Heat Shock Proteins Flashcards
BRAIN AMYLOIDOSES
Protein aggregates with typical amyloid structure are common among many different neurodegenerative disorders, collectively defined BRAIN AMYLOIDOSES (AD, PD, ALS, HD, polyQ diseases, etc)
Amyloid structure & toxicity
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
Structure of amyloid aggregates over time
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)
Fibrillary structures
Fibrillary structures often display the properties of amyloid (~10 nm-wide fibrils
with crossed B-pleated sheet structures)
Toxic pore structure
pores insert into membrane –> leakage of material causing toxicity
Ideal protein folding and what dictates it
Primary structure (aa’s) dictate protein folding and favours the most energetically/thermodynamically stable conformation
BUT cellular enviro/crowding can alter this folding
How the cellular enviro can alter folding
- 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
T/F only partially folded/unfolded proteins risk misfolding
FALSE
even proteins that are already folded risk unfolding–esp when under cellular stress
Chaperone–roles
- 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)
Chaperone–role brief
refolding AND removal of misfolded proteins (proteostasis)
Molecular chaperones
enhance the efficiency of de novo protein
folding and promote the refolding of misfolded proteins
Chaperones in healthy cells
In healthy cells, misfolded proteins are refolded correctly through the intervention of molecular chaperones or are degraded by the proteosomal system or by autophagy
Chaperones as we age
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.
Molecular chaperones have roles in
- proteosomal degradation
- macroautophagy
- non-conventional secretion
= removal of misfolded proteins to decrease toxic burden
And folding and refolding
Monomers misfolding
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
Molecular chaperones and direction of monomer folding
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
heat shock protein HSP) families
Five main families of ATP-binding, heat shock proteins (HSP):
HSP100, HSP90, HSP70, HSP40 and small HSPs (sHSPs)
HSP90
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.
HSP70
assists in the stabilization and folding of many substrates and is found in most cellular compartments.
HSP40
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.
HSP40 and 70
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
HSP90 Cycle process (6 steps)
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
How does a protein get refolded vs degradated
Depending on associated proteins and chaperones the proteins that bind (i.e. the client) can either be:
- stabilized and refolded
- directed to proteosomal degradation
HSF 1
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.
HSF-1 response is ____ due to
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
HSF-1 under normal conditions
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
HSF-1 in cellular stress conditions
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
Negative feedback of HSF-1
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
HSPs in neurodegeneration: AD
- HSP70 overexpression decreases aggregation of hyperphosphorylated tau in tangles
- Inhibition of HSP70 expression has the opposite effect
HSPs in neurodegeneration: PD
HSP70 promotes degradation of α-synuclein
HSPs in neurodegeneration: ALS
HSP70 decreases toxicity of mutant SOD1
HSPs in neurodegeneration: HD
Overexpression of HSP40 and HSP70 in HD models decreases mutant Htt toxicity.
HSPs in neurodegeneration: other HSPs
HSPs other than HSP40/70 have shown protective effects in a variety of
protein-misfolding diseases
Geldanamycin: what is it
- 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
17-AAG
a compound with similar biological
activity to Geldanamycin, but decreased toxicity
GM analogue w/o the toxicity
Geldanamycin/17-AAG: mechanism
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.
Why inhibit HSP90: stabilized proteins
- 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
Why inhibit HSP90: chaperone production
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
Benefits of inhibiting HSP90
- 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)
Geldanamycin in transgenic flies
Geldanamycin treatment prevents dopaminergic cell loss in α-synuclein transgenic flies
due to upregulation of HSP70
Clinical use of HSP90 inhibitors: 17AAG
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
Barriers to 17-AAG use in NDDS
- 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
SNX-0723: what is it
- 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
Synthetic inhibitors of HSP90
13 are in clinical trials for cancer
treatment
SNX-0723: in PD
- SNX0723 increases HSP70 levels and decreases alpha-synuclein oligomerization and toxicity in vitro
SNX-0723 vs. 17-AAG
- SNX-0723 is more potent than 17-AAG
- can cross the BBB (unlike 17AAG) and therefore is a potential drug candidate for PD
Drugs that increas HSF1 activity
17AAG, SNX-0723
Riluzole
NSAIDs
Arimoclomol
Riluzole
Increases the steady-state levels of HSF1, by inhibiting its degradation.
Riluzole is better known as glutamate modulator (see lecture on excitotoxicity).
NSAIDs
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.
Arimoclomol (BRX-220)
- 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.
Arimoclomol: clinical uses
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
