Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.

The structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client maturation state.

The Hsp90 chaperone promotes folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during folding of kinases, nuclear receptors, and tau. Here we determined the cryoelectron microscopy (cryo-EM) structure of the human Hsp90:FKBP51:p23 complex to 3.3 Å, which, together with mutagenesis and crosslinking analyses, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent to Hsp90 client binding sites, whereas a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 is positioned to act on specific client residues presented during Hsp90-catalyzed remodeling.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Exploration of the binding determinants of protein phosphatase 5 (PP5) reveals a chaperone-independent activation mechanism.

The protein phosphatase 5 (PP5) is normally recruited to its substrates by the molecular chaperones, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90). This interaction requires the tetratricopeptide repeat (TPR) domain of PP5, which binds to an EEVD motif at the extreme C termini of cytosolic Hsp70 and Hsp90 isoforms. In addition to bringing PP5 into proximity with chaperone-bound substrates, this interaction also relieves autoinhibition in PP5's catalytic domain, promoting its phosphatase activity. To better understand the molecular determinants of this process, we screened a large, pentapeptide library for binding to PP5. This screen identified the amino acid preferences at each position, which we validated by showing that the optimal sequences bind 4- to 7-fold tighter than the natural EEVD motifs and stimulate PP5's enzymatic activity. The enhanced affinity for PP5's TPR domain was confirmed using a protein-adaptive differential scanning fluorimetry assay. Using this increased knowledge of structure-activity relationships, we re-examined affinity proteomics results to look for potential EEVD-like motifs in the C termini of known PP5-binding partners. This search identified elongator acetyltransferase complex subunit 1 (IKBKAP) as a putative partner, and indeed, we found that its C-terminal sequence, LSLLD, binds directly to PP5's TPR domain in vitro. Consistent with this idea, mutation of elongator acetyltransferase complex subunit 1's terminal aspartate was sufficient to interrupt the interaction with PP5 in vitro and in cells. Together, these findings reveal the sequence preferences of PP5's TPR domain and expand the scope of PP5's functions to include chaperone-independent complexes.

iPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death.

BACKGROUND: Tau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. METHODS: To understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. RESULTS: FTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. CONCLUSIONS: Our results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

Chemical Features of Polyanions Modulate Tau Aggregation and Conformational States.

The aggregation of tau into insoluble fibrils is a defining feature of neurodegenerative tauopathies. However, tau has a positive overall charge and is highly soluble; so, polyanions, such as heparin, are typically required to promote its aggregation in vitro. There are dozens of polyanions in living systems, and it is not clear which ones might promote this process. Here, we systematically measure the ability of 37 diverse, anionic biomolecules to initiate tau aggregation using either wild-type (WT) tau or the disease-associated P301S mutant. We find that polyanions from many different structural classes can promote fibril formation and that P301S tau is sensitive to a greater number of polyanions (28/37) than WT tau (21/37). We also find that some polyanions preferentially reduce the lag time of the aggregation reactions, while others enhance the elongation rate, suggesting that they act on partially distinct steps. From the resulting structure-activity relationships, the valency of the polyanion seems to be an important chemical feature such that anions with low valency tend to be weaker aggregation inducers, even at the same overall charge. Finally, the identity of the polyanion influences fibril morphology based on electron microscopy and limited proteolysis. These results provide insights into the crucial role of polyanion-tau interactions in modulating tau conformational dynamics with implications for understanding the tau aggregation landscape in a complex cellular environment.

Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models.

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4-2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

The interactions of molecular chaperones with client proteins: why are they so weak?

The major classes of molecular chaperones have highly variable sequences, sizes, and shapes, yet they all bind to unfolded proteins, limit their aggregation, and assist in their folding. Despite the central importance of this process to protein homeostasis, it has not been clear exactly how chaperones guide this process or whether the diverse families of chaperones use similar mechanisms. For the first time, recent advances in NMR spectroscopy have enabled detailed studies of how unfolded, "client" proteins interact with both ATP-dependent and ATP-independent classes of chaperones. Here, we review examples from four distinct chaperones, Spy, Trigger Factor, DnaK, and HscA-HscB, highlighting the similarities and differences between their mechanisms. One striking similarity is that the chaperones all bind weakly to their clients, such that the chaperone-client interactions are readily outcompeted by stronger, intra- and intermolecular contacts in the folded state. Thus, the relatively weak affinity of these interactions seems to provide directionality to the folding process. However, there are also key differences, especially in the details of how the chaperones release clients and how ATP cycling impacts that process. For example, Spy releases clients in a largely folded state, while clients seem to be unfolded upon release from Trigger Factor or DnaK. Together, these studies are beginning to uncover the similarities and differences in how chaperones use weak interactions to guide protein folding.

Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism.

Overexpression and aggregation of α-synuclein (ASyn) are linked to the onset and pathology of Parkinson's disease and related synucleinopathies. Elevated levels of the stress-induced chaperone Hsp70 protect against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations, and inhibitors, we confirm that Hsp70 interacts with ASyn via an as yet unidentified, noncanonical interaction site in the C-terminal domain. Finally, we report a biological role for a similar mode of action in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target the Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70's canonical action. Additionally, these results raise the question of whether other misfolded proteins might also engage Hsp70 via the same noncanonical mechanism.