Heat Shock Proteins

Question 1

BRAIN AMYLOIDOSES

Answer 1

Protein aggregates with typical amyloid structure are common among many different neurodegenerative disorders, collectively defined BRAIN AMYLOIDOSES (AD, PD, ALS, HD, polyQ diseases, etc)

Question 2

Amyloid structure & toxicity

Answer 2

A common feature of all forms of amyloid aggregates is the initial acquisition of a b-sheet secondary structure that may be responsible for shared mechanisms of toxicity

Question 3

Structure of amyloid aggregates over time

Answer 3

Natively unfolded can go into protofibril –> fibril or toxic pore
Or natively unfolded proteins can form alternative conformations (with increased/decreased toxicity depending on additional factors)

Question 4

Fibrillary structures

Answer 4

Fibrillary structures often display the properties of amyloid (~10 nm-wide fibrils
with crossed B-pleated sheet structures)

Question 5

Toxic pore structure

Answer 5

pores insert into membrane –> leakage of material causing toxicity

Question 6

Ideal protein folding and what dictates it

Answer 6

Primary structure (aa’s) dictate protein folding and favours the most energetically/thermodynamically stable conformation

BUT cellular enviro/crowding can alter this folding

Question 7

How the cellular enviro can alter folding

Answer 7

• the cytosol is packed with proteins and this enviro can lead partially folding proteins to misfold leading to misfolding and aggregation
• but chaperones can help direct proteins into certain conformations

Question 8

T/F only partially folded/unfolded proteins risk misfolding

Answer 8

FALSE

even proteins that are already folded risk unfolding–esp when under cellular stress

Question 9

Chaperone–roles

Answer 9

• can help with refolding or may push proteins to wither a disordered aggreagtes or amyloid fibrils
• can also help deliver misfolded proteins for degradation (proteasomal or autophagy)

Question 10

Chaperone–role brief

Answer 10

refolding AND removal of misfolded proteins (proteostasis)

Question 11

Molecular chaperones

Answer 11

enhance the efficiency of de novo protein

folding and promote the refolding of misfolded proteins

Question 12

Chaperones in healthy cells

Answer 12

In healthy cells, misfolded proteins are refolded correctly through the intervention of molecular chaperones or are degraded by the proteosomal system or by autophagy

Question 13

Chaperones as we age

Answer 13

With aging, the induction and/or activity of molecular chaperones and the function of the proteosomal system decrease, favoring accumulation of toxic misfolded proteins (impaired proteostasis).
This may account for the late onset of neurodegenerative diseases that are linked to protein aggregation.

Question 14

Molecular chaperones have roles in

Answer 14

• proteosomal degradation
• macroautophagy
• non-conventional secretion
  = removal of misfolded proteins to decrease toxic burden

And folding and refolding

Question 15

Monomers misfolding

Answer 15

While acquiring a misfolded conformation is often thermodynamically favourable the conversion back may require more energy to occur
- molecular chaperones can intervene and help return the protein to its native conformation

Question 16

Molecular chaperones and direction of monomer folding

Answer 16

by stabilizing a native or misfolded monomeric conformation chaperones, incl. HSP 40/70, might prevent the intramolecular transition that gives rise to spherical and annular oligomers WHILE simultaneously stabilizing those that lead to inclusion body formation (amorphous aggregates and fibrils) –> likely less toxic

Question 17

heat shock protein HSP) families

Answer 17

Five main families of ATP-binding, heat shock proteins (HSP):
HSP100, HSP90, HSP70, HSP40 and small HSPs (sHSPs)

Question 18

HSP90

Answer 18

Cytosolic dimeric chaperone that stabilizes misfolded proteins and regulates the activity of various signalling proteins:

• steroid hormone receptors
• tyrosine kinases
• nitric oxide synthase
• calcineurin, etc.

Question 19

HSP70

Answer 19

assists in the stabilization and folding of many substrates and is found in most cellular compartments.

Question 20

HSP40

Answer 20

co-chaperones that bind misfolded proteins and target them to HSP70.
In complex with HSP70, HSP40 proteins stimulate ATP hydrolysis, resulting in a conformational switch that facilitates the folding of non-native protein substrates.

Question 21

HSP40 and 70

Answer 21

misfolded proteins first bind to HSP40 then are delivered to HSP70
binding of ATP to HSP40 and subsequent ATP hydrolysis causes a conformational change allowing refolding of the non-native protein substrate

Question 22

HSP90 Cycle process (6 steps)

Answer 22

1) Hop binds to HSP90 dimer
2) Hop recruits ‘Hsp40/70-client complex’
3) ATP binds to HSP90 + changes HSP90 conformation to ‘closed’
4) p23 binds HSP90 in ‘closed’ confirmation and promates client protein activation (i.e. changes folding of client)
5) activated client released with ATP hydrolysis
6) ADP released and HSP90 reverts to ‘open’ conformation

Question 23

How does a protein get refolded vs degradated

Answer 23

Depending on associated proteins and chaperones the proteins that bind (i.e. the client) can either be:

• stabilized and refolded
• directed to proteosomal degradation

Question 24

HSF 1

Answer 24

Transcriptional activator that controls the cellular response to cytoplasmic proteotoxic stimuli (elevated temperature, oxidative stress, viral infections etc.)

HSF1 controls the expression of chaperone proteins in response to cellular stress.

Question 25

HSF-1 response is ____ due to

Answer 25

Transient; negative feedback from HSP40/70 - Even under conditions of chronic proteotoxic stress, HSF1-dependent transcription of chaperone proteins is transient, because HSF1 is negatively regulated via a feedback loop by HSP70 and HSP40 binding

Question 26

HSF-1 under normal conditions

Answer 26

some extra HSP40/70/90 is present in the cytosol as inactive complex with HSF-1 - always some HSPs that are actively folding BUT excess is sequestered into these complexes

Question 27

HSF-1 in cellular stress conditions

Answer 27

When exposed to stress/increased conc of misfolded proteins need more HSPs to deal --> HSPs are removed from complex with HSF-1 --> HSPs go help fold, HSF-1 trimerziess and enters the nucleus --> increased transcription of HSP genes --> more HSP40/70 etc. to help cope with stress

Question 28

Negative feedback of HSF-1

Answer 28

Following increased transcription of HSP genes and increased HSP production --> elevated cytosolic HSPs will lead to the reformation of inactive complexes with HSF-1 leading to negative feedback onto HSP40/70/90

Question 29

HSPs in neurodegeneration: AD

Answer 29

- HSP70 overexpression decreases aggregation of hyperphosphorylated tau in tangles - Inhibition of HSP70 expression has the opposite effect

Question 30

HSPs in neurodegeneration: PD

Answer 30

HSP70 promotes degradation of α-synuclein

Question 31

HSPs in neurodegeneration: ALS

Answer 31

HSP70 decreases toxicity of mutant SOD1

Question 32

HSPs in neurodegeneration: HD

Answer 32

Overexpression of HSP40 and HSP70 in HD models decreases mutant Htt toxicity.

Question 33

HSPs in neurodegeneration: other HSPs

Answer 33

HSPs other than HSP40/70 have shown protective effects in a variety of protein-misfolding diseases

Question 34

Geldanamycin: what is it

Answer 34

- Geldanamycin (GM) is a natural product originally developed as antifungal agent. - an anticancer drug with neuroprotective action - Initial studies demonstrated antitumor effects of GM, but liver toxicity

Question 35

17-AAG

Answer 35

a compound with similar biological activity to Geldanamycin, but decreased toxicity GM analogue w/o the toxicity

Question 36

Geldanamycin/17-AAG: mechanism

Answer 36

inserts into the ATP binding pocket of HSP90 --> blocks the HSP90 cycle --> leads to proteosomal degradation of client protein = anti-tumoral if client was a cancer-protein = anti-NDD if the client was mutant tau etc.

Question 37

Why inhibit HSP90: stabilized proteins

Answer 37

- HSP-90 is involved in stabilization of many misfolded protein conformations incl. tau, alpha-syn, PINK1 --> promotes disease progression in NDDS - therefore blocking HSP90 blocks stabilization of these and promotes their degradation --> decreases cellular stress

Question 38

Why inhibit HSP90: chaperone production

Answer 38

inhibition of HSP-90 results in the release of HSF-1 from the complex with inactive chaperones (likely due to increased recruitment of these chaperones following HSP90 inhibition) once released HSF1 trimerizes, enters the nucleus and increases chaperone transcription

Question 39

Benefits of inhibiting HSP90

Answer 39

- prevent stabilization of pathological clients and therefore promote their degradation - activation of HSF1 and the production of more chaperones (which promotes proper folding, refolding or degradation of pathological protein conformations)

Question 40

Geldanamycin in transgenic flies

Answer 40

Geldanamycin treatment prevents dopaminergic cell loss in α-synuclein transgenic flies due to upregulation of HSP70

Question 41

Clinical use of HSP90 inhibitors: 17AAG

Answer 41

17-AAG and other synthetic inhibitors of HSP90 are in clinical trials (Phase I/II) for the treatment of various types of cancers. No clinical trials in neurodegenerative conditions

Question 42

Barriers to 17-AAG use in NDDS

Answer 42

- 17-AAG is a substrate of P-glycoprotein and its passage through the BBB might be a problem (can't accumulate in the brain --> not effective for neurological treatments) - Also shows some liver toxicity, but not with the use of synthetic inhibitors

Question 43

SNX-0723: what is it

Answer 43

- a synthetic inhibitor of HSP90 - a benzamide derivative and its analogues are a novel class of synthetic small molecule HSP90 inhibitors that emerged from the screening of compounds that selectively bind to the ATP pocket of HSP90

Question 44

Synthetic inhibitors of HSP90

Answer 44

13 are in clinical trials for cancer | treatment

Question 45

SNX-0723: in PD

Answer 45

- SNX0723 increases HSP70 levels and decreases alpha-synuclein oligomerization and toxicity in vitro

Question 46

SNX-0723 vs. 17-AAG

Answer 46

- SNX-0723 is more potent than 17-AAG | - can cross the BBB (unlike 17AAG) and therefore is a potential drug candidate for PD

Question 47

Drugs that increas HSF1 activity

Answer 47

17AAG, SNX-0723 Riluzole NSAIDs Arimoclomol

Question 48

Riluzole

Answer 48

Increases the steady-state levels of HSF1, by inhibiting its degradation. Riluzole is better known as glutamate modulator (see lecture on excitotoxicity).

Question 49

NSAIDs

Answer 49

High concentrations of non-steroidal anti-inflammatory drugs such as sodium salicylate and indomethacin synergize with cellular stress and increase binding of HSF1 to DNA during cellular stress.

Question 50

Arimoclomol (BRX-220)

Answer 50

- Hydroxylamine derivative that prolongs stress-induced activation of HSF1 by binding to the active form of HSF1 and stabilizing it. - acts as a “smart drug” as it acts increasing the heat shock response only in cells that are already under stress.

Question 51

Arimoclomol: clinical uses

Answer 51

Arimoclomol is in Phase III clinical trial in patients with SOD1-positive ALS. SOD1 + placebo = decreased motor neurons SOD1 + Arimoclomol = increased motor neurons, mice survived longer