Chaperones Flashcards
What does it mean for a chaperone to be inducibly expressed vs Constitutively expressed?
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
What is the Heat Shock Response? What happens?
- 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
How long does the Heat Shock Response continues after a heat shock of 1h at 42˚C ?
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
What is HSF1?
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
How does regulation of HSF occurs?
- Monomeric HSF1 is folded, but mimics unfoleded protein (exposed hydrophobic patches) and is bound by Hsp90
- After heat shock, unfolded proteins compete with HSF1 for Hsp90 binding
- Free HSF1 trimerizes → activates transcription
- Increased transcription of chaperons including Hsp90
- New Hsp90 can bind to HSF1 so found in monomer form (inactivated)
What is the difference between ATP-dependent and ATP-independent chaperones?
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!!
What are the 3 families of chaperones we studied in the class?
What is common to all of them?
They are all ATP-dependent
Hsp70 family, Hsp90 family and Chaperonins (Hsp60)
Where are found Hsp70 chaperones?
Cytosol: HSC70 (constitutive) and HSP70 (inducible)
Endoplasmic Reticulum: BiP (induced by ER Unfolded Protein Response)
Also in the mitochondria and in ribosomes
Where are found Hsp90 chaperones?
Cytosol: HSP90 alpha and beta
ER: GRP94 (induced by ER Unfolded Protein Response)
Also in mitochondria
*Constitutively expressed + induced by Heat shock response
Where are found Hsp60 / Chaperonins?
Cytosol: TRiC
ER: none
There also HSP60 in mitochondrias
*Constitutively expressed + induced by Heat shock response
What are the characteristics of HSP70 family?
- 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
What are the HSP70 co-chaperones?
DNAJ (Hsp40) family promotes HSP70 substrate binding
Nucleotide Exchange Factors (NEFs) promote substrate release
Describe the functional cycle of HSP70.
- Hsp40-mediated delivery of substrate to ATP-bound Hsp70
- Hydrolysis of ATP to ADP mediated by Hsp40 → closing of alpha-helical lid and tight binding of substrate by Hsp70
- NEF catalyzes exchange of ADP for ATP
- ATP-binding → Opening of alpha-helical lid → substrate release
- Released substrate either fold to Native state of is substrate for another chaperone
What is the structure of DNAJ co-chaperone?
- 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
How does Nuclear Exchange Factors (NEFs) work with HSP70?
- 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
How does HSP70 help protein folding?
- 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
What are the characteristics of HSP90 family?
- 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
Explain the functional cycle of HSP90.
- Substrate + Hsp70 + Hsp40 + HOP enter dimer
- Substrate bound wealky in open nucleotide-free state
- ATP binding allows dimer to close and bind substrate tightly (helped by FKBP52 and p23 co-chaperone)
- ATP hydrolysis to ADP compacts the dimer and releases substrate (opens the clamp to release the substrate)
How does the HSP70-HSP90-Cochaperone system work?
- 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
What do HSP70 and HSP90 have in common?
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
What are TPR Co-chaperones?
Name 3 co-chaperones that have TPR domains.
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
What is FKBP52?
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
How does HSP90 get involved in signaling?
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
Explain how v-src is an example of HSP90 regulation
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
- v-src expressed in epithelial cells → become cancerous
- 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
What are the characteristics of Chaperonins (HSP60 family)?
- 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
How does the GroEL cavity in chaperonines work?
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)
How does groEL help with protein folding ?
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
What are the similarities / difference between E. Coli GoEL/GroES and human Chaperonins (HSP60)?
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
What tool is used as a metaphore for HSP70, HSP90 and HSP60?
HSP60/Chaperonins = Cage
HSP70 family = Locking piler
HSP90 = Nutcracker
By what characteristic are Heat Shock Proteins identified/named?
Molecular weight
Where in the cell does Ubiquitin-mediated degradation occur?
In the cytosol
Can occur for proteins in the ER → kicked out of the ER to the cytosol
What are the main characteristics of Ubiquitin?
- 8 kDa protein
- 76 AA
- Can be covalently linked to lysine side chains
What is the role / characteristics of E1?
- 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
What is the role / characteristics of E2?
- E2 conjugating enzyme transfers Ub to its own Cys from E1
- Thioester bond
*Not ATP-dependent?
What is the general role / characteristic of E3?
E3 ligase selects target protein to be modified
- triggers Ub transfer from E2 → Lysine 48 side chain on substrate
*Multiple times for poly-Ub
What are the 2 family types of E3?
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
What are the different post-translational modifications that can affect Lysine?
- Methylation
- Acetylation
- Ubiquinitation
For Ubiquitination to be perceived as a degredation signal by the cell, K48 has tu be Ubiquitinated on target protein
What are the characteristics of Ubiquitin linkage?
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
How is Ubiquinitation different from Phosphorylation, Acetylation and Methylation? (as a PTM)
It is the addition of a full protein to another protein, instead of being the addition of a molecule
How many different E1, E2, E3 enzymes and proteasome subunits have genes coding for in the human genome?
~ 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
What are different mechanisms of Susbtrate selection by E3 ligase?
- All proteins continually degraded, but at different rates
- Degredation of substrate controlled by selectivity of E3 lygase, not by the proteasome
- Quality control degradation of a misfolded protein
- Constitutive degradation of a native protein to control its level (for short half-life proteins) → N-end Rule
- Degredation of a native protein in response to a signal
*Each case = different E3 ligase recognition mechanism
How does quality control degradation of a misfolded protein work?
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
Explain the CHIP mechanism.
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
What is the N-End Rule?
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
Which AA have sides chains that are recognized by N-End Rule E3 ligases?
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
What are the possible N-end rule modifications?
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
What are the components of Regulated Degradation E3 / SCF E3?
- 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
By what system does Regulated degradation E3 / SCF E3 transfer Ub from E2 → target protein
- E3 Ub ligase complex (Skp1 / Cullin / F-box)
- Cullin (scaffold) binds E2 and F-box (susbtrate-binding protein)
- F-box protein binds phosphorylated substrate
- Susbtrate is presented to the E2 for ubiquitination (Skp1 = enzymatic core), brought into close proximity
- De-Ubiquitinated E2 released and new ubiquitinated E2 binds to Cullin
- *F-box stays bound to phosphorylated substrate
How does degradation regulation occur for SCF E3?
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
What are the main characteristics of the proteasome?
General function (where)?
- 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
What are the main characteristics of the 20S core of the proteasome?
- 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
What are the main characteristics of the 19S regulator (cap) of the proteasome?
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
What are the functions of Ub receptors forming the lid of the 19S regulator cap of the proteasome?
- Increase efficiency of targeting
- Select only K48 chains
- Protect against premature DUB activity (if chain is too short, won’t select for it)
What are the 2 types of Ub receptors?
And they functions.
- Intrinsic receptors: cap subunits Rpn10 and Rpn13 bind to poly-Ub
- 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
What does Rpn13 of Rpn10 binds to?
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
What are the differences between the 3 active subunits of each inner beta-rings in the proteasome core (6 total)?
- 1 cuts basic AAs
- 1 cut acidic AAs
- 1 cut hydrophobic AAs
What happens to peptides after they are cut in the proteasome core?
They diffuse out and are digested into AA by peptidases
Is it possible to have a polypeptide sequence that is not degraded by proteasomes?
Yes, If it doesn’t have Lysine, can be ubiquitinated → can’t be degraded
What is the co-chaperone of GroES Chaperonin?
GroEL cap (10 kDa)
