are the potential outcomes for a partially-folded protein bound to Hsc70?
A
- Productive
○ Partially folded Hsc70 protein may be
§ Released n find stable conformation
§ Passed onto other chaperones for further folding and/or assembly into multimeric complexes- Destructive
○ Transported to lysosome
○ Passed to proteasomes for degradation
- Destructive
9
Q
How are protein clients released from Hsc70?
A
- Nucleotide exchange factor (NEF) binds to Hsc70-client complex
- Removes ADP from the nucleotide-binding site -> promotes nucleotide exchange -> allows ATP entry into the nucleotide binding site of Hsc70
Hsc70-ATP adopts an OPEN conformation -> releases first client person
- Removes ADP from the nucleotide-binding site -> promotes nucleotide exchange -> allows ATP entry into the nucleotide binding site of Hsc70
10
Q
What are Hsc70 co-chaperones n what do they do?
A
11
Q
How is the protein’s fate determined?
A
- Concentrations of Hsc70 n Hsp90 determine the proportion of an unfolded/misfolded protein that can gain stable conformation n the proportion that is destroyed
TLDR: Co-chaperones make decisions by competing to release chaperone clients
12
Q
What do chaperones do (4)?
A
- Prevent aggregation of unfolded proteins
○ HSc70 binds hydrophobic regions of a client -> delaying folding of these regions until hydrophilic parts of the target protein hv gained structure- Provide a controlled environment for folding
○ Chaperonins form a cage that encloses the target protein, allowing folding in a protected environment, away from the cytosol – they may even aid folding directly - Permit assembly and disassembly of multimeric complexes:
○ Histone complexes, clathrin cages, α-synuclein fibres, etc. - Can direct proteins with folding problems for destruction
○ Hsc70 co-chaperone BAG-1 can engage a Hsc70:client complex with the proteasome and the lysosome
- Provide a controlled environment for folding
13
Q
What is the architecture of the proteasome?
A
- In the center there are 4 rings, each of 7 subunits
○ 2 beta rings followed by an alpha- Beta rings hv catalytic activity at the core
- Chymotrypsin like activity
- Trypsin like activity
In effect we hv 3 different proteolytic activities at the core
14
Q
What is the process by which a protein that has failed Hsc70-mediated folding is targeted for degradation?
A
- Ub is activated by E1 activating enzyme (kept in a reduced state)
○ Following oxidation, get ubiquitin addition- Activated Ub transferred to an E2 Ub-conjugating enzyme covalently via conjugating enzyme
- Ub-conjugating enzymes take the Ub from the E1
- E2-Ub conjugate associates w an E3 ubiquitin ligase
- Complex is targeted to the Hsc70 molecule
○ Holding onto the protein that’s failed to fold - E3 ligase transfers the Ub to the target n marks the target at the destructive pathway
15
Q
Describe the variation in specificity of E1s, E2s n E3s
A
- E1s
○ ~9
○ Mammalian cells
○ Vital enzymes [can’t KO]- E2s
○ >30
○ Each can select their own E3s -> provide some substrate specificity - E3s
○ 100s of different #3s
○ Each type selects its target proteins by recognizing some specific feature:
§ Extended residence in a chaperone system
§ N-terminus
§ Misfolded regions
§ Exposure of a degradation signal
○ E3s effectively control the stability of proteins involved in key cellular processes
§ Timing the key transitions in the cell cycle
§ Circadian rhythms
§ Development
§ Signaling
§ Immunity
- E2s
16
Q
How is a protein targeted to the proteasome?
A
- Ubiquitin: conserved 76 AA protein found in all eukaryotic cells
- Cytosolic proteins destined for proteasomal degradation are marked for destruction by covalent addition of a chain of Ub molecules (polyubiquitylation) -> allow them to be bound by the 19S RP
- Ub is added covalently to side chain of available Lys residue on target molecule
- Process is repeated using side chains of Lys residues in Ub until chain of at least 4 Ub is completed [tetra-Ub is a degradation signal]
- Polyubiquitylated proteins can be bound by the proteasomal 19S RSP
17
Q
How are polyubiquitylated proteins degraded?
A
- Polyubiquitylated proteins bind the 19S regulatory particle of the proteasome
- RP uses ATP to generate energy to unfold the target protein and feed it into the 20S core.
- Deubiquitylases (DUBs) remove Ub molecules and return them to a common pool for recycling
- 3 proteolytic activities are encoded by the β subunits of the 20S core
- 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
18
Q
What other function does proteasome have besides degradation?
A
- Fail-safe mechanism
- Can also bind to proteins
- One of the RP subunits acts as chaperone that directs some clients for destruction n allow RE-FOLDING of others back into native conformation
19
Q
What happens when the ubiquitin-proteasome system fails?
A
- When proteasomes or E3 fails
○ Proteins that would normally be destroyed accumulate -> aggregates (e.g. Parkinson’s, Alzheimer’s)
○ Cell cycle proteins not degraded -> cell proliferation (i.e. cancer)- Overactive proteasomes
○ Autoimmune diseases
§ Systemic lupus erythematosus
§ Rheumatoid arthritis
- Overactive proteasomes
20
Q
What is the cytosolic post-translation modification of proteolytic cleavage?
A
- Effector proteases of apoptosis are stored in an inactive condition
- Targeted specific proteolytic cleavage
Proteolytic cleavage of inactive precursor procaspase 3 (can be cleaved by CASP8,9,10)-> subunit rearrangement (active CASP3) -> apoptosis (lecture 20)
- Targeted specific proteolytic cleavage
21
Q
What is the cytosolic post-translation modification of adding lipids?
A
- Rabs bound to GDP are inactive [Rabs are double prenylated]
- GDI (GDP dissociation inhibitor protein) bindis to GDP -> masks Rab’s double prenylation
- Nucleotide exchange occurs (GDP -> GTP)
○ GDI can no longer bind to GDP
○ Prenyl groups are exposed - RESULT: Rab-GTP prenyl groups enter target membrane [prenyl groups are hydrophobic n bury into nearest membrane]
22
Q
What is the cytosolic post-translation modification of phosphorylation?
A
- Phosphates are large negatively charged group hence alter size, shape n charge of proteins they are attached
- Tend to alter activity of target protein (can be activated/inactivated)
- Cyclin CDK interact -> cyclin-CDK complex (heterodimer)
- CAK phosphorylates CDK -> active complex of cyclin-CDK
- Inactivated by Wee1 kinase’s phosphorylation
Can be activated by dephosphorylation of cdc25 phosphatase
23
Q
What is the cytosolic post-translation modification of ADP ribosylation and methylation?
A
- ADP ribosylation
○ Adding 1 or more ADP-ribose to protein
○ APPLICATION: cell signaling, DNA repair, apoptosis
○ Cholera toxin + diphtheria toxin (Lecture 8) are ADP-ribosyls- Methylation
○ Protein methylation takes place on R or K in protein sequence (lecture 4-5)
- Methylation
