Read with caution!
This post was written during early stages of trying to understand a complex scientific problem, and we didn't get everything right. The original author no longer endorses the content of this post. It is being left online for historical reasons, but read at your own risk.
“Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases,” Neef et al., Nature Reviews Drug Discovery 10, 930-944 (Dec 2011)
Studies in cell culture, fruitfly, worm and mouse models of protein misfolding-based neurodegenerative diseases indicate that enhancing the protein-folding capacity of cells, via elevated expression of chaperone proteins, has therapeutic potential. Here, we review advances in strategies to harness the power of the natural cellular protein-folding machinery through pharmacological activation of heat shock transcription factor 1 — the master activator of chaperone protein gene expression — to treat neurodegenerative diseases.
“Modulation of heat shock transcription factor 1 as a therapeutic target for small molecule intervention in neurodegenerative disease,” Neef et al., PLoS Biology (2009)
Neurodegenerative diseases such as Huntington disease are devastating disorders with no therapeutic approaches to ameliorate the underlying protein misfolding defect inherent to poly-glutamine (polyQ) proteins. Given the mounting evidence that elevated levels of protein chaperones suppress polyQ protein misfolding, the master regulator of protein chaperone gene transcription, HSF1, is an attractive target for small molecule intervention. We describe a humanized yeast-based high-throughput screen to identify small molecule activators of human HSF1. This screen is insensitive to previously characterized activators of the heat shock response that have undesirable proteotoxic activity or that inhibit Hsp90, the central chaperone for cellular signaling and proliferation. A molecule identified in this screen, HSF1A, is structurally distinct from other characterized small molecule human HSF1 activators, activates HSF1 in mammalian and fly cells, elevates protein chaperone expression, ameliorates protein misfolding and cell death in polyQ-expressing neuronal precursor cells and protects against cytotoxicity in a fly model of polyQ-mediated neurodegeneration. In addition, we show that HSF1A interacts with components of the TRiC/CCT complex, suggesting a potentially novel regulatory role for this complex in modulating HSF1 activity. These studies describe a novel approach for the identification of new classes of pharmacological interventions for protein misfolding that underlies devastating neurodegenerative disease.
Given that HSF1 coordinately activates the expression of multiple protein chaperones and other cytoprotective genes –, HSF1 activation could be of potential therapeutic value in neurodegenerative diseases associated with a wide array of manifestations of protein misfolding.
Here, we demonstrate in cultured neuronal precursor cells, and in a fruit fly model of polyQ disease, that HSF1A can suppress the aggregation of a polyQ protein and cytotoxicity associated with polyQ expression. Interestingly, in both the cell culture and fly polyQ model, the concentration of HSF1A required to reduce cytotoxicity is significantly less than the concentration required for maximum Hsp70 expression. Similar results have been described for the Hsp90 inhibitor geldanamycin, which was able to rescue cell viability in a Drosophila model of Parkinson disease at concentrations significantly lower than that required to induce Hsp70 expression maximally ,. Together, these results suggest that relatively small yet chronic increases in protein chaperone expression are sufficient to significantly stabilize misfolded polyQ proteins and thereby reduce cytotoxicity. It is also possible that low levels of HSF1A reduce the threshold of HSF1 activation in response to the accumulation of unfolded proteins at physiological temperatures. This hypothesis is supported by our results demonstrating that HSF1A can reduce the temperature threshold required for HSF1 activation. Previous studies have shown that misfolded polyQ proteins are turned over by autophagy and ubiquitin-dependent proteosomal degradation and that protein chaperones can impinge on both processes ,. In addition, HSF1 and protein chaperones have been suggested to be potentiators of oncogenic transformation . Although we do not yet fully understand whether HSF1A-mediated protein chaperone expression affects these processes, these hypotheses are currently under investigation.
“Small molecule proteostasis regulators for protein conformational diseases,” Calamini et al., Nature Chemical Biology (2011)
Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of ∼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1–dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.
There is increasing evidence that misfolded proteins expressed in diseases of protein conformation are not efficiently counterbalanced by a compensatory induction of cellular stress responses such as the HSR11. Enhancing the activity of HSF-1 and the concentrations of molecular chaperones by genetic techniques or pharmacological manipulation has been shown to restore proteostasis in several models of disease12, 13, 14, 15, 16, 17, 18, 19, 20.
In this study we describe the results of a large-scale small-molecule screen in human cells for HSF-1–dependent activators of chaperone expression. We identified 263 proteostasis regulators that chemically induce the HSR and result in the activation of HSF-1 and the elevated expression of multiple chaperone gene families. The proteostasis regulators described here are previously unidentified chemical series and, compared to previously identified small-molecule activators of the HSR, do not cause protein misfolding, proteasome inhibition or Hsp90 inhibition. A more in-depth understanding of these proteostasis regulators and their ability to activate the HSR and restore protein folding in multiple disease models offers new opportunities and strategies for small-molecule chaperone therapeutics for protein conformational diseases with new specificities and reduced toxicity.
Our proteostasis regulator strategy is based on the idea that small molecules can mimic the molecular signals recognized by the cell that are associated with a proteostatic imbalance (Supplementary Scheme 1). This activation of stress-signaling pathways in turn restores the stability and functionality of the proteome. The ability of these proteostasis regulators to activate one or more stress-response pathways suggests a therapeutic approach that uses the cell’s biological response to damaged proteins to protect cells against chronic disease. Through this approach, we applied our growing understanding of stress biology to promote the health of the cell. In doing so, we used compounds to enhance the properties of biological pathways that are already employed by the cell to manage proteostasis, even when challenged by stress and disease. We suggest that this systems and network approach could be an alternative method for drug discovery as we harness the protective abilities of cellular stress responses to protect the cell against the multitude of deficiencies that occur during chronic proteotoxicity and stress.
The central role for HSF-1 in maintaining and restoring proteostasis makes this transcription factor and the HSR attractive targets for therapeutic intervention in conformational diseases. The observation that diverse chemical types have in common the ability to induce the HSR, despite their broad range of activities, supports our proposal that HSF-1 is a stress network hub that integrates multiple stress signaling pathways to coordinate regulatory responses to maintain proteostasis in health, aging and disease. Of the 263 hits identified in this study that activate HSF-1, we focused our attention on the seven major clusters represented by the chemical series: β-aryl-α,β-unsaturated-carbonyls (cluster A), β–nitrostyrenes (cluster B), β-Cl−α,β-unsaturated-carbonyls (cluster C), nitrobenzofurazans (cluster D), nitrofuranylamides (cluster E), unsaturated barbituric acids (cluster F) and 2-cyanopentadienamide (cluster G). These chemical series have a broad range of pharmacological indications and diverse mechanisms of action and, to our knowledge, have not been previously linked to proteostasis and the HSR. For example, compounds in cluster A are chalcone and curcumin analogs and have antibacterial, antioxidant and cancer chemopreventive activities38, 39. These compounds are known to inhibit NF-κB and modulate the Keap1-Nfr2 complex40, 41. Nitrobenzofurans (cluster E) have antitubercular activity, and nitrofuran antibiotics (nitrofurantoin) are currently used as second-line agents for urinary tract infections. The nitroimidazole antibiotics are structurally related; for example, metronidazole is a widely used antibiotic for the treatment of anaerobic bacterial and protozoan infections42. Compounds belonging to cluster F are barbiturate analogs associated with antiinflammatory side effects; in particular, thiobarbiturates reduce the activation of NF-κB. Thiopental, but not the oxy-analog pentobarbital, is the only barbiturate that has been suggested to activate the HSR, and this property has been attributed to the reactivity of thiopental with protein thiols43. Notably, barbiturate analogs have been previously reported as potentiators of defective ΔF508-CFTR channel gating44. Our results reveal unexplored mechanisms by which these chemical classes exert their beneficial effects and suggest new pathways that are involved in the activation of HSF-1. We propose that the ability of barbiturate analogs to rescue defective mutant ΔF508-CFTR channel gating can now be linked to the activation of the HSR, the UPR or both. Likewise, the neuroprotective effects attributed to curcumin45, a chalcone analog, may be caused by the induction of chaperone expression46. Taken together with the data presented here, we propose that compounds of the same chemical classes identified in our HTS can be reclassified as proteostasis regulators.
Considering that the pathogenesis of many diseases, such as Alzheimer’s disease, Parkinson’s disease, ALS and cystic fibrosis disorders, is also associated with oxidative stress, the activation of the ARE pathway in conjunction with the HSR may be highly beneficial in the treatment of these disorders. In support of this concept, we here show that the small-molecule proteostasis regulator F1, which simultaneously induced both stress-protective pathways, was the only proteostasis regulator that restored proteostasis in distinct cellular compartments.
In conclusion, we propose that the adjustment of the proteostasis network by small-molecule proteostasis regulators of the HSR provides a previously unexploited and potentially powerful approach to obtaining proteome balance in both loss-of-function and gain-of-function diseases by providing a corrective environment based on the principle of proteome balance that is superior to that found in the upregulation of one single pathway. In addition to their usefulness in potential therapeutic development, small-molecule HSR inducers can be used as pharmacological tools for further dissecting the multistep activation pathway of HSF-1. We believe that a better understanding of the regulation of the HSF-1 activation pathway and its signaling mechanisms could lead to the discovery of compounds with stress signatures that are HSF-1 selective or could activate multiple stress pathways that may be effective in the control of diseases of protein conformation.
“HspB5/alphaB-crystallin: properties and current progress in neuropathy,” Hu et al., Curr Neurovasc Res, Vol. 5 Iss. 2 p. 143 (2008)
HspB5/alphaB-crystallin (alphaBC), one of the most representative member of mammalian small heat shock protein family (sHsp), shares the common features with other nine members HspB1-B4 and HspB6-10. Meanwhile, it has a strong antiapoptic effect; its interaction with cytoskeleton participates in maintaining cell structure and plays an important role in cytoprotection. Recent studies reveal that HspB5 has a strong relationship with neurological diseases acting as a protective molecular chaperone or in certain conditions, a pathogenic factor. This review gives a brief introduction on properties of HspB5, its current progress in neurological diseases and potential therapeutic intervention in demyelinating disease, neurodegenerative disease, myopathy and cerebrovascular disease.
“The effects of trimethylamine n-oxide on the structural stability of prion protein,” Yang et al. in “Advanced Understanding of Neurodegenerative Diseases,” ed. Chang, InTech (2011)
TMAO is a well known protective osmolyte that can increase structural stability of proteins against chemical of temperature-induced denaturation (Baskakov et al. 1998; Bolen & Baskakov 2001; Celinski & Scholtz 2002; Gursky 1999; Qu et al. 1998). TMAO has an extraordinary ability to force thermodynamically unstable proteins to fold (Baskakov & Bolen 1998). In contrast, TMAO destabilizes proteins at a low pH (Singh et al. 2005).
At pH 6.9, 20 C, the a-helical conformation of MoPrP is unfolded by TMAO. When the concentration of TMAO is low at ≤ 1.0 M, the degree of unfolding is small and the fibril conversion takes place on the mostly folded a-helical structures. In contrast to low concentration of TMAO, high concentration of TMAO at ≥ 1.0 M largely unfolds the a-helix and this unfolding inhibits amyloid formation completely. In other words, low concentration of TMAO decelerates the prion nucleation represented by lag phase in nucleation-dependent polymerization model, whereas TMAO enhances the growth of prion fibrils. This contrast effect indicates that the structural requirement of prion proteins in nucleation and in fibril elongation is different. The importance of partial unfolding of prion in amyloid formation has been suggested (Apreti et al. 2004; Kuwata et al. 2002; Nicholson et al. 2002). The structure of partially folded prion protein that initiates amyloid formation remains unclear.
Taking together these findings, a certain amount of a-helical conformation seems to be required in the structural conversion of prion proteins in nucleation, and a hydrophobic interaction is essential in fibril elongation.
“Chemical chaperones assist intracellular folding to buffer mutational variations,” Bandyopadhyay et al., Nature Chemical Biology (2012)
Protein folding has been implicated in chaperone-mediated mutational buffering12. Here, using small-molecule modulators of in vivo protein folding, we demonstrate that modifiers of protein folding may generally buffer mutational variations in vivo. Furthermore, our work uncovers differential mutational buffering by different osmolytes.
We show that each of the chemical chaperones tested has a subset of mutations that it can buffer in vivo, possibly because of differences in the mechanism of action of the different osmolytes. Although TMAO (and possibly trehalose) may buffer mutations that destabilize the core substantially or lead to a drastic increase in the flexibility of nonnative conformations, other osmolytes such as proline or glycerol may buffer mutations that are on the protein surface. Thus, the complex milieu of physiological osmolytes in the cell may be associated with an unappreciated spectrum of genetic buffering. This source of canalization adds to the processes already known to be involved in imparting robustness to cellular phenotypes, including processes linked to protein folding.
We find that trehalose has a mechanism of folding assistance similar to that of TMAO. In light of evidence that certain pathogenic prokaryotes, such as Mycobacterium tuberculosis, accumulate large concentrations of the osmolyte trehalose41, we speculate that it may promote access to a larger amino acid sequence space, thereby increasing the probability of generating drug-resistant variations. Trehalose also extends the lifespan of nematodes and is more predominant in mutants that have extended lifespans42. As aging is linked to the collapse of proteostasis43, it will be interesting to find out whether trehalose decelerates aging by assisting protein folding. It is also important to note that in E. coli, chronic heat shock or chaperone depletion is able to decrease the cellular pool of trehalose; we posit that a small but considerable fraction of the mutants that have been reported to be heat sensitive could have been trehalose-sensitive mutants. Further work on the role of metabolites in protein folding and mutational buffering will be needed to understand whether metabolic shifts can alter both mutational buffering and proteostasis. Thus, understanding small molecule–mediated modulation of proteostatic potential, whether it occurs directly or through molecular chaperones44, 45, will be crucial for appreciating the principles of proteostasis-driven protein evolution as well as for therapeutic interventions in misfolding disorders.