Lecture 10: Cytosolic events

Question 1

Where does protein synthesis occur in eukaryotic cells?

Answer 1

• Cytosol starting on free cytosolic ribosomes
• Except tin amounts that occur in mitochondria and plastids

Question 2

What are the stages of protein synthesis in a eukaryotic cell?

Answer 2

• 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

Question 3

What are the characteristics that distinguish unfolded or misfolded proteins from folded proteins?

Answer 3

• Unfolded proteins and misfolded proteins
  ○ Protease-sensitive
  ○ Non-functional
  ○ Prone to aggression
  
  • Folded proteins
    ○ Stable
    ○ Resistant to proteases
    ○ Functional

Question 4

What is the effect does overcrowding of macromolecules in cells hv?

Answer 4

• Promotes rapid biochemistry: overrides diffusion and affinity
  
  • Favours aggregation of proteins: many proteins making inappropriate contacts

Question 5

How do nascent proteins find their stable conformations?

Answer 5

• 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

Question 6

Describe an experiment that allows us to study chaperones

Answer 6

• 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

Question 7

Explain how Hsc70 reduces the aggregation of nascent/unfolded proteins

Answer 7

• Hsc70 shields the hydrophobic regions of its clients

Question 8

What are the potential outcomes for a partially-folded protein bound to Hsc70?

Answer 8

• 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

Question 9

How are protein clients released from Hsc70?

Answer 9

• 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

Question 10

What are Hsc70 co-chaperones n what do they do?

Question 11

How is the protein’s fate determined?

Answer 11

• 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

Question 12

What do chaperones do (4)?

Answer 12

• 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

Question 13

What is the architecture of the proteasome?

Answer 13

• 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

Question 14

What is the process by which a protein that has failed Hsc70-mediated folding is targeted for degradation?

Answer 14

• 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

Question 15

Describe the variation in specificity of E1s, E2s n E3s

Answer 15

• 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

Question 16

How is a protein targeted to the proteasome?

Answer 16

• 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

Question 17

How are polyubiquitylated proteins degraded?

Answer 17

• 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

Question 18

What other function does proteasome have besides degradation?

Answer 18

• 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

Question 19

What happens when the ubiquitin-proteasome system fails?

Answer 19

• 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

Question 20

What is the cytosolic post-translation modification of proteolytic cleavage?

Answer 20

• 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)

Question 21

What is the cytosolic post-translation modification of adding lipids?

Answer 21

• 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]

Question 22

What is the cytosolic post-translation modification of phosphorylation?

Answer 22

• 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

Question 23

What is the cytosolic post-translation modification of ADP ribosylation and methylation?

Answer 23

• 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)