Chaperone Machine Research Articles

Role and mechanism of the Hsp70 molecular chaperone machines in bacterial pathogens.

Heat shock proteins are highly conserved, stress-inducible, ubiquitous proteins that maintain homeostasis in both eukaryotes and prokaryotes. Hsp70 proteins belong to the heat shock protein family and enhance bacterial survival in hostile environments. Hsp70, known as DnaK in prokaryotes, supports numerous processes such as the assembly and disassembly of protein complexes, the refolding of misfolded and clustered proteins, membrane translocation and the regulation of regulatory proteins. The chaperone-based activity of Hsp70 depends on dynamic interactions between its two domains, known as the ATPase domain and the substrate-binding domain. It also depends on interactions between these domains and other co-chaperone molecules such as the Hsp40 protein family member DnaJ and nucleotide exchange factors. DnaJ is the primary chaperone that interacts with nascent polypeptide chains and functions to prevent their premature release from the ribosome and misfolding before it is targeted by DnaK. Adhesion of bacteria to host cells is mediated by both host and bacterial Hsp70. Following infection of the host, bacterial Hsp70 (DnaK) is in a position to initiate bacterial survival processes and trigger an immune response by the host. Any mutations in the dnaK gene have been shown to decrease the viability of bacteria inside the host. This review will give insights into the structure and mechanism of Hsp70 and its role in regulating the protein activity that contributes to pathogenesis.

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo. Using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproducts production and flux rearrangements, we demonstrate that DnaK significantly impacts the hierarchical order of carbon sources utilization, the excretion of main coproducts and the distribution of metabolic fluxes, thus revealing a multilevel interaction of DnaK with the central metabolism.

The diverse roles of Hsp90 and where to find them.

The Eighth International Conference on the Hsp90 Chaperone Machine took place in November 2016 at the Seeon Abbey in Germany. This year's program focused on a variety of topics, reflecting Hsp90's diverse roles in cellular and physiological function. The highlights included structural insights into the Hsp90 folding mechanism and conformational dynamics, post-translational modifications, client protein maturation, Hsp90 cochaperone function and Hsp90's role in disease physiology.

HOP expression is regulated by p53 and RAS and characteristic of a cancer gene signature.

The Hsp70/Hsp90 organising protein (HOP) is a co-chaperone essential for client protein transfer from Hsp70 to Hsp90 within the Hsp90 chaperone machine. Although HOP is upregulated in various cancers, there is limited information from in vitro studies on how HOP expression is regulated in cancer. The main objective of this study was to identify the HOP promoter and investigate its activity in cancerous cells. Bioinformatic analysis of the -2500 to +16bp region of the HOP gene identified a large CpG island and a range of putative cis-elements. Many of the cis-elements were potentially bound by transcription factors which are activated by oncogenic pathways. Luciferase reporter assays demonstrated that the upstream region of the HOP gene contains an active promoter in vitro. Truncation of this region suggested that the core HOP promoter region was -855 to +16bp. HOP promoter activity was highest in Hs578T, HEK293T and SV40- transformed MEF1 cell lines which expressed mutant or inactive p53. In a mutant p53 background, expression of wild-type p53 led to a reduction in promoter activity, while inhibition of wild-type p53 in HeLa cells increased HOP promoter activity. Additionally, in Hs578T and HEK293T cell lines containing inactive p53, expression of HRAS increased HOP promoter activity. However, HRAS activation of the HOP promoter was inhibited by p53 overexpression. These findings suggest for the first time that HOP expression in cancer may be regulated by both RAS activation and p53 inhibition. Taken together, these data suggest that HOP may be part of the cancer gene signature induced by a combination of mutant p53 and mutated RAS that is associated with cellular transformation.

The HSP70 chaperone machines of Chlamydomonas are induced by cold stress

The responses of Chlamydomonas reinhardtii cells to low temperatures have not been extensively studied compared with other stresses. Like other organisms, this green alga has heat shock protein 70s (HSP70s) that are located in chloroplast, mitochondrion and cytosol. To test whether temperature downshifts affected HSP70s synthesis, we used real-time PCR and protein gel blot analysis. C. reinhardtii cells exposed to cold stress show increased HSP70s mRNA levels. Genes encoding other components of HSP70 chaperone machines (e.g. CGE1, CDJ1, HSP90C and HSP90A) are also up-regulated in response to decreased temperature. We demonstrated that the accumulation of all analyzed mRNA occur more slowly and with reduced amplitude in cells exposed to cold than in cells treated with heat. Furthermore, C. reinhardtii cells display the splicing of the CGE1 transcript that was dependent on low temperature. Finally, the transcription regulator of C. reinhardtii HSF1 is also cold-responsive, suggesting its role in the transcriptional regulation of HSP genes at low temperature.

A Remodeled Hsp90 Molecular Chaperone Ensemble with the Novel Cochaperone Aarsd1 Is Required for Muscle Differentiation.

Hsp90 is the ATP-consuming core component of a very abundant molecular chaperone machine that handles a substantial portion of the cytosolic proteome. Rather than one machine, it is in fact an ensemble of molecular machines, since most mammalian cells express two cytosolic isoforms of Hsp90 and a subset of up to 40 to 50 cochaperones and regulate their interactions and functions by a variety of posttranslational modifications. We demonstrate that the Hsp90 ensemble is fundamentally remodeled during muscle differentiation and that this remodeling is not just a consequence of muscle differentiation but possibly one of the drivers to accompany and to match the vast proteomic changes associated with this process. As myoblasts differentiate into myotubes, Hsp90α disappears and only Hsp90β remains, which is the only isoform capable of interacting with the novel muscle-specific Hsp90 cochaperone Aarsd1L. Artificially maintaining Hsp90α or knocking down Aarsd1L expression interferes with the differentiation of C2C12 myotubes. During muscle differentiation, Aarsd1L replaces the more ubiquitous cochaperone p23 and in doing so dampens the activity of the glucocorticoid receptor, one of the Hsp90 clients relevant to muscle functions. This cochaperone switch protects muscle cells against the inhibitory effects of glucocorticoids and may contribute to preventing muscle wasting induced by excess glucocorticoids.

Modeling the UPR in neurodegenaration

Event Abstract Back to Event Modeling the UPR in neurodegenaration Georgia Theocharopoulou1* and Catherine Bobori1* 1 Ionian University, Informatics, Greece Neurodegenerative diseases are characterized by problems in protein synthesis causing misfolded proteins. Unfolded protein response (UPR), a cellular stress response, has a role in the accumulation of unfolded or misfolded proteins, using four basic functions. In Huntington’s Disease it has been observed that there is an amount of misfolded huntingtin in the cytoplasm, as well as reduced amount of molecular chaperones leading to the reduction of their role. Molecular chaperones, are proteins, which act in a dynamic manner in cases of stress or misfolding, and are involved in the quality control system of the cell. Age –related changes in the mechanisms of chaperones action, lead to the eventual failure of protein defense system. Deciphering protein-protein interactions will help to the identification of specific causal pathways in protein aggregation and better understanding of the unfolded protein response. This work will use neuroinformatics’ methods in order to design in vitro techniques that will examine the density of chaperones in HD cells, as well as the effectiveness of the UPR. The elucidation of the complex response of UPR will help to test new therapeutic strategies against neurodegenerative diseases. References Saibil, Helen. "Chaperone machines for protein folding, unfolding and disaggregation." Nature reviews Molecular cell biology 14.10 (2013): 630-642. Kalathur, Ravi Kiran Reddy, et al. "The unfolded protein response and its potential role in Huntington ́s disease elucidated by a systems biology approach." F1000Research 4 (2015). Mittal, Jeetain, and Robert B. Best. "Dependence of protein folding stability and dynamics on the density and composition of macromolecular crowders." Biophysical journal 98.2 (2010): 315-320. Kumaraswamy, Priyadharshini, Swaminathan Sethuraman, and Uma Maheswari Krishnan. "Designing In Vitro Strategies and In Vivo Models for Alzheimer's Disease-Promises & Challenges." Current Alzheimer Research 11.6 (2014): 588-607. Braakman, Ineke, and Daniel N. Hebert. "Protein folding in the endoplasmic reticulum." Cold Spring Harbor perspectives in biology 5.5 (2013): a013201. Bortolussi, Luca, and Alberto Policriti. "Modeling biological systems in stochastic concurrent constraint programming." Constraints 13.1-2 (2008): 66-90. Kaufman, Randal J. "Orchestrating the unfolded protein response in health and disease." The Journal of clinical investigation 110.10 (2002): 1389-1398. Wetie, Armand G. Ngounou, et al. "Protein–protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches." Cellular and Molecular Life Sciences 71.2 (2014): 205-228. Keywords: Neurodegenaration, Huntington Disease, Computational models, chaperones, unfolded protein response (UPR) Conference: Neuroinformatics 2016, Reading, United Kingdom, 3 Sep - 4 Sep, 2016. Presentation Type: Poster Topic: Computational neuroscience Citation: Theocharopoulou G and Bobori C (2016). Modeling the UPR in neurodegenaration. Front. Neuroinform. Conference Abstract: Neuroinformatics 2016. doi: 10.3389/conf.fninf.2016.20.00066 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 31 May 2016; Published Online: 18 Jul 2016. * Correspondence: Mrs. Georgia Theocharopoulou, Ionian University, Informatics, CORFU, 49100, Greece, zeta.theo@ionio.gr Mrs. Catherine Bobori, Ionian University, Informatics, CORFU, 49100, Greece, tero-nana@windowslive.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Georgia Theocharopoulou Catherine Bobori Google Georgia Theocharopoulou Catherine Bobori Google Scholar Georgia Theocharopoulou Catherine Bobori PubMed Georgia Theocharopoulou Catherine Bobori Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chaperone DnaJ Influences the Formation of Biofilm by Escherichia coli

DnaJ chaperone, a member of the so called DnaK-DnaJ-GrpE chaperone machine plays an important role in cell physiology. The ability of Escherichia coli ΔdnaJ mutant to form biofilm was studied. It was shown that this mutant is impaired in biofilm development when exposed to 42 degrees C for 2 h. The impairment in biofilm development was observed when the heat shock was applied either at the onset of biofilm formation or 2 h later. The biofilm formed was thinner and its structure was changed as compared to wild-type strain. This defect could be complemented by the introduction of a wild-type gene on a low-copy plasmid.

Cooperation of Hsp70 and Hsp100 chaperone machines in protein disaggregation.

Unicellular and sessile organisms are particularly exposed to environmental stress such as heat shock causing accumulation and aggregation of misfolded protein species. To counteract protein aggregation, bacteria, fungi, and plants encode a bi-chaperone system composed of ATP-dependent Hsp70 and hexameric Hsp100 (ClpB/Hsp104) chaperones, which rescue aggregated proteins and provide thermotolerance to cells. The partners act in a hierarchic manner with Hsp70 chaperones coating first the surface of protein aggregates and next recruiting Hsp100 through direct physical interaction. Hsp100 proteins bind to the ATPase domain of Hsp70 via their unique M-domain. This extra domain functions as a molecular toggle allosterically controlling ATPase and threading activities of Hsp100. Interactions between neighboring M-domains and the ATPase ring keep Hsp100 in a repressed state exhibiting low ATP turnover. Breakage of intermolecular M-domain interactions and dissociation of M-domains from the ATPase ring relieves repression and allows for Hsp70 interaction. Hsp70 binding in turn stabilizes Hsp100 in the activated state and primes Hsp100 ATPase domains for high activity upon substrate interaction. Hsp70 thereby couples Hsp100 substrate binding and motor activation. Hsp100 activation presumably relies on increased subunit cooperation leading to high ATP turnover and threading power. This Hsp70-mediated activity control of Hsp100 is crucial for cell viability as permanently activated Hsp100 variants are toxic. Hsp100 activation requires simultaneous binding of multiple Hsp70 partners, restricting high Hsp100 activity to the surface of protein aggregates and ensuring Hsp100 substrate specificity.

Structural Rigidity Regulates Functional Interactions in the Hsp40-Hsp70 Molecular Machine

Protein interactions commonly involve lock-and-key associations between interacting domains. Structural features of these binding partners regulate the nature and extent of their interactions. We report our investigations on specific physical characteristics required of the Hsp40 J-domain to stimulate ATP hydrolysis in the Hsp40-Hsp70 molecular chaperone machine. Characterization of functional and dysfunctional Hsp40-Hsp70 interactions using isothermal titration calorimetry (ITC), and nuclear magnetic resonance (NMR) spectroscopy reveals the importance of structural rigidity for Hsp40 function. Our results suggest that the functional J-domain acts like a semi-elliptical spring, whose resistance to bending in Hsp40-Hsp70 interactions modulates the ATPase domain conformational change and promotes ATP hydrolysis.

The role of HSP70 and its co-chaperones in protein misfolding, aggregation and disease.

Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein folding, quality control and function. In particular, the HSP70 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and its co-chaperones have been recognised as potent modulators of inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. In has become evident that the HSP70 chaperone machine functions not only in folding, but also in proteasome mediated degradation of neurodegenerative disease proteins. Thus, there has been a great deal of interest in the potential manipulation of molecular chaperones as a therapeutic approach for many neurodegenerations. Furthermore, mutations in several HSP70 co-chaperones and putative co-chaperones have been identified as causing inherited neurodegenerative and cardiac disorders, directly linking the HSP70 chaperone system to human disease.

Protein folding in the cell, from atom to organism.

Proper cell function requires proper protein folding. Misfolding of specific proteins, caused either by mutation or environmental stress, underlies many human diseases, including cancer and diabetes and Parkinson’s, Huntington’s, and Alzheimer’s disease. Protein folding has been studied for decades in the test tube, initially spearheaded by the classical studies of Anfinsen and colleagues (1). Whereas in vitro studies continue to provide invaluable contributions toward developing a complete understanding of the fundamental physical and chemical forces that govern folding, there has been a concomitant and growing appreciation that the cellular environment presents unique challenges—as well as unique support systems—for protein folding that cannot be captured easily in testtube folding experiments. To understand how to keep cells healthy and how to correct diseased cells, we must understanding how proteins fold and misfold in vivo and define the mechanism of action of the cellular protein quality-control machinery (Fig. 1). Since 1990, the biennial FASEB Protein Folding in the Cell conference has been devoted to bridging the gap between our understanding of how proteins fold in the test tube and the consequences for the cell should they misfold. The Protein Folding in the Cell conference occupies a unique niche among summer conferences because of its ongoing success at drawing together cell biologists, geneticists, biophysicists, biochemists, theorists, and computational biologists to tackle the complex problems of protein folding and misfolding and their implications for human disease. The extent to which this meeting has played a central role in the development of the field cannot be overstated. Historically, biophysical chemists/theoreticians and cell biologists/geneticists who study protein folding rarely crossed paths. However, the complexity of the protein folding “problem” and the paucity of methods available to study it in detail require a deep and multidisciplinary attack. Recent progress has shown the value of this approach in tackling some of the major outstanding questions in the field. At the 2014 meeting, held July 20–25 at the Vermont Academy in Saxton’s River, VT, the nine sessions and one Keynote Address covered every aspect of the protein-folding problem and its consequences for proper cell function. The titles of the scientific sessions were: “Principles of Protein Folding,” “Functions of Chaperone Machines,” “Cellular Responses and Protein Biogenesis,” “Protein Folding and Trafficking Networks,” “Biology and Biophysics of Amyloids and Aggregation,” “Evolution of Proteins and Protein Machines,” “Protein Quality Control and Degradation,” “Stress Responses, Molecular Chaperones, and Signaling,” and “Protein Maturation and Folding Decisions.” In addition to the didactic sessions, another dynamic aspect of the conference was its three-poster sessions that featured 93 submitted abstracts. The meeting was attended by nearly 150 scientists from academia, industry, and publishing houses and began with a Keynote Lecture from Lila Gierasch (University of Massachusetts, Amherst). In her lecture, Dr. Gierasch elegantly illustrated the synergy created by combining in vitro and in vivo studies of protein folding, focusing on her recent nuclear magnetic resonance and other biophysical studies (3, 4) to understand the allosteric regulation of the heat shock protein (Hsp)70 class of molecular chaperones and the impact of our improved understanding of how misfolded proteins are routed through protein quality-control networks in vivo (5). Many of the scientific presentations were buoyed by recent methodological and conceptual advances, often

The Hsp90 ensemble: coordinated Hsp90-cochaperone complexes regulate diverse cellular processes.

The Seventh International Conference on the Hsp90 Chaperone Machine took place in October 2014, in Seeon, Germany. The program highlighted recent findings in a variety of areas, including structures of heat-shock protein 90 (Hsp90)–client protein complexes, coordination of Hsp90 with cochaperones, new cellular and physiological roles for Hsp90 and therapeutic targeting of the Hsp90 ensemble for the treatment of disease and prevention of infection.

A Direct Regulatory Interaction between Chaperonin TRiC and Stress-Responsive Transcription Factor HSF1

Heat shock transcription factor 1 (HSF1) is an evolutionarily conserved transcription factor that protects cells from protein-misfolding-induced stress and apoptosis. The mechanisms by which cytosolic protein misfolding leads to HSF1 activation have not been elucidated. Here, we demonstrate that HSF1 is directly regulated by TRiC/CCT, a central ATP-dependent chaperonin complex that folds cytosolic proteins. A small-molecule activator of HSF1, HSF1A, protects cells from stress-induced apoptosis, binds TRiC subunits in vivo and in vitro, and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation, and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. These results demonstrate a direct regulatory interaction between the cytosolic chaperone machine and a critical transcription factor that protects cells from proteotoxicity, providing a mechanistic basis for signaling perturbations in protein folding to a stress-protective transcription factor.

A Chaperome Subnetwork Safeguards Proteostasis in Aging and Neurodegenerative Disease

Chaperones are central to the proteostasis network (PN) and safeguard the proteome from misfolding, aggregation, and proteotoxicity. We categorized the human chaperome of 332 genes into network communities using function, localization, interactome, and expression data sets. During human brain aging, expression of 32% of the chaperome, corresponding to ATP-dependent chaperone machines, is repressed, whereas 19.5%, corresponding to ATP-independent chaperones and co-chaperones, are induced. These repression and induction clusters are enhanced in the brains of those with Alzheimer's, Huntington's, or Parkinson's disease. Functional properties of the chaperome were assessed by perturbation in C. elegans and human cell models expressing Aβ, polyglutamine, and Huntingtin. Of 219 C. elegans orthologs, knockdown of 16 enhanced both Aβ and polyQ-associated toxicity. These correspond to 28 human orthologs, of which 52% and 41% are repressed, respectively, in brain aging and disease and 37.5% affected Huntingtin aggregation in human cells. These results identify a critical chaperome subnetwork that functions in aging and disease.

HSPA1A-Independent Suppression of PARK2 C289G Protein Aggregation by Human Small Heat Shock Proteins

The C289G mutation of the parkin E3-ubiquitin protein ligase (PARK2) is associated with autosomal recessive juvenile onset Parkinson's disease and was found to be associated with protein aggregation. Members of the human small heat shock proteins (HSPBs) have been implicated in protein degradation and prevention of protein aggregation. In this study, we show that of the 10 HSPB members, individual overexpression of HSPB1, HSPB2, HSPB4, and HSPB7 suppresses PARK2 C289G-associated protein aggregation. Intriguingly, the protective actions of these HSPBs are not impaired upon inactivation of the ATP-dependent HSP70 chaperone machines. Depending on the HSPB member the protective actions involve either autophagic or proteasomal degradation pathways.

Purification and biochemical characterization of DnaK and its transcriptional activator RpoH from Neisseria gonorrhoeae.

DnaK plays a central role in stress response in the important human pathogen Neisseria gonorrhoeae. The genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ(32))-dependent promoters. In this study, we cloned, purified and biochemically characterised N. gonorrhoeae DnaK (NgDnaK) and RpoH. The NgDnaK and RpoH sequences are 73 and 50 % identical to the sequences of their respective E. coli counterparts. Similar to EcDnaK, nucleotide-free NgDnaK exists as a mix of monomers, dimers and higher oligomeric species in solution, and dissociates into monomers on addition of ATP. Like E. coli σ(32), RpoH of N. gonorrhoeae is monomeric in solution. Kinetic analysis of the basal ATPase activity of purified NgDnaK revealed a V max of 193 pmol phosphate released per minute per microgram DnaK (which is significantly higher than reported basal ATPase activity of EcDnaK), and the turnover number against ATP was 0.4 min(-1) under our assay conditions. Nucleotide-free NgDnaK bound a short model substrate, NR-peptide, with micromolar affinity close to that reported for EcDnaK. Our analysis showed that interaction between N. gonorrhoeae RpoH and DnaK appears to be ATP-dependent and non-specific, in stark contrast to the E. coli DnaK system where σ(32) and DnaK interact as monomers even in the absence of ATP. Sequence comparison showed that the DnaK-binding site of σ(32) is not conserved in RpoH. Our findings suggest that the mechanism of DnaK/RpoH recognition in N. gonorrhoeae is different from that in E. coli.

Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes.

Progress and prospects for targeting Hsp90 to treat fungal infections

Fungal pathogens pose a major threat to human health worldwide. They infect billions of people each year, leading to at least 1·5 million deaths. Treatment of fungal infections is difficult due to the limited number of clinically useful antifungal drugs, and the emergence of drug resistance. A promising new strategy to enhance the efficacy of antifungal drugs and block the evolution of drug resistance is to target the molecular chaperone Hsp90. Pharmacological inhibitors of Hsp90 function that are in development as anticancer agents have potential to be repurposed as agents for combination antifungal therapy for some applications, such as biofilm infections. For systemic infections, however, effective combination therapy regimens may require Hsp90 inhibitors that can selectively target Hsp90 in the pathogen, or alternate strategies to compromise function of the Hsp90 chaperone machine. Selectively impairing Hsp90 function in the pathogen could in principle be achieved by targeting Hsp90 co-chaperones or regulators of Hsp90 function that are more divergent between pathogen and host than Hsp90. Antifungal combination therapies could also exploit downstream effectors of Hsp90 that are critical for fungal drug resistance and virulence. Here, we discuss the progress and prospects for establishing Hsp90 as an important therapeutic target for life-threatening fungal infections.

High-throughput screen of natural product libraries for hsp90 inhibitors.

Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.