Identification of Cellular Protein Targets of a Half-Sandwich Iridium(III) Complex Reveals Its Dual Mechanism of Action via Both Electrophilic and Oxidative Stresses.

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C∧N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high-affinity targets. Upon iridium complex treatment, the folding activity of Heat Shock Protein HSP90 was inhibited in vitro and major cytoskeleton disorganization was observed. A wide array of imaging and biochemical methods validated selected targets and provided a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity. The proposed methodological workflow can open innovative avenues in metallodrug discovery.

Novel Strategy for In Vitro Validation of Babesia orientalis Heat Shock Proteins Chaperone Activity and Thermostability.

BACKGROUND: Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro. METHODS: BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide). RESULTS: Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock. CONCLUSION: Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host-parasite interaction.

Drugs targeting the particle for arrangement of quaternary structure (PAQosome) and protein complex assembly.

INTRODUCTION: The PAQosome is a 12-subunit complex that acts as a co-factor of the molecular chaperones HSP90 and HSP70. This co-chaperone has been shown to participate in assembly and maturation of several protein complexes, including nuclear RNA polymerases, RNA processing factors, the ribosome, PIKKs, and others. Subunits of the PAQosome, adaptors, and clients have been reported to be involved in various diseases, making them interesting targets for drug discovery. AREA COVERED: In this review, the authors cover the detailed mechanisms of PAQosome and chaperone function. Specifically, the authors summarize the status of the PAQosome and some related chaperones and co-chaperones as candidate targets for drug discovery. Indeed, a number of compounds are currently being tested for the development of treatments against diseases, such as cancers and neurodegenerative conditions. EXPERT OPINION: Searching for new drugs targeting the PAQosome requires a better understanding of PAQosome subunit interactions and the discovery of new interaction partners. Thus, PAQosome subunit crystallization is an important experiment to initiate virtual screening against new target and the development of in silico tools such as AlphaFold-multimer could accelerate the search for new interaction partner and determine more rapidly the interaction pocket needed for virtual drug screening.

The AhR Signaling Mechanism: A Structural Point of View.

The Aryl hydrocarbon Receptor (AhR) is a well-known sensor of xenobiotics; moreover, it is considered a promising drug target as it is involved in the regulation of many patho-physiological processes. For these reasons the study of its ligand-activated transcription mechanism has stimulated several studies for over twenty years. In this review we highlight the key role of molecular structural information in understanding the different steps of the signaling mechanism. The architecture of the AhR cytosolic complex, encompassing the hsp90 chaperone protein and the XAP2 and p23 co-chaperones, has become available in the last year thanks to Cryo-EM experiments. The structure of the AhR ligand-binding (PAS-B) domain has remained elusive for a long time; it has been predicted by homology modelling, based on known PAS systems, and its ligand-bound forms were modelled through ligand molecular docking. Although very recently some structural information on this domain has become available, considerable efforts are still needed to determine the binding geometries of the AhR key ligands by experimental high-resolution studies. On the other hand, the dimeric structure of AhR with the ARNT protein, bound to the specific DNA responsive element, was partially determined by X-ray crystallography and it was completed by homology modelling. On the whole the current structural knowledge of the main protein complexes that form over the AhR mechanism opens the way to confirm and further investigate the main steps of the proposed ligand-activated transcription mechanism of the AhR.

Cryo-EM reveals how Hsp90 and FKBP immunophilins co-regulate the glucocorticoid receptor.

Hsp90 is an essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins, including the glucocorticoid receptor (GR). Previously, we revealed that Hsp70 and Hsp90 remodel the conformation of GR to regulate ligand binding, aided by co-chaperones. In vivo, the co-chaperones FKBP51 and FKBP52 antagonistically regulate GR activity, but a molecular understanding is lacking. Here we present a 3.01 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP52 complex, revealing how FKBP52 integrates into the GR chaperone cycle and directly binds to the active client, potentiating GR activity in vitro and in vivo. We also present a 3.23 Å cryogenic electron microscopy structure of the human GR:Hsp90:FKBP51 complex, revealing how FKBP51 competes with FKBP52 for GR:Hsp90 binding and demonstrating how FKBP51 can act as a potent antagonist to FKBP52. Altogether, we demonstrate how FKBP51 and FKBP52 integrate into the GR chaperone cycle to advance GR to the next stage of maturation.

Human Aha1's N-terminal extension confers it holdase activity in vitro.

Molecular chaperones are key components of protein quality control system, which plays an essential role in controlling protein homeostasis. Aha1 has been identified as a co-chaperone of Hsp90 known to strongly accelerate Hsp90's ATPase activity. Meanwhile, it is reported that Aha1 could also act as an autonomous chaperone and protect stressed or disordered proteins from aggregation. Here, in this article, a series of in vitro experiments were conducted to verify whether Aha1 has a non-Hsp90-dependent holdase activity and to elucidate the associated molecular mechanism for substrate recognition. According to the results of the refolding assay, the highly conserved N-terminal extension spanning M1 to R16 in Aha1 from higher eukaryotes is responsible for the holdase activity of the protein. As revealed by the NMR data, Aha1's N-terminal extension mainly adopts a disordered conformation in solution and shows no tight contacts with the core structure of Aha1's N-terminal domain. Based on the intrinsically disordered structure feature and the primary sequence of Aha1's N-terminal extension, the fuzzy-type protein-protein interactions involving this specific region and the unfolded substrate proteins are expected. The following mutation analysis data demonstrated that the Van der Waals contacts potentially involving two tryptophans including W4 and W11 do not play a dominant role in the interaction between Aha1 and unfolded maltose binding protein (MBP). Meanwhile, since the high concentration of NaCl could abolish the holdase activity of Aha1, the electrostatic interactions mediated by those charged residues in Aha1's N-terminal extension are thus indicated to play a crucial role in the substrate recognition.

Insight into the Nucleotide Based Modulation of the Grp94 Molecular Chaperone Using Multiscale Dynamics.

Grp94, an ER-localized molecular chaperone, is required for the folding and activation of many membrane and secretory proteins. Client activation by Grp94 is mediated by nucleotide and conformational changes. In this work, we aim to understand how microscopic changes from nucleotide hydrolysis can potentiate large-scale conformational changes of Grp94. We performed all-atom molecular dynamics simulations on the ATP-hydrolysis competent state of the Grp94 dimer in four different nucleotide bound states. We found that Grp94 was the most rigid when ATP was bound. ATP hydrolysis or nucleotide removal enhanced mobility of the N-terminal domain and ATP lid, resulting in suppression of interdomain communication. In an asymmetric conformation with one hydrolyzed nucleotide, we identified a more compact state, similar to experimental observations. We also identified a potential regulatory role of the flexible linker, as it formed electrostatic interactions with the Grp94 M-domain helix near the region where BiP is known to bind. These studies were complemented with normal-mode analysis of an elastic network model to investigate Grp94's large-scale conformational changes. SPM analysis identified residues that are important in signaling conformational change, many of which have known functional relevance in ATP coordination and catalysis, client binding, and BiP binding. Our findings suggest that ATP hydrolysis in Grp94 alters allosteric wiring and facilitates conformational changes.

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and ß1-ß2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and ß1-ß2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.

Despite their structural similarities, the cytosolic isoforms of human Hsp90 show different behaviour in thermal unfolding due to their conformation: An FTIR study.

Heat shock proteins 90 (Hsp90) are chaperones that promote the proper folding of other proteins under high temperature stress situations. Hsp90s are highly conserved and ubiquitous proteins, and in mammalian cells, they are localized in the cytoplasm, endoplasmic reticulum, and mitochondria. Cytoplasmic Hsp90 are named Hsp90α and Hsp90ß and differ mainly in their expression pattern: Hsp90α is expressed under stress conditions, while Hsp90ß is a constitutive protein. Structurally, both share the same characteristics by presenting three well-conserved domains, one of which, the N-terminal domain, has a binding site for ATP to which various drugs targeting this protein, including radicicol, can bind. The protein is mainly found in dimeric form and adopts different conformations depending on the presence of ligands, co-chaperones and client proteins. In this study, some aspects of structure and thermal unfolding of cytoplasmic human Hsp90 were analysed by infrared spectroscopy. The effect on Hsp90ß of binding with a non-hydrolysable ATP analogue and radicicol was also examined. The results obtained showed that despite the high similarity in secondary structure the two isoforms exhibit substantial differences in their behaviour during thermal unfolding, as Hsp90α exhibits higher thermal stability, slower denaturation process and different event sequence during unfolding. Ligand binding strongly stabilizes Hsp90ß and slightly modifies the secondary structure of the protein as well. Most likely, these structural and thermostability characteristics are closely related to the conformational cycling of the chaperone and its propensity to exist in monomer or dimer form.

TRAP1 S-nitrosylation as a model of population-shift mechanism to study the effects of nitric oxide on redox-sensitive oncoproteins.

S-nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S-nitrosothiol (SNO) adduct. S-nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S-nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S-nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S-nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S-nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows ( https://github.com/ELELAB/SNO_investigation_pipelines ) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S-nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S-nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field.

Saccharomyces cerevisiae as a tool for deciphering Hsp90 molecular chaperone function.

Yeast is a valuable model organism for their ease of genetic manipulation, rapid growth rate, and relative similarity to higher eukaryotes. Historically, Saccharomyces cerevisiae has played a major role in discovering the function of complex proteins and pathways that are important for human health and disease. Heat shock protein 90 (Hsp90) is a molecular chaperone responsible for the stabilization and activation of hundreds of integral members of the cellular signaling network. Much important structural and functional work, including many seminal discoveries in Hsp90 biology are the direct result of work carried out in S. cerevisiae. Here, we have provided a brief overview of the S. cerevisiae model system and described how this eukaryotic model organism has been successfully applied to the study of Hsp90 chaperone function.

Pan-HSP90 ligand binding reveals isoform-specific differences in plasticity and water networks.

Isoforms of heat shock protein 90 (HSP90) fold oncoproteins that facilitate all 10 hallmarks of cancer. However, its promise as a therapeutic target remains unfulfilled as there is still no FDA-approved drug targeting HSP90 in disease. Among the reasons hindering progress are side effects caused by pan-HSP90 inhibition. Selective targeting of the four isoforms is challenging due to high sequence and structural similarity. Surprisingly, while decades of drug discovery efforts have produced almost 400 human HSP90 structures, no single ligand has been structurally characterized across all four human isoforms to date, which could reveal structural differences to achieve selectivity. To better understand the HSP90 landscape relevant for ligand binding and design we take a three-pronged approach. First, we solved the first complete set of structures of a single ligand bound to all four human isoforms. This enabled a systematic comparison of how side-chains and water networks respond to ligand binding across isoforms. Second, we expanded our analysis to publicly available, incomplete isoform-ligand series with distinct ligand chemistry. This highlighted general trends of protein and water mobility that differ among isoforms and impact ligand binding. Third, we further probed the Hsp90α conformational landscape for accommodating a congeneric series containing the purine scaffold common to HSP90 inhibitors. This revealed how minor ligand modifications flip ligand poses and perturb water and protein conformations. Taken together, this work illustrates how a systematic approach can shed new light on an "old" target and reveal hidden isoform-specific accommodations of congeneric ligands that may be exploited in ligand discovery and design.

Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.

Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90•apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.

Diptoindonesin G, a new Hsp90 drug.

Hsp90 is a molecular chaperone that participates in protein folding, activation, and stabilization of substrate proteins. Since many diseases, including cancer, neurodegenerative diseases, and metabolic diseases, are caused by protein misfolding, drugs that inhibit Hsp90 are being pursued as potential targets for treatments. In the recent JBC Editor's Pick by Donahue et al., the authors show that diptoindonesin G is a new Hsp90 inhibitor that promotes degradation of the estrogen receptor, an Hsp90 client, without inducing the heat shock response.

Structural basis of the key residue W320 responsible for Hsp90 conformational change.

The 90-kDa heat shock protein (Hsp90) is a homodimeric molecular chaperone with ATPase activity, which has become an intensely studied target for the development of drugs for the treatment of cancer, neurodegenerative and infectious diseases. The equilibrium between Hsp90 dimers and oligomers is important for modulating its function. In the absence of ATP, the passive chaperone activity of Hsp90 dimers and oligomers has been shown to stabilize client proteins as a holdase, which enhances substrate binding and prevents irreversible aggregation and precipitation of the substrate proteins. In the presence of ATP and its associated cochaperones, Hsp90 homodimers act as foldases with the binding and hydrolysis of ATP driving conformational changes that mediate client folding. Crystal structures of both wild type and W320A mutant Hsp90αMC (middle/C-terminal domain) have been determined, which displayed a preference for hexameric and dimeric states, respectively. Structural analysis showed that W320 is a key residue for Hsp90 oligomerization by forming intermolecular interactions at the Hsp90 hexameric interface through cation-π interactions with R367. W320A substitution results in the formation of a more open conformation of Hsp90, which has not previously been reported, and the induction of a conformational change in the catalytic loop. The structures provide new insights into the mechanism by which W320 functions as a key switch for conformational changes in Hsp90 self-oligomerization, and binding cochaperones and client proteins.Communicated by Ramaswamy H. Sarma.

Functions of the Hsp90-Binding FKBP Immunophilins.

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Cdc37 as a Co-chaperone to Hsp90.

The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.

p23 and Aha1: Distinct Functions Promote Client Maturation.

Hsp90 is a conserved molecular chaperone regulating the folding and activation of a diverse array of several hundreds of client proteins. The function of Hsp90 in client processing is fine-tuned by a cohort of co-chaperones that modulate client activation in a client-specific manner. They affect the Hsp90 ATPase activity and the recruitment of client proteins and can in addition affect chaperoning in an Hsp90-independent way. p23 and Aha1 are central Hsp90 co-chaperones that regulate Hsp90 in opposing ways. While p23 inhibits the Hsp90 ATPase and stabilizes a client-bound Hsp90 state, Aha1 accelerates ATP hydrolysis and competes with client binding to Hsp90. Even though both proteins have been intensively studied for decades, research of the last few years has revealed intriguing new aspects of these co-chaperones that expanded our perception of how they regulate client activation. Here, we review the progress in understanding p23 and Aha1 as promoters of client processing. We highlight the structures of Aha1 and p23, their interaction with Hsp90, and how their association with Hsp90 affects the conformational cycle of Hsp90 in the context of client maturation.

Design, synthesis, and biological evaluation of 4-(1H-1,2,3-triazol-1-yl)benzamides as HSP90 inhibitors.

Heat shock protein 90 (HSP90) is a promising anticancer drug target, which could be employed to construct HSP90 inhibitors-based drug conjugates for selective tumor therapy. Herein, a series of 4-(1H-1,2,3-triazol-1-yl)benzamides were rationally designed, synthesized as HSP90 inhibitors, and their structures were characterized by 1H NMR, 13C NMR, and HR-MS. Preliminary HSP90 binding assay showed that compounds 6b, 6l, 6m, 6n, 6t, and 6u exhibited significant HSP90α binding affinity. Among these selected compounds, 6u displayed the most potent anti-proliferative activities and particularly in Capan-1 cell line. Molecular modeling studies also confirmed possible mode of interaction between 6u and the binding sites of HSP90 by hydrogen bond and hydrophobic interactions. Above all, these encouraging data indicated that 6u could be used as a HSP90 inhibitor for further study and helped the recognition of the 4-(1H-1,2,3-triazol-1-yl)benzamide motif as a new scaffold for HSP90 inhibitors.

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo.

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.