Andrea D. Thompson

Binding of a Small Molecule at a Protein–Protein Interface Regulates the Chaperone Activity of Hsp70–Hsp40

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

Pharmacological chaperone for α-crystallin partially restores transparency in cataract models

A visionary approach to transparency Cataracts are the most common cause of vision loss, especially in our ever-increasing elderly population. Cataracts arise when crystallin, a major protein component of the eye lens, begins to aggregate, which causes the lens to become cloudy. Makley et al. explored whether small molecules that reverse this aggregation might have therapeutic potential for treating cataracts, which normally require surgery (see the Perspective by Quinlan). They used a screening method that monitors the effect of ligands on temperature-dependent protein unfolding and identified several compounds that bind and stabilize the soluble form of crystallin. In proof-of-concept studies, one of these compounds improved lens transparency in mice. Science, this issue p. 674; see also p. 636 A compound that reverses the molecular cause of cataract formation improves eye lens transparency in mice. [Also see Perspective by Quinlan] Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.

The heat shock protein 70 (Hsp70) family of molecular chaperones has important functions in maintaining proteostasis under stress conditions. Several Hsp70 isoforms, especially Hsp72 (HSPA1A), are dramatically upregulated in response to stress; however, it is unclear whether these family members have biochemical properties that are specifically adapted to these scenarios. The redox-active compound, methylene blue (MB), has been shown to inhibit the ATPase activity of Hsp72 in vitro, and it promotes degradation of the Hsp72 substrate, tau, in cellular and animal models. Here, we report that MB irreversibly inactivates Hsp72 but not the nearly identical, constitutively expressed isoform, heat shock cognate 70 (Hsc70; HSPA8). Mass spectrometry results show that MB oxidizes Cys306, which is not conserved in Hsc70. Molecular models suggested that oxidation of Cys306 exposes Cys267 to modification and that both events contribute to loss of ATP binding in response to MB. Consistent with this model, mutating Cys267 and Cys306 to serine made Hsp72 largely resistant to MB in vitro, and overexpression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Furthermore, mutating Cys267 and Cys306 to the pseudo-oxidation mimic, aspartic acid, mirrored MB treatment: the C267D and C306D mutants had reduced ATPase activity in vitro, and overexpression of the C267/306D double mutant significantly reduced tau levels in cells. Together, these results suggest that redox sensing by specific cysteine residues in Hsp72, but not Hsc70, may be an important component of the chaperone response to oxidative stress.

Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/μg/min−1. We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of ΔdnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding.

Analysis of the Tau-Associated Proteome Reveals that Exchange of Hsp70 for Hsp90 Is Involved in Tau Degradation

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 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 min 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 data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas 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 Alzheimers 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.

Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK)

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.

Analysis of the interaction of BCL9 with β-catenin and development of fluorescence polarization and surface plasmon resonance binding assays for this interaction

The transcriptional activator beta-catenin is the primary mediator of the canonical Wnt signaling pathway and is frequently upregulated in many types of human cancer. Recent studies have suggested that the interaction of beta-catenin and its cofactor, B-cell lymphoma 9 (BCL9), is crucial for its transcriptional activity. Targeting this interaction using small molecules will improve our understanding of the beta-catenin/Wnt signaling pathway and may lead to the development of a new class of anticancer drugs. In this study, we developed a fluorescence polarization (FP)-based BCL9 binding assay. Using our initial FP assay, we performed extensive mutational analysis on four critical hydrophobic residues in the BCL9 peptide and determined the precise region in BCL9 responsible for binding to beta-catenin. These results led to further optimization of our FP assay, making it amenable for high-throughput screening (HTS). We also developed and validated a complementary surface plasmon resonance (SPR)-based binding assay and showed that our synthetic BCL9-based peptides are capable of fully inhibiting the binding of beta-catenin to wild-type BCL9 protein. Our studies provide not only further insight into the interaction between BCL9 and beta-catenin but also quantitative and reliable biochemical binding assays for the discovery of potent and specific small-molecule inhibitors of this interaction.

Identification of Key Hinge Residues Important for Nucleotide-Dependent Allostery in E. coli Hsp70/DnaK

DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaKs nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.

The three cornerstones of chemical biology: innovative probes, new discoveries, and enabling tools.

New Discoveries, and Enabling Tools Andrea D. Thompson,† Leah N. Makley,† Kathryn McMenimen,‡ and Jason E. Gestwicki*,† †Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States ‡Department of Chemistry, Mt. Holyoke College, South Hadley, Massachusetts 01075, United States K Symposia have a long history of serving as catalysts for the advancement of biomedical and life sciences by connecting scientists within and across disciplines. Thus, it was a natural extension of the central Keystone mission to hold the first ever Keystone Symposia on “Chemical Biology and Novel Tools in Pharmacology” on February 12−16, 2012. Organized by Laura Kiessling (University of Wisconsin, Madison), Jason Gestwicki (University of Michigan), and Kevan Shokat (University of California, San Francisco), this conference brought scientists from the international chemical biology community together in Santa Fe, New Mexico to communicate new ideas and technologies as well as recent progress in research. Unlike the majority of Keystone Symposia, this meeting was not focused on a particular aspect of biology but rather nucleated chemists and biologists across a wide range of disease areas and biological topics, providing an especially vibrant forum for the discussion of cross-platform ideas. As evidence of the breadth of the community, representatives from 10 countries, including academic, industrial, and government researchers and more than 40 graduate students, came together for this meeting. The conference schedule was filled with impressive presentations from experts across multiple areas of chemical biology (Figure 1). Throughout the program’s various sessions, three overarching themes emerged: (A) Design and Discovery of New Molecules and Probes, (B) New Advances in Biology Identified Using Chemical Approaches, and (C) Development of Novel, Enabling Methods (Figure 2). Select highlights are described here to illustrate the breadth and depth of these concepts.

Rescued diagnostic quality by motion correction of dynamic cardiac positron emission tomography (PET) perfusion images

Cardiac positron emission tomography (PET) imaging is widely utilized to measure myocardial blood flow and perfusion. Patient motion is a well-recognized cause of artifacts and techniques to correct for this have been developed, primarily for single photon imaging. Herein, we describe a case where gross patient motion during stress imaging resulted in non-diagnostic static images summed between 2 and 7 minutes. Respiratory gated images did not improve image quality (not shown). However, manual adjustment of dynamic images to correct for gross patient motion yielded diagnostic quality images and allowed detection of a proximal LAD stenosis. A 63-year-old man, with a medical history of tuberculous meningitis, normal pressure hydrocephalus status post ventriculoperitoneal shunt, and mild cognitive impairment, presented with exertional substernal chest pain. Normal plasma cardiac troponin ruled out acute myocardial infarction. Due to new T-wave inversions on electrocardiography, additional risk stratification was sought. The patient is obese and unable to exercise. Consequently, he underwent rest and regadenoson stress cardiac PET/CT imaging using rubidium82 as the perfusion tracer. Gross patient motion artifact rendered the study non-diagnostic by standard static reconstructions (Figure 1A). However, by manually adjusting the dynamic images acquired during this period (2 to 7 minutes) for patient motion, we resolved a completely reversible defect in the territory of proximal left anterior descending (LAD) coronary artery (Figure 1B). The patient underwent coronary angiography which confirmed proximal LAD disease and was percutaneously revascularized (Figure 2). This case highlights the utility of motion correction of the dynamic image series. The motion artifact in this case was a nearly 16 mm gross patient shift early during