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BIOC 212 > Chaperones > Flashcards

Chaperones Flashcards

1
Q

What does it mean for a chaperone to be inducibly expressed vs Constitutively expressed?

A

Inducible chaperones: heat shock proteins
- Upregulation induced by stress conditions

Constitutive chaperones: Assisted protein folding
- Hold or stabilize hydrophobic residues
- Assist folding in normal conditions
- Universal mechanism or protein homeostasis

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2
Q

What is the Heat Shock Response? What happens?

A
  • Activated by unfolded cytosolic proteins: heat stress, oxidative damage, proteasome inhibition
  • Transcription of Heat Shock Proteins is up-regulated; other genes down regulated
    *Many Hsp are chaperones
  • Response continues after stress, chaperones continue refolding proteins
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3
Q

How long does the Heat Shock Response continues after a heat shock of 1h at 42˚C ?

A

After 1h, increased transcription of Hsp stops, but the translated proteins are stable and stay to keep refolding proteins (recovery from heat shock)
~12h = HSP expression highest point
~24h = return to normal

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4
Q

What is HSF1?

A

Heat Strock Factor 1 → activates transcription of HSPs

Has DNA binding domain, Regulatory domains, Transcription-Activation domain

Inactive HSF1 = monomeric; active = trimeric
Active HSF1 recognizes heat shock element promotors to promote transcription of heat shock element genes

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5
Q

How does regulation of HSF occurs?

A
  1. Monomeric HSF1 is folded, but mimics unfoleded protein (exposed hydrophobic patches) and is bound by Hsp90
  2. After heat shock, unfolded proteins compete with HSF1 for Hsp90 binding
  3. Free HSF1 trimerizes → activates transcription
  4. Increased transcription of chaperons including Hsp90
  5. New Hsp90 can bind to HSF1 so found in monomer form (inactivated)
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6
Q

What is the difference between ATP-dependent and ATP-independent chaperones?

A

ATP-dependent:
- Actively promote folding
- Substrate binding and release are regulated by ATPase cycles

ATP-independent:
- Prevent agregagtion + can catalyze some folding steps
- Good at holding substrates!!

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7
Q

What are the 3 families of chaperones we studied in the class?
What is common to all of them?

A

They are all ATP-dependent
Hsp70 family, Hsp90 family and Chaperonins (Hsp60)

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8
Q

Where are found Hsp70 chaperones?

A

Cytosol: HSC70 (constitutive) and HSP70 (inducible)
Endoplasmic Reticulum: BiP (induced by ER Unfolded Protein Response)
Also in the mitochondria and in ribosomes

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9
Q

Where are found Hsp90 chaperones?

A

Cytosol: HSP90 alpha and beta
ER: GRP94 (induced by ER Unfolded Protein Response)
Also in mitochondria
*Constitutively expressed + induced by Heat shock response

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10
Q

Where are found Hsp60 / Chaperonins?

A

Cytosol: TRiC
ER: none
There also HSP60 in mitochondrias
*Constitutively expressed + induced by Heat shock response

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11
Q

What are the characteristics of HSP70 family?

A
  • 70 kDa monomers
  • 2 domains: ATPase domain controls substrate-binding domain
  • ATP-bound = no substrate peptide binding
  • ADP-bound = substrate domain closed tightly on peptide
  • Binds short hydrophobic sequences
  • Function with help fo Co-Chaperones
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12
Q

What are the HSP70 co-chaperones?

A

DNAJ (Hsp40) family promotes HSP70 substrate binding

Nucleotide Exchange Factors (NEFs) promote substrate release

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13
Q

Describe the functional cycle of HSP70.

A
  1. Hsp40-mediated delivery of substrate to ATP-bound Hsp70
  2. Hydrolysis of ATP to ADP mediated by Hsp40 → closing of alpha-helical lid and tight binding of substrate by Hsp70
  3. NEF catalyzes exchange of ADP for ATP
  4. ATP-binding → Opening of alpha-helical lid → substrate release
  5. Released substrate either fold to Native state of is substrate for another chaperone
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14
Q

What is the structure of DNAJ co-chaperone?

A
  • Regulates HSP70 function
  • many DNAJs - at least 53 different genes in human cells

J domain (conserved):
- Bind transiently to Hsp70
- Activates hydrolysis of ATP → binding of polypeptide (but DNAJ is ATP-INdependent)
- Doesn’t bind substrate

Other specific domains determine their specific biological function:
Some DNAJs bind substrate through specific domains → act as ATP-independent chaperons
Some DNAJ do not bind substrate:
- specific domains attach DNAK to a protein complex or intracellular membrane → recruit HSP70 to the complex or membrane
Some have a dimerization domain

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15
Q

How does Nuclear Exchange Factors (NEFs) work with HSP70?

A
  • NEF removes ADP from HSP70 and allow ATP to bind
  • NEF binding opens up HSP70 ATPase domain and weakens interactions with nucleotides
  • ATP binds when NEF dissociates
  • ATP-bound HSP70 release polypeptide
    *Several NEF families in human
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16
Q

How does HSP70 help protein folding?

A
  • binds to hydrophobic regions of folding intermediates and prevents incorrect contacts from forming
  • Release of polypeptide form HSP70 → change to fold
  • Balance between DNAJs and NEFs → optiml rate of HSP70 substrate binding and release
  • Substrate-binding DNAJs may provide additional assistance / hold the protein
  • Can form multi-chaperone complex with HSP90
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17
Q

What are the characteristics of HSP90 family?

A
  • Homodimers → 2 subunits joined at C-terminus, 2x90kDa = 180 kDa
  • N-terminus = ATP binding domains
  • ATP controls opening and closing of dimer
  • Co-chaperone p23 stabilizes closed form by binding on the N-terminus
  • Binds to hydrophobic AND POLAR surfaces → stabilizes intermediate folded states
  • Different substrates can bind different sites on the sides unlike HSP70 and Chaperonins which 1 binding site for substrate

Co-chaperones = TRP, p23

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18
Q

Explain the functional cycle of HSP90.

A
  1. Substrate + Hsp70 + Hsp40 + HOP enter dimer
  2. Substrate bound wealky in open nucleotide-free state
  3. ATP binding allows dimer to close and bind substrate tightly (helped by FKBP52 and p23 co-chaperone)
  4. ATP hydrolysis to ADP compacts the dimer and releases substrate (opens the clamp to release the substrate)
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19
Q

How does the HSP70-HSP90-Cochaperone system work?

A
  • In the cytosol
  • Substrate released from HSP70 and bound by HSP90 in coordination
  • HOP co-chaperone has recognition domains for HSP70 and HSP90 so assists complex formation
  • HSP70 dissociates with HSP90 binds ATP
  • many co-chaperones also act to provide flexibility, folding and non-folding functions
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20
Q

What do HSP70 and HSP90 have in common?

A

Have similar C-terminal EEVD motif
HSP70: PTIEEVD-COO-
HSP90: MEEVD-COO-

TPR domains on HOP co-chaperones recognize EEVD motifs, can be specific to HSC70, HSP90 or both

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21
Q

What are TPR Co-chaperones?
Name 3 co-chaperones that have TPR domains.

A

TPR domains = adaptors for HSP70 and HSP90

TPR co-chaperones often have other domains which interact with substrate directly

HOP: domain for HSP70 and HSP90 specifically
FKBP52: HSP90-binding TPR domain + PPlase domain
CHIP: binds to either HSP70 or HSP90 + ubiquitin ligase domain helps degrading protein if refolding unsuccessful

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22
Q

What is FKBP52?

A

TPR co-chaperone:
- TPR domain only specific to Hsp90 (MEEVD-COO-)
- 2 PPlase domains = peptidyl-prolyl isomerase → chaperone specific to proline that help transition from cis to trans configuration on polypeptide

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23
Q

How does HSP90 get involved in signaling?

A

Many HSP90 substrates are transduction signal proteins
- Kinases, receptors, transcription factors
- many also require HSC70
exception: HSP90 binds to kinases without need of HSC70

Mutations in signaling proteins are causes of cancer → HSP90 and HSC70 = drug targets for cancer treatment

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24
Q

Explain how v-src is an example of HSP90 regulation

A

c-src (cellular) = normal kinase involved in signaling cell growth, auto-regulated
v-src (viral) = mutant kinase → causes cancer, doesn’t downregulate so cell division never stops, needs HSP90 to keep active conformation

  1. v-src expressed in epithelial cells → become cancerous
  2. Treat cell with HSP90 inhibitor → can’t chaperone v-src anymore → cells revert from cancer to normal growth

Problem: But if HSP90 is inhibited, can’t bind HSF1, so trimerizes → transcribes more HSP90

25
Q

What are the characteristics of Chaperonins (HSP60 family)?

A
  • Large oligomeric complexes with typical double-ring structure
  • E.coli GroEL: 2 rings x 7 indentical subunits x 60 kDa = 840 kDa → has substrate binding domain + ATPase domain
  • E.Coli GroES cap co-chaperone (x1): 7 subunit x 10 kDa = 70 kDa
  • E.coli = homologous of human mitochondiral Hsp60 and Hsp10
26
Q

How does the GroEL cavity in chaperonines work?

A

2 rings are identical and work in alternating cycles:
- Down position (no nucleotide) → hydrophobic residues facing inside to bind hydrophobic patches in polypeptides
- Up position (ATP-bound) → subunit bind to GroES cap instead of substrate
- Large cavity with a POLAR surface is formed (hydrophilic AA facing inside of cavity)
- Susbtrate enclosed inside cavity, no longer bound

Domains:
- ATPase domain = interface with opposite ring (upside down)
- Movement of substrate binding domain controlled by ATPase in both rings → either binds to substrate (down, no nucleotide) or GroES (up, ATP-bound)

27
Q

How does groEL help with protein folding ?

A

Substrate enclosed inside polar cavity:
- Provides chance to fold
- confinement favours more compact conformations → promotes folding
- ATP hydrolysis acts as a timer for substrate release

28
Q

What are the similarities / difference between E. Coli GoEL/GroES and human Chaperonins (HSP60)?

A

Human mitochondrial HSP60 functions like GroEL

Human chaperonin in cytosol:
- TriC (TCP1 Ring Complex)
- does not have cap co-chaperone
- long substrate-binding domains from the cavity themselves

29
Q

What tool is used as a metaphore for HSP70, HSP90 and HSP60?

A

HSP60/Chaperonins = Cage
HSP70 family = Locking piler
HSP90 = Nutcracker

30
Q

By what characteristic are Heat Shock Proteins identified/named?

A

Molecular weight

31
Q

Where in the cell does Ubiquitin-mediated degradation occur?

A

In the cytosol
Can occur for proteins in the ER → kicked out of the ER to the cytosol

32
Q

What are the main characteristics of Ubiquitin?

A
  • 8 kDa protein
  • 76 AA
  • Can be covalently linked to lysine side chains
33
Q

What is the role / characteristics of E1?

A
  • E1 Ub activating enzyme attaches Ub to itself (E1)
  • E1 Cysteine side chain with C-teminus of Ub → Thioester bond
    *2x ATP-dependent steps for formation of high energy thioester bond
34
Q

What is the role / characteristics of E2?

A
  • E2 conjugating enzyme transfers Ub to its own Cys from E1
  • Thioester bond
    *Not ATP-dependent?
35
Q

What is the general role / characteristic of E3?

A

E3 ligase selects target protein to be modified
- triggers Ub transfer from E2 → Lysine 48 side chain on substrate
*Multiple times for poly-Ub

36
Q

What are the 2 family types of E3?

A

RING E3:
Direct transfer, E3 is never ubiquitinilated itself, most abundant

HECT E3:
Indirect, E3 transfers Ub from E2 → itself → Lysine 48 on target protein

37
Q

What are the different post-translational modifications that can affect Lysine?

A
  • Methylation
  • Acetylation
  • Ubiquinitation
    For Ubiquitination to be perceived as a degredation signal by the cell, K48 has tu be Ubiquitinated on target protein
38
Q

What are the characteristics of Ubiquitin linkage?

A

C-terminus carboxyl of Ub is covalently linked to the side chain NH3+ of Lysine → ISOPEPTIDE BOND → adds an additional N-terminal to the protein (the one of Ub)

  • Ub C-term can be linked to Lys63, 48 or 11 of another Ub, not all perceived as degredation signal
  • Long K48 poly-Ub chains = target protein signal for degradation
  • Mono-Ub or K63 poly-Ub = other signals

A susbtrate (protein) can have multiple ubiquitination sites, but not all lysines are Ubiquitination sites depending on accessibility

39
Q

How is Ubiquinitation different from Phosphorylation, Acetylation and Methylation? (as a PTM)

A

It is the addition of a full protein to another protein, instead of being the addition of a molecule

40
Q

How many different E1, E2, E3 enzymes and proteasome subunits have genes coding for in the human genome?

A

~ 12 genes for E1
~ 50 genes for E2
> 600 genes for E3
Most proteasome subunits only have 1 genes

*So many different E3 enzymes for each degradation situation, all use same proteasome → More efficient than having different proteases with different specifies

41
Q

What are different mechanisms of Susbtrate selection by E3 ligase?

A
  • All proteins continually degraded, but at different rates
  • Degredation of substrate controlled by selectivity of E3 lygase, not by the proteasome
  1. Quality control degradation of a misfolded protein
  2. Constitutive degradation of a native protein to control its level (for short half-life proteins) → N-end Rule
  3. Degredation of a native protein in response to a signal
    *Each case = different E3 ligase recognition mechanism
42
Q

How does quality control degradation of a misfolded protein work?

A

Regulated by CHIP co-chaperone:
- TPR domain binds HSC70 or HSP90 (recognize exposed hydrophobic patches)
- E3 ligase domain binds E2 (that has Ub bound to it)

Chaperone + CHIP + E2 = complete E3 ligase complex
Chaperone-bound substrate selectively ubiquitinated

43
Q

Explain the CHIP mechanism.

A

CHIP (co-chaperone) interactions with chaperones are transient → relatively fast binding and release

Balance between chaperone-mediated folding and degradation:
- Substrate bound by chaperones for long times more likley to for complex with CHIP and be ubiquitinated (tried refolding multiple times and not working)
- Substrate bound to chaperones for short period of time likely to escape Ubiquitination

44
Q

What is the N-End Rule?

A

For Ubiquitination of short-life proteins

  • All proteins are translated with N-terminal Met
  • Many proteins processed by cleavage → different AA becomes N-terminus
  • Some N-terminal residues bound by N-end rule E3 ligases which ubiquitinates the proteins
  • Degrades proteins rapidly, whether folded or not
45
Q

Which AA have sides chains that are recognized by N-End Rule E3 ligases?

A

Basic: Arg, Lys, His
Polar: Tyr
Hydrophobic: Phe, Trp, Leu, Ile
(All the ones with rings → Try, His, Top, Phe)
RIK Will FLY High (8)

Each of these amino acids are recognized by a specific E3 ligase

46
Q

What are the possible N-end rule modifications?

A

Aspartate, Glutamate (acidic) → Arg added to N-terminus
Asparagine, Glutamine (polar amides) → side chains converted to Asp, Glu by removal of amide → Arg added to N-terminus

47
Q

What are the components of Regulated Degradation E3 / SCF E3?

A
  • Skp1 = adaptor (between F-box and Cullin)
  • Cullin = Scaffold protein (binds E2 and specific F-box)
  • F-box = Substrate-binding protein (binds to phosphorylated substrate)
    *classes of proteins, there are many different F-boxes
48
Q

By what system does Regulated degradation E3 / SCF E3 transfer Ub from E2 → target protein

A
  1. E3 Ub ligase complex (Skp1 / Cullin / F-box)
  2. Cullin (scaffold) binds E2 and F-box (susbtrate-binding protein)
  3. F-box protein binds phosphorylated substrate
  4. Susbtrate is presented to the E2 for ubiquitination (Skp1 = enzymatic core), brought into close proximity
  5. De-Ubiquitinated E2 released and new ubiquitinated E2 binds to Cullin
  6. *F-box stays bound to phosphorylated substrate
49
Q

How does degradation regulation occur for SCF E3?

A

By PHOSPHORYLATION

  • Many F-box proteins recognize phosphorylated peptide sequences
  • Phosphorylation by kinase = degradation signal
  • De-phosphorylation prevents degradation
  • SCF ligases degrade native, functional proteins to stop function (not bc misfolded or short-life)

For proteins that are needed for specific phases of the cell cycle, but no longer needed

50
Q

What are the main characteristics of the proteasome?
General function (where)?

A
  • Large oligomeric complex with central 20S core particle + 2x 19S regulatory caps
  • Core + 2x caps = 26s proteasome → ~2.5MDa
  • Responsible for general protein degradation in cytosol and nucleus, and from the endoplasmic reticulum
51
Q

What are the main characteristics of the 20S core of the proteasome?

A
  • 2 outer rings of 7 similar alpha subunits → 19s cap attaches to outer rings
  • 2 inner rings of 7 similar beta subunits → 3 of each of them have protease activity on inside surface
52
Q

What are the main characteristics of the 19S regulator (cap) of the proteasome?

A

2 components: Base + Lid

Base = 6 AAA-family proteins ATPase subunits → protein “unfoldase” → unfolds target proteins and passes it down to the core

Lid = non-ATPase subunits, poly-Ub receptors (Rpn10/13), deubiquitinases (DUBs → Rpn8/11)
*Rpn = Regulatory particle non-ATPase

53
Q

What are the functions of Ub receptors forming the lid of the 19S regulator cap of the proteasome?

A
  • Increase efficiency of targeting
  • Select only K48 chains
  • Protect against premature DUB activity (if chain is too short, won’t select for it)
54
Q

What are the 2 types of Ub receptors?
And they functions.

A
  1. Intrinsic receptors: cap subunits Rpn10 and Rpn13 bind to poly-Ub
  2. Extrinsic (shuttling) Ub receptors:
    - In the cytosol, separate from proteasome
    Have 2 domains:
    - Ub-associated domain (UBA) → bind poly-Ub
    - Ub-like domain (UBL) → recognized by cap
    - Brings poly-Ub protein to the proteasome
    ex: hHR23a, scDdi1, PLIC-1
55
Q

What does Rpn13 of Rpn10 binds to?

A

They are domains of the proteasome
Binds to poly-Ub or UBL domains of shuttling receptors →responsible for targetting of the proteasome

Rpn = regulatory protein non-ATPase

56
Q

What are the differences between the 3 active subunits of each inner beta-rings in the proteasome core (6 total)?

A
  • 1 cuts basic AAs
  • 1 cut acidic AAs
  • 1 cut hydrophobic AAs
57
Q

What happens to peptides after they are cut in the proteasome core?

A

They diffuse out and are digested into AA by peptidases

58
Q

Is it possible to have a polypeptide sequence that is not degraded by proteasomes?

A

Yes, If it doesn’t have Lysine, can be ubiquitinated → can’t be degraded

59
Q

What is the co-chaperone of GroES Chaperonin?

A

GroEL cap (10 kDa)

BIOC 212 (15 decks)

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