5 - Molecular Chaperones and Protein Quality Control

Question 1

By what mechanisms do molecular chaperones act on proteins?

Answer 1

by stabilising them, by fully unfolding them for translocation across membranes or for degradation, or by assisting in their correct folding and assembly. Chaperones such as the trigger factor stabilise proteins as they exit the ribosome.

Question 2

Why are some chaperones used to bind to unfolded protein chains?

Answer 2

Unfolded protein chains are otherwise vulnerable to proteolysis.

Question 3

What happens to chaperones once a protein is folded?

Answer 3

The chaperones do not interact with native, folded, proteins nor form any part of the final structure.

Question 4

How does the specificity of chaperones vary?

Answer 4

Some chaperones are non-specific, and interact with a wide variety of proteins, while others are more specific.

Question 5

These are the dumbest cue cards in this deck aren’t they?

Answer 5

Yes, but its kinda difficult to make cue cards on this topic so it isn’t totally your fault.

Question 6

Why might chaperones have ATPase activity?

Answer 6

Some molecular chaperones have catalytic activity that may induce conformation change or catalyse structural changes such as proline cis-trans isomerisation, reduction or formation of disulphide bridges or post-translational modification. As a result of this, chaperones often couple ATP binding and hydrolysis to their action

Question 7

How do chaperones act on misfolded proteins?

Answer 7

Chaperones not only prevent mis-folding but also act as ‘rescuers’ for already misfolded or aggregated proteins, allowing them to dissociate and refold.

Question 8

What are PPIs?

Answer 8

The deity of the cold-caller cult.

Peptidyl Prolyl Isomerases (PPIs) are the catalytic chaperones involved in reversible proline cis-trans isomerisation. This is often a rate limiting step in protein folding so PPIs are used to accelerate the process.

Question 9

What are PDIs?

Answer 9

Protein Disulphide Isomerases (PDIs) play a similar role by catalysing the correct formation of disulphide bonds and shuffling the existing ones within a protein until they are in the correct and most stable conformation. They are localised to the endoplasmic reticulum.

Question 10

What are heat shock proteins?

Answer 10

These make up the majority of chaperones, and are expressed as a response to cellular stress. Examples include Hsp60, 70, 90, 100 and small Hsps including homologues of lens α-crystallin.

Question 11

What does nucleoplasmin do?

Answer 11

It is involved in nucleosome assembly.

Question 12

What are prosequences?

Answer 12

Intramolecular chaperones such as Subtilisin or α-lytic protease. In contrast to the action of molecular chaperones, pro-sequences appear to catalyse the protein-folding reaction directly.

Question 13

What are the chaperone families?

Answer 13

• Small Heat Shock Proteins
• Hsp60 System
• Hsp70 System
• Hsp90
• Hsp100/clp
• Calnexin/Calreticulin
• Folding Catalysts
• Prosequences

Question 14

What does the small heat shock family do?

Answer 14

These act as sponges for misfolded proteins, preventing aggregation and protecting against cellular stress.

Examples include Hsp25 and α-crystallin, which prevents aggregation in the eyes (cataracts)

Question 15

What do the Hsp60 system family chaperones do?

Answer 15

These are ATPases that unfold the protein to enable it to re-fold correctly.

Examples include GroEL and cpn60

Question 16

What do the Hsp70 system family chaperones do?

Answer 16

Like Hsp60 family chaperones, these are ATPases that unfold the protein to enable it to re-fold correctly. However these also stabilise extended protein chains and are involved in membrane translocation, as well as being part of the heat shock response.

Examples include DnaK and BiP

Question 17

What do the Hsp90 family chaperones do?

Answer 17

These are ‘holding chaperones that bind and stabilise proteins, particularly steroid receptors and protein kinases.

These proteins are thought to act as a buffer for genetic variation.

Question 18

What do Hsp100/clp family chaperones do?

Answer 18

These are ATPase unfolding chaperones that provide thermotolerance and are involved in proteolysis and resolubilisation of aggregates.

Question 19

What do Calnexin/Calreticulin family chaperones do?

Answer 19

These are involved in quality control and glycoprotein maturation in the ER.

Question 20

What do folding catalyst family chaperones do?

Answer 20

This family contains PDI and PPI, which actively isomerise the structure of the protein.

Question 21

What do prosequence family chaperones do?

Answer 21

These directly catalyse the folding of proteins, particularly proteases.

eg Subtilisin, α-lytic protease

Question 22

How vulnerable are misfolded proteins to proteolysis?

Answer 22

Very vulnerable, unless they form aggregates or are deposited in aggresomes at which point they become more immune to proteolysis.

Question 23

What is the general structure of a Hsp70 family protein?

Answer 23

An ATPase domain is linked to a substrate binding domain. The substrate binding domain has a region which performs the chaperone binding, which is covered by a lid region.

ATP hydrolysis by the ATPase domain favours lid opening and subsequent domain docking with the target, as it stimulates a conformation change which allows the ATPase domain to bind to both regions of the binding domain on different faces.

Question 24

Which Hsp70 chaperones are found in the prokaryotic cytosol and what is their role?

Answer 24

DnaK and its cofactors DnaJ and GrpE.

Stabilises newly synthesised polypeptides and preserves folding competence;
reactivates heat-denatured proteins; controls heat-shock response

Question 25

Which Hsp70 chaperones are found in the yeast and mammalian cytosol and what is their role?

Answer 25

SSA1, SSB1 (yeast), Hsc/hsp70, hsp40 (mammalian)

Protein transport across organelle membranes,
binds nascent polypeptides,
dissociates clathrin from coated vesicles,
promotes lysosomal degradation of cytosolic proteins

Question 26

Which Hsp70 chaperones are found in the ER and what is their role?

Answer 26

KAR2, BiP/Grp78

Protein translocation into the ER.

Question 27

Which Hsp70 chaperones are found in the mitochondria and chloroplasts and what is their role?

Answer 27

SSC1, ctHsp70

Protein translocation into mitochondria.
Insertion of light-harvesting complex into thylakoid membrane.

Question 28

What is Hsp90 and what is its role?

Answer 28

Hsp90 is an inherent ATPase that exists as a dimer, as well as employing accessory proteins known as cochaperones to regulate the protein folding by conformation optimisation. It is also linked to proteasome degradation.

Question 29

What is the structure of Hsp90?

Answer 29

Each Hsp90 monomer is composed of three domains; the C-terminal domain is responsible for dimerisation, the middle domain for substrate binding and the N-terminal domain for ATP binding/hydrolysis. There are unstructured linkers between each of the domains that give the protein great flexibility

Question 30

What is the Hsp90 cycle?

Answer 30

When ATP is not bound at the N-terminal domain the chaperone is linked only at the C-terminus, leading to a very open and flexible structure.

Hsp90 can dimerise at both ends when ATP is bound. This allows it to bind its substrate.

ATP hydrolysis leaves an ADP bound and when the substrate dissociates this leaves a state more compact than unbound but not dimerised at the N-terminal domain.

Question 31

What are AAA proteins?

Answer 31

A large and diverse family of ATPases associated with
unfolding, unwinding, assembly and disassembly of protein and nucleic acid complexes (ATPases Associated with various cellular Activities)

Worst. Name. Ever.

Question 32

Provide some examples of AAA proteins?

Answer 32

Hsp100/Clp family of chaperones/proteases,
components in DNA replication, recombination and restriction,
the NSF protein in vesicle fusion,
dynein motor proteins

Question 33

What is the mechanism of Hsp100 chaperones?

Answer 33

Hsp100 proteins can totally unfold their substrate
proteins in order to deliver them to associated proteases.

They can also dissolve large aggregates in cooperation withthe Hsp70 system

Question 34

What is the structure of the Hsp100 chaperone-protease complex?

Answer 34

A tall, hollow cylinder comprising two protease HsIU ATPase domains with a HsIV protease domain sandwiched in between.

The ATPases feed the unfolded protein chain into the proease region.

Question 35

How are chaperonins different from chaperones?

Answer 35

Chaperonins assist folding without imparting steric information.

There are two groups of chaperonins, group I and group II.

Question 36

What are Group I chaperonins?

Answer 36

Group I are found in eubacteria, mitochondria and chloroplasts. They are very abundant and non-specific, and consequently will interact with most non-native proteins. It includes GroEL/Hsp60 (14 x 60 kD subunits) and GroES/Hsp10 (7 x 10kD subunits).

Question 37

What is the structure of Group II chaperonins and what were are they found?

Answer 37

Group II chaperonins have 16 or 18 x 55kD subunits.

TF55 and thermosomes are found in archaea.

TCP-1 is found in eukaryotic cytosol, with over 8 related gene products. This is not very abundant and folds actin, tubulin, transducin and WD-40 domain proteins.

Question 38

What do small Hsps bind and how?

Answer 38

They bind and stabilise denatured protein under conditions of cellular stress, ageing and degenerative disease. However, they don’t appear to have unfolding and refolding activity.

Small heat shock proteins have a high capacity for protein binding, up to 2 denatured proteins per subunit.

Question 39

What is the structure of small Hsps?

Answer 39

In most smHSPs, a dimer of β-sandwich folds held together by edge strand exchange forms the conserved building block.

However, this is different in α-crystallin. Crystal and solid state NMR structures of α-crystallin show a curved dimer without strand exchange.

Question 40

How many classes of chaperones are there - and what on earth do they do?

Answer 40

Class I - bind to nascent chains as they emerge from the ribosome and stabilise the elongating chain, e.g. Trigger Factor
Class II - otherwise known as chaperonins, provide a favourable environment for proteins to fold within e.g. GroEL/GroES