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Protein Misfolding - Coggle Diagram

- - - - Folding pathways in vivo are influenced by ribosomes because they limit access to the polypeptide sequence and the NTD is available prior to the CTD
        
        Different stretches are also synthesised at different speeds, which can influence folding - non-optimal codons are synthesised slower (Kimchi-Sarfaty, 2007, Science)
      - Most have little substrate specificity and often recognise hydrophobic bundles of aa sidechains. Some function actively (hydrolysing ATP to drive conformational changes) or have a 'handover' role where they just temporarily protect some sidechains
      - Prokaryotes use DnaK/J and GroEL chaperones (latter for ~10% of cytosolic proteins) which sometimes are preceded by trigger factors binding
        
        GroES forms a cap over GroEL
        Proteins enter as a result of hydrophobic residues facing inwards. The complex turns inside out upon GroEL and ATP binding (exposing polar residues which encourage folding and protection of hydrophobic residues) and after ~10s the correctly folded protein is released
      - Eukaryotes have many more chaperones;
        NAC binds the polypeptide whilst bound to the ribosome
        At the ribosome exit Hsp70 and RAC are present
        Hsc70/Hsp40 bind when ~50aa are exposed
        TRiC mediates the folding of a small number of proteins which are trafficked to it by GIMC or Prefoldin
        
        Trigger factors bind the ribosome to form a cradle over the ribosome exit tunnel to provide a protective environment
        It can actively unfold misfolded structures using its tight-binding surface (Preissle & Deuerling, 2012)
        
        Hsp70 = most abundant chapeone and is an important target in infection, cancer, neurodegenerative diseases and immunity
        
        In the ADP-bound/nt-free states the lid locks the substrate into the binding cleft
        ATP binding creates a binding site for the linker which causes the lid to open and release the substrate
        
        Hsp90 is for late-stage protein folding and assembly
        
        Functions on 5-10% of the proteome and is activated by heat shock
        Promising drug target as its substrates are typically degraded when it is inhibited and such inhibitors have been shown to have efficacy on cancers (Workman, Clarke and Al-Lazikani, Oncotarget)
        
        Enables proteins to assemble with other proteins, with ligands and with cofactors
        
        ATP binding causes dimerisation at the N-terminus to form a closed ring to mediate the action on the substrates
- - - - p97 (AAA ATPase), which is involved in ERAD by promoting retranslocation of proteasome substrates across the ER membrane, has been targeted (Anderson, Cancer Cell, 2015)
    - - FL-CDB3 has shown efficacy in p53-mutant lung cacner cells (Issaeva, PNAS, 2003)
      - PRIMA-1 has shown efficacy in reactivating mutant p53 (Lambert, Cancer Cell, 2009)
  - - - ERT (doesn't work for CNS because can't pass BBB)
      - Substrate reduction therapy - only Zavesca is approved (inhibits the enzyme responsible for glycosphingolipid synthesis)
      - Pharmacological chaperones (won't work for mutations that cause gross alterations)
        
        Clinical candidates are available for Fabry, Gaucher and Pompe diseases and all drugs weakly bind the active site
        
        Migalastat was developed with rational drug design (Valenzano, Assay & Drug Dev Tech, 2011) and has been shown to only work in the latter stages of the disease (Benjamin, J Inherit Metab Dis, 2009) for certain mutations (covers 30-50% of the population)
        
        Migalastat could theoretically be used with ERT to stabilise the healthy protein (Amicus/slide 44 lec2 show this has some benefit)
        
        Pre-clin studies by Amicus show that migalastat + fabrazyme increases fabrazyme tissue uptake and reduces [GL-3] in the kidneys
      - Proteostasis regulators have shown proof-of-principle in LSD (Mu, Cell, 2008) - celastrol & MG-132
  - - - Amyloid fibrils may not be the toxic species (fibrils are stable) so it is thought that it is the precursors (which are poorly understood/studied) that are toxic
        
        Determining the fibril formation pathway small molecule inhibitors have since been developed
        
        There is a theory that all proteins can be induced to form amyloid fibrils under the right conditions (Singh, Sci Rep, 2014)
    - - Lysozyme amyloidosis results in kgs of amyloid in the liver, spleen and kidneys and cannot be fixed by organ transplant
      - Diabetes-related amyloidosis is produced as a result of chronic dialysis and is induced by Cu2+ ions stimulated beta2-microglobulin aggregation
    - - AD (Ab), PD (a-syn), Dementia with Lewy bodies (a-syn), Frontotemporal dementia with Parkinsonism (Tau) etc
      - Fibrils tend to be formed from natively disordered/unfolded proteins
      - AD is caused by improper processing of APP - beta-celavage occurs instead of alpha-cleavage (which produces P3 and AICD
      - Kuru disease (linked to cannibalism in PNG) is similar to 'Scrapie' disease in sheep and so experiments were done
        
        Injecting three chimps with Kuru brain tissue produced no effects in first two years and then in 1965 (2yrs after) they began to show all the Kuru symptoms)
        
        Both are 100% fatal (very uncommon) and cause no immune response with high incubation times
        
        Was thought that DNA/RNA was required because multiple 'strains' existed but EM suggested that if the infectious agent contained DNA it would have to be 1000x smaller than the smallest known virus at the time
        
        Hence it was theorised that it could be transmitted by proteins
        
        Prusiner (1982) did the key work here and isolated two forms of Prion Protein (C & Sc), the latter of which tracked with infectivity
        
        Prion/misfolded protein seems capable of inducing natively folded proteins to adopt the prion fold too, resulting in a cascade - these large fibrils can fragment and hence spread infection
        
        Was rejected as unprecedented as proteins were known to have only one functional fold
        Existence of strains went counter to understanding of proteins as it would have to mean that proteins can take on numerous folds
        There was also no proof - protein needed to be purified and then be able to infect another animal by itself
        
        In 1994 Wickner showed that yeast had evidence of prions URE2 and this was later confirmed by Colby (PNAS, 2009) in mammals
        
        Crystal images show that PrPC --> PrPSc involves a conformational change that increases beta-sheet content
        
        There are multiple similar disease in humans (and other animals) and are called transmissable spongifrom encephalopathies (TSEs)
      - One big question brought up by TSEs is why aren't all amyloids infectious? - Some people have said that they are (Abbott, Nature, 2016)
        
        Some murine experiments seem to back up this theory for AD (Eisele, 2010, Science)
    - - Large deposits unsurprisingly disrupt organ function
      - They are often sticky and may aggregate with other essential proteins such as chaperones
      - Excessive aggregation stimulates apoptosis through upregulation of caspases
      - They may be overloading the ER/autophagy-lysosome pathway
      - They may overactivate the UPR (unfolded protein response) pathway which shuts down protein synthesis through PERK
        
        Increased levels of p-PERK (active form) have been observed in AD, PD, prion disease and ALS
      - Oligomers may also be inducing toxicity as they often have increased hydrophobic surfaces, with spherical pore-like structures (Chen, 2015, PNAS)
        
        Some theories of how this works in AD are laid out in Kayed & Lasagna-Reves' review (J Alz Dis, 2013)
        
        May disrupt the cell membrane, stimulate receeptors that induce cell death (frizzled receptor, NGF receptor, NMDA receptor), intracellular uptake may induce pathways that result in cell death (APOE receptor-mediated entry)
        
        Bolognesi (ACS Chem Bio, 2010) showed eivdence that oligomers are more toxic than fibrils - sort of confirming the oligomer theory
        
        Cell-to-cell transmission is not well understood but may result from aggregates released by dying cells or exomes secreted by living cells - there seems to be a prion-like process going on though
        
        E22G 'arctic' mutation in Drosophila has been countered with F4D and I31E mutations (could be a route to a pathway if chaperones can target these mutations) (Luheshi, PLoS Bio, 2007)
        
        Kayed (2003) demonstrated that an antibody (A11) is capable of recognising the oligomeric state of Ab and various other amyloidoses-inducing oligomers
        
        Does this mean that there is a possibility for a one-size-fits-all therapy for amyloidoses diseases?
    - - Proteostasis regulators can be used (potentially as generic drugs) to increase the activity of chaperones or activate the UPS/autophagy pathways
      - If the protein has a folded native structure then pharmacological chaperones could be used (won't work for AD & PD though)
      - Transthyretin amyloidoses are caused by mutations in the thryoxine transporter transthyretin and are familial diseases which lead to organ dysfunction and death within 10 years of onset
        
        TTR tetramer dissociation into monomers is the RLD
        
        FAP, FAC (especially common in afro-americans), SSA & CNSA
        
        Early therapies used liver or liver & heart transplants but these didn't really work
        Or they focused on symptomatic relief
        
        Thyroxine was found to improve kinetic stability of TTR so a screen was done to repurpose a drug for TTR amyloidoses
        
        Diflusinal (NSAID) was found by TOjo's team in 2006
        
        Since then tafamidis has been developed and is much more effective than diflusinal and works for all mutants (V30M and V122I) by Bulawa's group in 2012 and as of 2019 is approved for use in US, Japan and EU for treatment of FAP
        
        Further analysis by Sant'Anna (Nature Comm, 2016) identified tolcapone as a candidate drug (was previously used in PD therapy) and is more effective than tafamidis in vitro
        
        Stabilises the dimer-dimer interface and has much lower Kd2, especially when comparing CARDIAC isoforms of TTR
        
        Has shown in vitro and in vivo (mice and humans) efficacy over tafamidis (and is ~140x cheaper)
        
        Has passed PhII clinical trials and has orphan drug status
        Also has significant hepatotoxicity, limiting its clinical usefulness
      - Late-onset neurodegenerative disease often suffer from having targets that are initially unfolded (hence chaperones are useless)
        
        Aducanumab has shown promise, as has BACE1 inhibitors (but none are in patients currently)
        
        Efforts have been made to target the amyloid-forming proteins directly (PLoS, 2014, 87133) and indirectly (Mecozzi, Nature Chem Bio, 2014; Moreno, Science Trasnl Med, 2013)
      - Serpinopathies have been treated with transplants, ERT, drugs to inhibit serping polmerisation and modulate proteostasis machinery
        
        Some have suggested using stem-cell based therapy (Lomas & Rashid, 2012, Stem Cell Res & Ther)
        
        Small drugs that bind the A-sheet surface prevent reactive-centre loop insertion which may help treat this
        
        Modulation of proteostasis machinery may be used (carbamazepine was identified to enhance autophagy and the proteasome in a cell model) (Maurice & Perlmutter, Clin Transl Sci, 2012)
    - - There is poor correlation however between circulating [b2-m] and the fibril load suggesting additional factors are present (further emphasised by b2-m fibrils being unable to be induced in vitro at very high concs)
      - Addition of Cu2+ ions seems to be stimulating it (Cu2+ free filter membrane patients have 50% reduced incidence of DRA compared to those with them) (Eichner & Radford, 2011)
      - Partial unfolding of b2-m exposes aggregation-prone sequences
- - - - UPS
        
        There must be a colavent attachment of Ubq and an unstructure/loosely structured region for engagement of the proteasome (Ciechanover & Stanhill, Biophys Acta, 2014)
        
        Proteasome activity decreases with ageing, leading to retention of accumulation of misfolded proteins (potentially why older people have more Ndegen diseases)
        This is the result of decreases [ATP], [Ubq] and [E2] as well as down regulation of proteasome subunits (Vernace, 2007)
        
        Targeting the UPS system could provide therapeutic benefit:
        E2:E3 interface
        E3:substrate interface
        Deubiquitinating enzymes
        Proteasome targeting
        Proteasome activity
        
        E3:S interface can either be targeted inhibiting catalytic activity of E3 or by disrupting the interaction (e.g. stabilising p53 by competitively inhibiting association with the active site of MDM2 with nutlin)
        Stapled alpha-helix peptide is a potent inhibitor of MDM2 and MDMX (Chang, 2013, PNAS) - this is the first example of an MDMX inhibiting drug and showed p53-dependent tumour growth inhibition
        
        Blocking delivery of polyubiquitinated substrates to the proteasome with ubistatins is another technique - suggests that the Ubq chain itself can be used as a target for pharmacological intervention in UPS
        
        Direct proteasome inhibition (e.g. bortezomib - chemo/radiosensitising) surprisingly show selective inhibition for cancer, thought to be due to the increased load on cancer cells
        Similar drugs have shown promise for drug resistance malaria too (Stokes, 2019, PLoS Pathog)
        
        PROTACs hijack this machinery to target misfolded proteins (GoF mutations) for degradation
        Novel because it doesn't have to bind a functional site just needs to act as a delivery mechanism and can be used at low []s
        
        Arvinas are the leaders in this field (Chopra, Nature Rev, 2019) and have hit the clinic for oncologic indication
        
        The linker length is found empirically
        
        Potentially the PROTACs will guide the target to a chaperone or the proteasome instead of an E3 ligase
        
        Limitation is that many therapeutic targets have no small molecule ligand and only six E3 ligases have them
        
        Polyproxins use peptides rather than small molecule targets (polyprox therapeutics) and should get round this issue
        
        There are natural precedents for polyproxin-like molecules such as Cks1 (Seeliger, 2003; Sitry, 2002) but the scaffold of such polyproxin is very important to put the epitopes in the correct conformation
        
        Ubiquitin is added through a three step process:
        E1 (ubiquitin activating enzyme) forms a covalent bond between its Cys and the C-terminus of Ubq
        E2 (ubiquitin carrier protein) binds E3 and Ubq
        E3 (ubiquitin ligase) catalyses transfer of Ubq from E2 onto the substrate and confers substrate specificity
        E4s promote poly-ubiquitination
        
        Substrate and E3 enzyme are unconventional, substrate binds very far from active site and E3 is very flexible to allow growth of substrate
  - - - Clinical examples areHs90inhibitors and proteasome inhibitors (anti-cancer therapies)
      - increase the levels of key molecular chaperones and can be generic across multiple diseases
    - - This can improve the amount of functional protein (CF) by shifting the equilbrium to favour correctly folded over unfolded
      - Clinical examples are migalastat for Fabry disease and tafamadis for transthyretin amyloidoses
      - increase stability/decrease degradation rate