Co-chaperone Aha1 Research Articles

Molecular chaperones: Guardians of tumor suppressor stability and function.

The term 'tumor suppressor' describes a widely diverse set of genes that are generally involved in the suppression of metastasis, but lead to tumorigenesis upon loss-of-function mutations. Despite the protein products of tumor suppressors exhibiting drastically different structures and functions, many share a common regulatory mechanism-they are molecular chaperone 'clients'. Clients of molecular chaperones depend on an intracellular network of chaperones and co-chaperones to maintain stability. Mutations of tumor suppressors that disrupt proper chaperoning prevent the cell from maintaining sufficient protein levels for physiological function. This review discusses the role of the molecular chaperones Hsp70 and Hsp90 in maintaining the stability and functional integrity of tumor suppressors. The contribution of cochaperones prefoldin, HOP, Aha1, p23, FNIP1/2 and Tsc1 as well as the chaperonin TRiC to tumor suppressor stability is also discussed. Genes implicated in renal cell carcinoma development-VHL, TSC1/2, and FLCN-will be used as examples to explore this concept, as well as how pathogenic mutations of tumor suppressors cause disease by disrupting protein chaperoning, maturation, and function.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1−/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Aha1 Is an Autonomous Chaperone for SULT1A1.

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Identification of the Hsp90 Isoforms and Co‐Chaperones that Repress Opioid Anti‐Nociception in the Spinal Cord

The various isoforms of the chaperone protein heat shock protein 90 (Hsp90) in conjunction with specific co‐chaperones are key regulators of signal transduction. We have recently identified a novel role for Hsp90 within downstream mu opioid receptor (MOR) signaling both within the brain and spinal cord. Within the brain, we found that Hsp90α and the co‐chaperones Cdc37 and p23 promote MOR‐induced ERK phosphorylation and subsequent anti‐nociception. However, in the spinal cord Hsp90 represses MOR‐induced ERK phosphorylation and subsequent anti‐nociception, so that Hsp90 inhibitor treatment in the spinal cord results in enhanced opioid pain relief. This study aimed to identify the Hsp90 isoforms and co‐chaperones necessary for this mechanism within the spinal cord, and whether they differ from the brain. We used novel isoform and co‐chaperone selective small molecule inhibitors and in vivo gene knockdown using CRISPR/Cas9 to selectively block each of the Hsp90 isoforms and co‐chaperones in the spinal cords of male and female CD‐1 mice. We then used opioid‐induced anti‐nociception in the tail flick assay as a readout, combined with immunohistochemistry to validate CRISPR/Cas9 knockdown in the spinal cord. In contrast to our earlier brain studies, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all acted to repress opioid anti‐nociception in the spinal cord, in that inhibiting these isoforms resulted in increased opioid anti‐nociception. We similarly found that the co‐chaperones Cdc37, p23, and Aha1 all similarly repressed opioid anti‐nociception in the spinal cord. These findings suggest that Hsp90β, Grp94, and Aha1 all act within the spinal cord but not the brain to regulate opioid anti‐nociception. These findings also suggest, in line with our earlier studies, that blocking these isoforms with systemic selective inhibitors could represent a novel therapeutic approach to enhance opioid therapy, increasing analgesic efficacy while reducing side effects like tolerance and reward/addiction.Support or Funding InformationThese studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS, and NIH R01CA213566 to BB. BB is a founder with an equity stake in Grannus Therapeutics, a virtual startup for developing novel Hsp90 inhibitors. No other author has any relevant conflicts to declare.

Dynamic Aha1 co-chaperone binding to human Hsp90.

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co‐chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214‐kDa complex forms by a two‐step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide‐binding site, providing a basis for its ATPase‐enhancing activity. Our data reveal important aspects of this pivotal chaperone/co‐chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.

Co-chaperones TIMP2 and AHA1 Competitively Regulate Extracellular HSP90:Client MMP2 Activity and Matrix Proteolysis

The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2.

Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.

Correction: Silencing of HSP90 Cochaperone AHA1 Expression Decreases Client Protein Activation and Increases Cellular Sensitivity to the HSP90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin.

AHA1 (activator of HSP90 ATPase) is a cochaperone of the ATP-dependent molecular chaperone, HSP90, which is involved in the maturation, stabilization/degradation, and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of cells with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) results in the degradation of client proteins via the ubiquitin-proteasome pathway. As AHA1 increases the ATPase activity of HSP90, we hypothesized that modulation of AHA1 expression could influence the activity of client proteins and/or the cellular response to 17-AAG. We show that the basal expression of AHA1 is different across a panel of human cancer cell lines, and that treatment with 17-AAG resulted in sustained AHA1 up-regulation. Increasing the expression of AHA1 did not affect the sensitivity to 17-AAG, but did increase C-RAF activity and the levels of phosphorylated MEK1/2 and ERK1/2 without affecting total levels of these proteins or of client proteins C-RAF, ERBB2, or CDK4. Conversely, small interfering RNA-selective knockdown of >80% of AHA1 expression decreased C-RAF activity and reduced the levels of MEK1/2 and ERK1/2 phosphorylation. Moreover, the AHA1 knockdown resulted in a significant (P < 0.05) increase in sensitivity to 17-AAG, due in part to a 2- to 3-fold increase in apoptosis. These results show that the reduction of AHA1 levels could decrease the phosphorylation of key signal transduction proteins, and for the first time, separate the activation and stabilization functions of HSP90. Furthermore, AHA1 knockdown could sensitize cancer cells to 17-AAG. We conclude that modulation of AHA1 might be a potential therapeutic strategy to increase sensitivity to HSP90 inhibitors.

Effects of Inhibitors on Hsp90's Conformational Dynamics, Cochaperone and Client Interactions.

The molecular chaperone and heat-shock protein Hsp90 has become a central target in anti-cancer therapy. Nevertheless, the effect of Hsp90 inhibition is still not understood at the molecular level, preventing a truly rational drug design. Here we report on the effect of the most prominent drug candidates, namely, radicicol, geldanamycin, derivatives of purine, and novobiocin, on Hsp90's characteristic conformational dynamics and the binding of three interaction partners. Unexpectedly, the global opening and closing transitions are hardly affected by Hsp90 inhibitors. Moreover, we find no significant changes in the binding of the cochaperones Aha1 and p23 nor of the model substrate Δ131Δ. This holds true for competitive and allosteric inhibitors. Therefore, direct inhibition mechanisms affecting only one molecular interaction are unlikely. We suggest that the inhibitory action observed in vivo is caused by a combination of subtle effects, which can be used in the search for novel Hsp90 inhibition mechanisms.

MP88-13 TUMOR SUPPRESSOR TSC1 IS A NEW HSP90 COCHAPERONE THAT FACILITATES FOLDING OF KINASE AND NON-KINASE CLIENTS

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2018MP88-13 TUMOR SUPPRESSOR TSC1 IS A NEW HSP90 COCHAPERONE THAT FACILITATES FOLDING OF KINASE AND NON-KINASE CLIENTS Mark Woodford, Rebecca Sager, Adam Blanden, Stewart Loh, David Gutmann, Oleg Shapiro, Dimitra Bourboulia, Michael Wong, Gennady Bratslavsky, and Mehdi Mollapour Mark WoodfordMark Woodford More articles by this author , Rebecca SagerRebecca Sager More articles by this author , Adam BlandenAdam Blanden More articles by this author , Stewart LohStewart Loh More articles by this author , David GutmannDavid Gutmann More articles by this author , Oleg ShapiroOleg Shapiro More articles by this author , Dimitra BourbouliaDimitra Bourboulia More articles by this author , Michael WongMichael Wong More articles by this author , Gennady BratslavskyGennady Bratslavsky More articles by this author , and Mehdi MollapourMehdi Mollapour More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2933AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mutation of either TSC1 or TSC2 causes tuberous sclerosis, a multisystem genetic syndrome that can lead to formation of benign tumors in the kidneys, lungs, and other organs and has also been linked to epilepsy and autism. The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however the precise mechanism remains elusive. Molecular chaperones such as heat shock protein-90 (Hsp90) are essential for the stability and activation of numerous signaling proteins, including tumor suppressors. The objective of this work was to determine the role of Hsp90 in the stability and activity of TSC. We hypothesize that Hsp90 mediates TSC formation and thus is essential for the stability of Tsc2. METHODS Tsc1-FLAG and Tsc2-FLAG were transiently expressed and isolated from HEK293 cells. Interacting proteins were identified by co-immunoprecipitation. Hsp90 chaperone function is coupled to its ATPase activity. We have developed an assay to measure this activity by determining the rate of ATP breakdown and inorganic phosphate production. Hsp90 was isolated from WT and TSC1-/- mice and assayed for ATPase activity in vitro. RESULTS Tsc2 interacts with the molecular chaperone Hsp90 both in vitro and in vivo. Treatment of HEK293 cells with Hsp90 inhibitors such as ganetespib or SNX2112 caused ubiquitination and proteasomal degradation of Tsc2; therefore Tsc2 is a new client of Hsp90. However, Tsc1 stability does not depend on Hsp90. Tsc1 potently decelerates Hsp90 ATPase activity and facilitates the interaction of Tsc2 and other clients with Hsp90. We also showed that Tsc1 competes with the activating co-chaperone Aha1 for binding to Hsp90, providing a reciprocal regulatory mechanism for chaperoning of kinase and non-kinase clients. CONCLUSIONS Tsc1 interacts with Tsc2 and protects Tsc2 from degradation. Many pathogenic mutations of Tsc2 destabilize the protein. Our data suggest that lack of interaction of pathogenic Tsc2 mutants with Tsc1 and consequently Hsp90 lead to their degradation. Further, these newly elucidated roles of Tsc1 and Tsc2 suggest a potential mechanism for the observed differences between TSC1 and TSC2 mutant disease. In the absence of Tsc2, Tsc1 will still perform its critical role as a co-chaperone of Hsp90. However when Tsc1 is lost, the stability of both Tsc2 and many other Hsp90 clients is dramatically reduced, leading to a different phenotypic manifestation. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1202 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Mark Woodford More articles by this author Rebecca Sager More articles by this author Adam Blanden More articles by this author Stewart Loh More articles by this author David Gutmann More articles by this author Oleg Shapiro More articles by this author Dimitra Bourboulia More articles by this author Michael Wong More articles by this author Gennady Bratslavsky More articles by this author Mehdi Mollapour More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Structural Insights into the Regulation Mechanism of HSP90 by Co-chaperone AHA1

Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that facilitates the folding and maturation of hundreds of “client” proteins, especially those involved in signaling and regulatory processes. This Hsp90 chaperone machinery is also essential for maintaining cellular protein homeostasis and for dealing with proteotoxic stress. The client maturation cycle is regulated by a large number of co-chaperones that sequentially interact with Hsp90 throughout its ATPase cycle. One of the most important, but poorly understood co-chaperones in eukaryotes is Activator of Hsp90 ATPase homolog 1 (Aha1). As a potent stimulator of the Hsp90 ATPase activity, Aha1 is thought to accelerate client protein remodeling. A knockout of Aha1 in yeast severely compromises the maturation of different Hsp90 client proteins. However, Aha1 overexpression inhibits the rate of Hsp90-dependent refolding of denatured luciferase, as well as cystic fibrosis associated protein CFTR. How Aha1, as an Hsp90 activator, facilitates ATP hydrolysis while inhibiting client maturation remains enigmatic. To better understand this question, we have determined the structure of the full-length 200-kDa Hsp90/Aha1 complex at 3.8 Å resolution using cryo-electron microscopy. When bound to Aha1, the Hsp90 was captured in the closed conformation, similar to the X-ray crystal structure of Hsp90 bound with inhibitory co-chaperone p23. However, the binding interface is completely different from that used by p23 as well as that observed by crystallography of a fragment of Aha bound to a fragment of Hsp90. In our structure, the C-terminal domain of Aha1 stabilizes the Hsp90 closed dimer by bridging the C-terminal domain of one protomer and the middle domain/N-terminal domain of the other protomer. This structure begins to explain the Aha1 inhibitory effect on Hsp90 and also provides a basis for designing co-chaperone specific inhibitors to regulate Hsp90 related diseases.

Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1

The function of the molecular chaperone Hsp90 depends on large conformational changes, the rearrangement of local motifs, and the binding and hydrolysis of ATP. The size and complexity of the Hsp90 system impedes the detailed investigation of their interplay using standard methods. To overcome this limitation, we developed a three-color single-molecule FRET assay to study the interaction of Hsp90 with a fluorescently labeled reporter nucleotide in detail. It allows us to directly observe the cooperativity between the two nucleotide binding pockets in the protein dimer. Furthermore, our approach disentangles the protein conformation and the nucleotide binding state of Hsp90 and extracts the kinetics of the state transitions. Thereby, we can identify the kinetic causes mediating the cooperativity. We find that the presence of the first nucleotide prolongs the binding of the second nucleotide to Hsp90. In addition, we observe changes in the kinetics for both the open and the closed conformation of Hsp90 in dependence on the number of occupied nucleotide binding sites. Our analysis also reveals how the co-chaperone Aha1, known to accelerate Hsp90’s ATPase activity, affects those transitions in a nucleotide-dependent and independent manner, thereby adding another layer of regulation to Hsp90.

A chemical compound inhibiting the Aha1–Hsp90 chaperone complex

The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins in vivo We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90.

Hsp90 Sensitivity to ADP Reveals Hidden Regulation Mechanisms

The ATPase cycle of the Hsp90 molecular chaperone is essential for maintaining the stability of numerous client proteins. Extensive analysis has focused on ATP-driven conformational changes of Hsp90; however, little is known about how Hsp90 operates under physiological nucleotide conditions in which both ATP and ADP are present. By quantifying Hsp90 activity under mixed nucleotide conditions, we find dramatic differences in ADP sensitivity among Hsp90 homologs. ADP acts as a strong ATPase inhibitor of cytosol-specific Hsp90 homologs, whereas organellular Hsp90 homologs (Grp94 and TRAP1) are relatively insensitive to the presence of ADP. These results imply that an ATP/ADP heterodimer of cytosolic Hsp90 is the predominant active state under physiological nucleotide conditions. ADP inhibition of human and yeast cytosolic Hsp90 can be relieved by the cochaperone aha1. ADP inhibition of bacterial Hsp90 can be relieved by bacterial Hsp70 and an activating client protein. These results suggest that altering ADP inhibition may be a mechanism of Hsp90 regulation. To determine the molecular origin of ADP inhibition, we identify residues that preferentially stabilize either ATP or ADP. Mutations at these sites can both increase and decrease ADP inhibition. An accounting of ADP is critically important for designing and interpreting experiments with Hsp90. For example, contaminating ADP is a confounding factor in fluorescence resonance energy transfer experiments measuring arm closure rates of Hsp90. Our observations suggest that ADP at physiological levels is important to Hsp90 structure, activity, and regulation.

The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles.

Heat Shock Protein 90 (Hsp90) is essential for tumor progression in humans and drug resistance in fungi. However, the roles of its many co-chaperones in antifungal resistance are unknown. In this study, by susceptibility test of Neurospora crassa mutants lacking each of 18 Hsp90/Calcineurin system member genes (including 8 Hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the Hsp90 co-chaperones Sti1 (Hop1 in yeast), Aha1, and P23 (Sba1 in yeast) were required for the basal resistance to antifungal azoles and heat stress. Deletion of any of them resulted in hypersensitivity to azoles and heat. Liquid chromatography–mass spectrometry (LC-MS) analysis showed that the toxic sterols eburicol and 14α-methyl-3,6-diol were significantly accumulated in the sti1 and p23 deletion mutants after ketoconazole treatment, which has been shown before to led to cell membrane stress. At the transcriptional level, Aha1, Sti1, and P23 positively regulate responses to ketoconazole stress by erg11 and erg6, key genes in the ergosterol biosynthetic pathway. Aha1, Sti1, and P23 are highly conserved in fungi, and sti1 and p23 deletion also increased the susceptibility to azoles in Fusarium verticillioides. These results indicate that Hsp90-cochaperones Aha1, Sti1, and P23 are critical for the basal azole resistance and could be potential targets for developing new antifungal agents.

Overexpressing target helper genes enhances secretion and glycosylation of recombinant proteins in Pichia pastoris under simulated microgravity

In this study, the potential helper genes were identified through the data analysis of transcriptomic and proteomic profiling in recombinant Pichia pastoris cultured under simulated microgravity (SMG). Co-expressing of four genes PRX1, YAP1, AHA1, and YPT6, involved in the oxidative stress response and protein folding, exhibited promising helper factor effects on the recombinant protein yields in engineered P. pastoris, respectively. When two of the above genes were co-expressed simultaneously, β-glucuronidase (PGUS) specific activity was further increased by 30.3-50.6% comparing with that of single helper gene, particularly when the oxidative stress response and protein folding genes were both present in the combinations. In addition, co-expressing co-chaperone AHA1 and transcription factor YAP1 not only enhanced PGUS secretion, but also affected its glycosylation. Thus, through deep "omics" analysis of SMG effects, our results provided combined impact of new helper factors to improve the efficacy of recombinant protein secretion and glycosylation in engineered P. pastoris.

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

Cooperation of local motions in the Hsp90 molecular chaperone ATPase mechanism

The Hsp90 chaperone is a central node of protein homeostasis activating a large number of diverse client proteins. Hsp90 functions as a molecular clamp that closes and opens in response to the binding and hydrolysis of ATP. Crystallographic studies define distinct conformational states of the mechanistic core implying structural changes that have not yet been observed in solution. Here, we engineered one-nanometer fluorescence probes based on photo-induced electron transfer into yeast Hsp90 to observe these motions. We found that the ATPase activity of the chaperone was reflected in the kinetics of specific structural rearrangements at remote positions that acted cooperatively. Nanosecond single-molecule fluorescence fluctuation analysis uncovered that critical structural elements that undergo rearrangement are mobile on a sub-millisecond time scale. We identified a two-step mechanism for lid closure over the nucleotide-binding pocket. The activating co-chaperone Aha1 mobilizes the lid of apo Hsp90, suggesting an early role in the catalytic cycle.

C-Abl Mediated Tyrosine Phosphorylation of Aha1 Activates Its Co-chaperone Function in Cancer Cells.

SummaryThe ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific “client” proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.

Hsp90 Oligomers Interacting with the Aha1 Cochaperone: An Outlook for the Hsp90 Chaperone Machineries.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.