J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play “generalist” roles, while others, such as Apj1 and Jjj2, are “specialists”, having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins.
In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and also each other to promote folding. This system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the large-scale, ternary interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ which determines whether they bind synergistically or competitively. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.
Protein-protein interactions (PPIs) control the assembly of multi-protein complexes and, thus, these contacts have enormous potential as drug targets. However, the field has produced a mix of both exciting success stories and frustrating challenges. Here, we review known examples and explore how the physical features of a PPI, such as its affinity, hotspots, off-rates, buried surface area and topology, may influence the chances of success in finding inhibitors. This analysis suggests that concise, tight binding PPIs are most amenable to inhibition. However, it is also clear that emerging technical methods are expanding the repertoire of “druggable” protein contacts and increasing the odds against difficult targets. In particular, natural product-like compound libraries, high throughput screens specifically designed for PPIs and approaches that favor discovery of allosteric inhibitors appear to be attractive routes. The first group of PPI inhibitors has entered clinical trials, further motivating the need to understand the challenges and opportunities in pursuing these types of targets.
high throughput screening; allostery; multi-protein complexes; Hsp70; Hsp90; fragment based drug discovery; natural products; protein-protein interactions
The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in fifteen neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 minutes after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the dataset was that tau binding to heat shock protein 70 (Hsp70) decreased while binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer’s disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.
New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.
polyomavirus; bithionol; hexachlorophene; T antigen; molecular chaperone; high throughput screen
Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units.
Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70’s ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify Hsp70’s role as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.
polyQ; protein misfolding; molecular chaperones; heat shock protein 70 (Hsp70); proteostasis; chemical genetics; chemical probes
Multi-protein complexes such as the transcriptional machinery, signaling hubs, and protein folding machines are typically comprised of at least one enzyme combined with multiple non-enzymes. Often the components of these complexes are incorporated in a combinatorial manner, in which the ultimate composition of the system helps dictate the type, location, or duration of cellular activities. Although drugs and chemical probes have traditionally targeted the enzyme components, emerging strategies call for controlling the function of protein complexes by modulation of protein-protein interactions (PPI). However, the challenges of targeting PPIs have been well documented and the diversity of PPIs makes a “one-size-fits-all” solution highly unlikely. These hurdles are particularly daunting for PPIs that encompass large buried surface areas and those with weak affinities. In this review, we discuss lessons from natural systems, in which allostery and other mechanisms are used to overcome the challenge of regulating the most difficult PPIs. These systems may provide a blueprint for identifying small molecules that target challenging PPIs and affecting molecular decision-making within multi-protein systems.
Multi-protein complexes: structures formed by non-covalent interactions between two or more proteins; Protein-protein interaction (PPI): occurs when proteins bind to one another via a direct physical interaction surface; Protein-ligand interface (PLI): the binding surface between a protein and its cognate ligand, typically smaller and more topologically defined than a PPI; PPI modulator: small “drug-like” molecule capable of either promoting or inhibiting the binding interaction between two proteins; Orthosteric inhibitor: a PPI modulator that inhibits protein interactions through direct binding competition; Allosteric binding: binding at one site that regulates function or binding at a distant site; Post-translational modifications (PTMs): covalent modifications of individual amino acid side chains that regulate the structure, localization and function of proteins including phosphorylation, glycosylation, acylation and ubiquitylation; Chemical probes: small molecules or chemical matter that can be utilized as research tools to study biology; High-throughput screening (HTS): a discovery experiment in which a rapid and efficient biological or chemical assay is employed to evaluate large numbers of chemical or biologicall agents for a particular activity
We sought novel strategies to reduce levels of the polyglutamine androgen receptor (polyQ AR) and achieve therapeutic benefits in models of spinobulbar muscular atrophy (SBMA), a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the Hsp90/Hsp70-based chaperone machinery, but mechanisms regulating the protein’s turnover are incompletely understood. We demonstrate that overexpression of Hip, a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of SBMA. These findings highlight the therapeutic potential of allosteric regulators of Hsp70, and provide new insights into the role of the chaperone machinery in protein quality control.
Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as congo red, thioflavin T and their derivatives, have been powerful tools for better understanding amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD.
Alzheimer’s disease; thioflavin T; congo red; curcumin; fibrils; protofibrils; oligomers; amyloid beta
More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.
autophagy; methylene blue; phosphorylation; tau; tissue culture; transgenic mouse
Aggregated amyloid-β (Aβ) peptide is implicated in the pathology of Alzheimer’s disease. In vitro and in vivo, these aggregates are found in a variety of morphologies, including globular oligomers and linear fibrils, which possess distinct biological activities. However, known chemical probes, including the dyes thioflavin T and Congo Red, appear to lack selectivity for specific amyloid structures. To identify molecules that might differentiate between these architectures, we employed a fluorescence-based interaction assay to screen a collection of 68 known Aβ ligands against pre-formed oligomers and fibrils. In these studies, we found that the fluorescence of five indole-based compounds was selectively quenched (~15%) in the presence of oligomers, but remained unchanged after addition of fibrils. These results suggest that indoles might be complementary to existing chemical probes for studying amyloid formation in vitro.
Alzheimer’s disease; neurodegeneration; protein misfolding; thioflavin T; fluorescence
Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications, to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses of these bifunctional constructs, owing to the abundance and favorable distribution of their target, FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syntheses and/or multiple protection-deprotection schemes prior to installation into CIDs. In this work, we describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated, cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified with an array of functional groups, including primary amines and carboxylic acids, which make the resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the synthesis of a heterobifunctional HIV protease inhibitor.
metathesis; cross-coupling; natural products; chemical inducers of dimerization; drug targeting; HIV protease
The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein’s life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery.
proteostasis; flavonoids; dihydropyrimidines; spergualin; sulfoglycolipids; geranylgeranyl acetone; protein folding; ATPase; protein-protein interactions
The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.
Electronic supplementary material
The online version of this article (doi:10.1007/s12192-011-0307-1) contains supplementary material, which is available to authorized users.
Electron microscopy; Chaperone; Allostery; Protein complexes; Oligomers
We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20–30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination with either the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.
The Hsp70 chaperones (Heat shock protein 70 kDa) are key to cellular protein homeostatis. However, they also have the ability to inhibit tumor apoptosis, and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer’s disease. Hence, Hsp70 are increasingly been identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer besides classical active site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anti-cancer compound MKT-077, is through differential interaction with the Hsp70 allosteric states. MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride) is therefore an “allosteric drug”. Using NMR spectroscopy, the compound’s binding site on human HSPA8 (Hsc70) is identified. The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular dynamics-based energy and solvation computations. Suggestions for improvement of the compound’s properties are made on the basis of the binding location and pose.
The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3.
Tau is a microtubule-associated protein that accumulates in at least 15 different neurodegenerative disorders, which are collectively referred to as tauopathies. In these diseases, tau is often hyperphosphorylated and found in aggregates, including paired helical filaments, neurofibrillary tangles and other abnormal oligomers. Tau aggregates are associated with neuron loss and cognitive decline, which suggests that this protein can somehow evade normal quality control allowing it to aberrantly accumulate and become proteotoxic. Consistent with this idea, recent studies have shown that molecular chaperones, such as heat shock protein 70 and heat shock protein 90, counteract tau accumulation and neurodegeneration in disease models. These molecular chaperones are major components of the protein quality control systems and they are specifically involved in the decision to retain or degrade many proteins, including tau and its modified variants. Thus, one potential way to treat tauopathies might be to either accelerate interactions of abnormal tau with these quality control factors or tip the balance of triage towards tau degradation. In this review, we summarize recent findings and suggest models for therapeutic intervention.
Spergualin is a natural product that exhibits immunosuppressive, anti-tumor and anti-bacterial activities. Its derivatives, such as 15-deoxyspergualin (15-DSG), have been clinically approved for acute allograft rejection. However, the reported syntheses are cumbersome (> 10 steps) and they suffer from low overall yields (~ 0.3 to 18%). Moreover, spergualin and its derivatives are chemically unstable and rapidly hydrolyzed in aqueous buffer. Here, we have re-explored these issues and report a modified synthetic route with significantly improved overall yield (~31 to 47%). The key transformation is a microwave-accelerated Ugi multi-component reaction that is used to generate the peptoid core in a single step. Using the products of this route, we found that modifications of the hemiaminal significantly increased chemical stability. Thus, we anticipate that this synthetic route will improve access to biologically active 15-DSG derivatives.
polyamine; chemical stability; combinatorial synthesis; immunosuppression; peptoid
DnaK is a molecular chaperone responsible for multiple aspects of proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors, we screened plant-derived extracts against a re-constituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK’s intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems.
A series of dihydropyridines were identified that have an effect on the accumulation of tau, an important target in Alzheimer's disease. The dihydropyridine collection was expanded using the Hantzsch multicomponent reaction to develop preliminary structure-activity relationships.
Alzheimer’s disease (AD) is characterized by the self-assembly of amyloid beta (Aβ) peptides. Recent models implicate some of the earliest Aβ oligomers, such as trimers and tetramers, in disease. However, the roles of these structures remain uncertain, in part, because selective probes of their formation are not available. Towards that goal, we generated bivalent versions of the known Aβ ligand, the pentapeptide KLVFF. We found that compounds containing sufficiently long linkers (~19 to 24 Å) recognized primarily Aβ trimers and tetramers, with little binding to either monomer or higher order structures. These compounds might be useful probes for early Aβ oligomers.