Molecular Chaperones

Question 1

Describe the energy landscapes of protein folding/misfolding?

Answer 1

Native state (and aggregation state) of a protein is more energetically favourable than its unfolded state
Protein folding and protein aggregation are competing reactions
During the folding process, protein may adopt energetically favourable but non-native conformations
Kinetically trapped intermediates, have a greater danger of misfolding due to non-native interactions 

This is very difficult inside the cell as there are other proteins/macromolecules, stress, pH, temperature etc…

Question 2

Describe molecular chaperones?

Answer 2

Molecular chaperones are molecular machines that stabilize, assist and maintain correct folding of polypeptides larger than 100 amino acids
They constitute up to 10% of the proteome and play important functions under normal conditions and during cellular cell responses

They prevent aggregation of partially folded proteins, to allow them to finish folding correctly
There is a competition between folding/unfolding, as hydrophobic residues in unfolded proteins are exposed to solvent and want to be buried
Without molecular chaperones - the partially folded protein will aggregate

Question 3

What are some types of molecular chaperones?

Answer 3

Heat Shock proteins constitute the majority of molecular chaperones 
Based on their molecular mass (of their monomer) and function, HSPs are classified into six families:
Small Heat Shock Proteins (sHsp)
Hsp40/J-class proteins (40 kDa)
Hsp60/Chaperonins (60 kDa)
Hsp70 (70 kDa)
Hsp90 (90 kDa)
Hsp100/Clp proteins (100kDa)

Question 4

How else can chaperones be classified?

Answer 4

Based on ATP dependence:
ATP dependent chaperones - Hsp70, Hsp90 and Hsp60
ATP independent chaperones - small heat shock proteins (sHsp)

Question 5

Describe the functions of molecular chaperones?

Answer 5

Assist de novo protein folding

Refolding of misfolded protein

Disaggregation of aggregates and degradation

Macromolecular-complex assembly/disassembly e.g. Hsc70-mediated disassembly of clathrin coats

Protein transport; e.g. Hsp70 BiP (or Grp78) is required to import polypeptides into the ER lumen/membrane

Question 6

Describe Hsp70 molecular chaperone?

Answer 6

There are 17 Hsp70 isoforms in human; some are inducible under stress conditions, some are constitutively expressed
Most conserved Hsp: 60-70%
Hsp70 is a 70 kDa protein that comprises of two domains: the nucleotide binding domain, 40 kDa (NBD) and substrate binding domain, 30 kDa (SBD)
SBD - very promiscuous, exposed to the solvent
Weak ATPase
Affinity for non native polypeptide – exposed hydrophobic sequences

Bacterial Hsp70 is called DnaK

Question 7

Describe the Hsp70 chaperone cycle?

Answer 7

The majority of Hsp70 functions rely on their ability to cycle between two functional conformations

ATP binding favours a compact, domain-docked conformation, which has low substrate affinity and fast substrate binding and release
ATP hydrolysis (converting ATP to ADP), results in stabilization of the domain-undocked state, which has high substrate affinity, but very slow and inefficient substrate binding and release

Therefore the chaperone only functions as fast as the ATP hydrolysis turnover

Question 8

Describe the Hsp70 chaperone system?

Answer 8

Other than the core molecule Hsp70, the Hsp70 chaperone system constitutes its essential co-chaperones such as Hsp40/J-protein and nucleotide exchange factors:

Hsp40 mediates the delivery of nascent or misfolded protein to ATP bound Hsp70 and accelerates hydrolysis of ATP, results in Hsp70 conformations changes (domain undocking)
J-protein - increases the ATP hydrolysis rate and they deliver the substrate to Hsp70 (not always the case - as very substrate specific - depending on the J-protein)

NEF (nucleotide exchange factor) binds to Hsp70, catalysing ADP dissociation and promoting ATP binding

Question 9

Describe the Hsp90 molecular chaperone?

Answer 9

Hsp90α and Hsp90β are the two major isoforms in the cytoplasm of mammalian cells
Hsp90α is inducible under stress conditions, while Hsp90β is constitutively expressed
Hsp90 analogues also exist in other cellular compartments (ER and mitochondria)

Hsp90 is a 90 kDa protein that consists of three regions: the ATP-binding domain (N-terminal domain), a conserved and structurally flexible middle domain (M-domain) and a C-terminal dimerization domain (C-domain)
Middle domain - responsible for interaction with substrate
Weak ATPase
Affinity for non native polypeptide

Question 10

Describe the Hsp90 chaperone cycle?

Answer 10

Hsp90 functions as a dimer
In the apo state (unbound/inactive), Hsp90 adopts a “V”-shaped form, termed “open conformation”
ATP binding triggers a series of conformational changes resulting in “closed conformation” in which the N-domain is dimerized
Hsp90 reaches the closed state in which ATP hydrolysis occurs
After ATP is hydrolysed, the N-domains dissociate, release ADP as well as inorganic phosphate (Pi), and Hsp90 returns to the open conformation again

Question 11

Describe the Hsp90 chaperone system?

Answer 11

Association with Hsp90 occurs at a later stage of the client folding process
The recruitment and assembly with client proteins requires collaboration of eukaryotic Hsp90 with Hsp70 and a multitude of the accessory proteins called ‘co-chaperones’ to form large dynamic multi-chaperone complexes

In eukaryotic cells, more than 20 co-chaperones have been identified to regulate the function of Hsp90 in different ways, such as the inhibition and activation of its ATPase activity as well as recruitment of specific client proteins
They are important for delivery of the specific substrate to Hsp90

Cochaperones: Hop, Cdc-37, p23 and Aha1
Client proteins - signalling kinases and hormone receptors

Question 12

What are some chaperonines?

Answer 12

Hsp60
GroEL/GroES (group I chaperonin) system
Group II chaperonins

Question 13

Describe chaperonins?

Answer 13

Chaperonins is a group of evolutionary conserved proteins consisting of subunit of 60 kDa molecular weight
Chaperonins form very large complexes - 800-1000 kDa double-ring complexes with seven to nine subunits per ring

The best-characterized chaperonin is GroEL from E. coli. GroEL-like HSP60 homologs have been found in mitochondrion and chloroplast of plant cells but not in cytosol

Question 14

Describe Hsp60 chaperonins?

Answer 14

The association with Hsp60 occurs at a later stage of the client folding process
Generally, chaperonin substrates have relatively slow folding kinetics - fold slowly
At normal growth temperatures (in the absence of stress), chaperonins interact with 10-15% of total newly synthesized cytosolic proteins
Under stress conditions, chaperonins interact with up to 30% of total newly synthesized cytosolic proteins

Question 15

Describe GroEL/DroES (group I chaperonin) system?

Answer 15

GroEL consists of two stacked ring with each ring containing 7 identical monomers

Each GroEL monomer is about 58 kDa
It can be divided into three separate domains: a nucleotide-binding equatorial domain, a flexible apical domain, and a hinge-like intermediate domain
Intermediate - responsible for conformational changes
Apical - binding to unfolded/partially folded proteins
The ring opening exposes hydrophobic amino acid residues for binding unfolded proteins

GroES forms a heptametric ring of 10 kDa subunits that lays like a lid on the ring opening of the GroEL
GroES preferentially binds to only one of the rings, that to the one containing the protein substrate

Question 16

Describe protein folding in the GroEL/GroES cage?

Answer 16

1. The substrate protein binds as a folding intermediate to the apical domains of the open GroEL ring
2. In an ATP-dependent step, GroES then binds via the ring opening and encloses the substrate protein
   This is accompanied by a conformational change of the GroEL, which increases its interior and changes its physical properties from hydrophobic to hydrophilic
3. The substrate protein remains trapped in the cage for about 10 seconds - this time is required to hydrolyse 7 ATP molecules on the GroEL ring
   During this time, the protein is free to fold
4. The binding of ATP to the opposite GroEL ring then leads to the dissociation of the GroES, the cage opens and the substrate is released

Question 17

What can happen at the end of protein folding in the GroEL/GroES cage?

Answer 17

Sometimes this isn’t enough so the protein goes back through the system again

It allows the partially folded protein enough time to be folded in isolation from other macromolecules

Question 18

Describe group II chaperonins?

Answer 18

TRiC - T-complex protein-1 ring complex
It is also known as CCT is an essential 1 MDa eukaryotic chaperonin
About 5-10% of all newly synthesized proteins require TRiC to fold
TRiC is a hetero-oligomeric chaperonins, which has eight homologous but distinct subunits in each of its two rings
With different monomers it allows more varied substrate binding to take place in the same place
Multisite substrate binding by TRiC that allows folding of its different substrates

Question 19

Describe small heat shock proteins (sHSPs)?

Answer 19

Under stress conditions substrate proteins are destabilized and begin to unfold
sHSPs bind these partially unfolded proteins in an energy independent manner and keep them in a folding-competent state
sHsps can be activated by several triggers such as substrate, stress situation (temperature, pH), post-translational modification (phosphorylation) or hetero-oligomerization

Bound substrates are subsequently refolded by the ATP-dependent Hsp70 chaperone system
They can prevent aggregation but not help folding directly

Question 20

Describe the structure of sHSPs?

Answer 20

Most sHSPs have a monomer molecular mass of 15-30 kDa
The evolutionarily conserved hallmark of the sHsp family is the a-crystallin domain (ACD)
It consists of 90 amino acids and adopts a compact β-sheet sandwich structure; isolated ACDs commonly form dimers
A striking feature of most sHsps is their ability to assemble into oligomers

Question 21

Describe the oligomerisation of sHSPs?

Answer 21

The majority of sHsps are found as large, often polydisperse, ensembles typically ranging from 12 to 32 or even more subunits

Under stress, the physiologic ensemble of sHsp oligomers are activated by a conformational shift to a higher content of smaller species (often dimers)
The unfolded substrate binds to and stabilizes by this activated ensemble of sHsps

Question 22

What can inefficient protein folding lead to?

Answer 22

Lack of chaperones = inefficient protein folding which can cause loss of function diseases e.g. Alzheimer’s, Huntington’s, Parkinson’s, cystic fibrosis and type 2 diabetes

But cancer cells require enhanced folding capacity (overexpression of Hsp70 and Hsp90 for example)

Question 23

What is proteostasis?

Answer 23

The protein quality control network maintains proper function of the cellular proteome or protein homeostasis

There are 3 points of control in proteostasis

1. Folding
2. Maintenance
3. Degradation and dis-aggregation

Question 24

What is involved in the chaperone action at the ribosome?

Answer 24

Ribosomal protein L23 is a specific chaperone binding site
Trigger Factor: a peptidyl-prolyl isomerase
NAC: Nascent chain associated complex => chaperones the nascent chain as it emerges from the ribosome
Hsp70/40 (DnaK in E.coli): also bind nascent chains

Question 25

What are the different role between molecular chaperones and chaperonins?

Answer 25

1. Molecular chaperones - bind & stabilize unfolded proteins (prevent misfolding)
2. Chaperonins - directly facilitate folding
   Both require energy from ATP hydrolysis

Hsp70 will fold nascent (new) polypeptide chains
Hsp70 normally targets kinases and transcription factors - allowing them to be kept in an activatable state
Hsp90 will then fold partially folded polypeptides from Hsp70

Question 26

Describe protein folding in the endoplasmic reticulum?

Answer 26

This is where proteins that need to be secreted are targeted
These proteins use co-translational synthesis and targeting/trafficking
The proteins are targeted due to a N-terminal signal sequence
This sequence gets cleaved during translocation into the ER membrane
They harbour many PTM = many different proteins

Chaperone BiP - binding immunoglobin protein, that is in the ER and helps folding of the protein

Question 27

What are some other chaperones that are involved in protein folding in ER?

Answer 27

PDI (protein disulphide isomerase) and ERp57 - formation of disulphide bonds
Hsc70 (yeast) or BiP (mammals) - transiently binds to prevent misfolding/aggregation
Peptidyl-prolyl isomerases - accelerate rotation about peptidyl-prolyl bonds (cis/trans)
This is often the rate limiting step in protein folding
Calnexin and calreticulin - bind to N-linked sugars with a single glucose residue, promotes folding and oligomerisation

Question 28

Describe dissaggregation and degradation pathways in the cell?

Answer 28

1. When proteins misfold in the cytosol:
   Chaperones that dis-aggregate, aggregated proteins (fibrils)
   Hsp70-Hsp104 (ClpB) bi-chaperone
   Hsp70-J protein - transfers aggregates to Hsp104
   Hsp104 - threading activity to refold aggregate
   Ubiquitin protein system (UPS) - a ubiquitin tail will tell the cell that the protein needs to be degraded by a proteasome
   Autophagy - the autophagosome merges with a lysosome - this activates proteases that can facilitates degradation of these proteins
2. In the ER
   ERAD - substrate recognition, retrotranslocation, polyubiquitination and proteosomal degradation in the cytosol
3. Compartmentalised degradation in
   The nucleus
   The mitochondria

Question 29

What are some mechanisms involved in compartmentalisation of degradation in the mitochondria?

Answer 29

1. Pim1: heptameric protease - cuts the protein
2. Further processing by two ATP dependent proteases in the inner membrane
3. Finally UPS: for retro-translocated misfolded substrates

Question 30

What is involved in compartmentalisation of degradation in the nucleus?

Answer 30

Contains its own 26S proteasome system
Some proteins are translocated into the nucleus for degradation as it offers an additional seclusion to shield from other cellular processes
Quality control through: SUMO and ubiquitination

Question 31

Describe maintenance of protein folding under stress?

Answer 31

Stress responses:
• External - heat, hypoxia, oxidative and osmotic
• Internal - protein synthesis/load e.g. Disease
There are stress response pathways that will up regulate some molecular chaperones in response
• The stress pathways have proteostasis detectors such as: a transcription factor in the cytosol or transmembrane protein in the ER lumen or ClPP-1 in the mitochondrial matrix
• Stress transducer - transduced to the nucleus where the transcription factor will transcribe and produce more molecular chaperones
They will then help/maintain homeostasis in the cell

Question 32

Describe the unfolded protein resonse (UPR) of the ER?

Answer 32

There are 3 branches of the UPR
ARF6, PERK and IRE1

IRE protein will misfold/oligomerisation in the ER and leading to the processing of XBP1 - a transcription factor
This heads into the nucleus to produce more molecular chaperones e.g. BiP