Hsp90 Complex Research Articles

Identification of Novel Cdc37 Interacting Proteins and Pathways in Human Alzheimer’ s Disease Brain Tissue Using Mass Spectrometry

Alzheimer’s disease (AD) is the most common form of dementia and the 6th leading cause of death in the United States. The major pathological hallmarks observed in AD include the formation of intracellular neurofibrillary tangles comprised of phosphorylated forms of the microtubule associated protein tau, and the deposition of extracellular plaques composed of amyloid beta. Cdc37 is a co-chaperone of Hsp90, which recruits client kinases to the Hsp90 complex for folding and stabilization. It has been previously shown that Cdc37 can not only bind and preserve tau, but also stabilize kinases that can phosphorylate tau. The goal of the current study was to identify novel Cdc37- interacting proteins in human AD tissue compared to normal tissue using an immunoprecipitation-based approach combined with mass spectrometry. We identified 39 unique proteins that interacted with Cdc37 in AD samples only and 7 proteins that interacted with Cdc37 in normal samples only. 39 proteins were found to bind Cdc37 in both AD and normal tissue. Of these, 18 showed increased interaction in AD tissue, 10 showed increased interaction in normal tissue and 11 showed equal nteraction in both samples. Ingenuity Pathway Analysis of the data indicates that these Cdc37-interacting proteins could signal through the p70S6K, PI3K / Akt, TGFs, ErbB, NF- kB, calmodulin, p38 MAPK and JNK pathways. Identification of these novel proteins and pathways linked to Cdc37 may indicate its role both as a non-kinase co-chaperone and in other pathways in the AD brain.

17-Allylamino-17-Demethoxygeldanamycin and the Enhancement of PS-341-Induced Lung Cancer Cell Death by Blocking the NF-κB and PI3K/Akt Pathways.

PS-341 is a highly selective and potent proteasome inhibitor that is cytotoxic to various types of cancer. However, no objective response was seen in a clinical trial with PS-341 as a single agent in non-small cell lung cancer. Its antitumor activity is limited by the simultaneously activated antiapoptosis pathway. Recently, PS-341-induced NF-κB activation via IκBα degradation has been suggested to be one of its antiapoptotic effects. In this study, we investigated the effects of a combined application of the heat shock protein (Hsp) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) with PS-341 in lung cancer cells. Hsp90 inhibition with 17-AAG was effective in enhancing PS-341-induced lung cancer cell death in vitro and in vivo. 17-AAG pretreatment induced the degradation of upstream regulators of IκB, IL-1R-associated kinase-1 (IRAK-1), and IκB kinases (IKKs), dose and time dependently, which resulted in blocking of PS-341-induced IκBα degradation, p65 nuclear translocation, transcriptional activity, and NF-κB-regulated antiapoptotic gene expressions such as COX-2. The concentrations of 17-AAG used for combinatorial treatment with PS-341 did not change cell viability or the activity of proteasome complex. Moreover, 17-AAG pretreatment decreased the level of phsophorylated Akt at serine 473 residue and suppressed active Akt-dependent inactivation of glycogen synthase kinase 3β. 17-AAG mediated the dissociation of its client proteins (IRAK-1, IKKs, and Akt) from the Hsp90 complex. As a result, it induced degradation of target proteins. Our results suggest that the combination of 17-AAG and PS-341 could be an effective anticancer therapy that overcomes the limited effects of PS-341.

Abstract 1733: Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells

Abstract Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today's proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733

A Novel Approach to Inhibit Heat Shock Response as Anticancer Strategy by Coumarine Compounds Containing Thiazole Skeleton.

Inhibition of the Hsp90 function is an essential therapeutic approach and several inhibitors were designed as anti-cancer agents. These inhibitors are ATPases and they aim to deregulate Hsp90 folding function. ATPase proteins are common in human metabolism but they form nonspecific targets. Hsp90 functions as dimer with coordinating chaperones. Heat Shock Organizing Protein (Hop) forms a bridge between Hsp90 and Hsp70-Hsp40 complex to form Hsp90-Hsp70 coordination. Perturbing conformational changes of these Hsp proteins, dimer formation, and protein-protein interactions inhibit Hsp90 substrate protein folding function. This approach does not target all ATPase proteins but targets Hsp90 function solely. For this purpose, we designed compounds to block Hsp90 function. Moreover, molecular docking studies as well as competition analysis of the compounds were performed with Hsp90. Novel thiazolyl coumarine compounds were determined as valuable C-terminal Hsp90 inhibitors and provide promising templates for the drug design. Anticancer activities of these novel compounds were tested by employing human colon (DLD-1) and liver cancer (HepG2) cell lines. Thiazolyl coumarine compounds are found to be significant and useful for the treatment of human colon and liver cancer as evidenced by in vitro and in silico results.

Targeting a stress-associated hsp90 complex as a novel therapeutic strategy to downregulate toxic tau in Alzheimer’s disease

Targeting a stress-associated hsp90 complex as a novel therapeutic strategy to downregulate toxic tau in Alzheimer’s disease

Oncogenic acidic nuclear phosphoproteins ANP32C/D are novel clients of heat shock protein 90

The acidic nuclear phosphoproteins (ANP32A-H) are an evolutionarily conserved family of proteins with diverse and sometimes opposing cellular functions. Here we show that the oncogenic family members ANP32C and ANP32D are associated in complexes containing the molecular chaperone Hsp90. The oncogenic ANP32C protein appears to be highly unstable with a rapid degradation (t1/2>30min) occurring upon treatment of cells with cycloheximide. ANP32C was also found to be associated with oncogenic Hsp90 complexes by virtue of its ability to interact and be immunoprecipitated by the Hsp90 inhibitor PU-H71. Further studies treating cells with the Hsp90 inhibitors PU-H71 and 17-AAG showed atypical increased protein stability and prevention of ANP32C degradation compared to the Hsp90 client AKT. Cells overexpressing ANP32C or its mutant ANP32CY140H showed enhanced sensitivity to treatment with PU-H71 as demonstrated by CCK-8 and colony formation assays. Our results highlight that certain malignancies with ANP32C/D overexpression or mutation might be specifically targeted using Hsp90 inhibitors.

Leishmania donovani P23 protects parasites against HSP90 inhibitor-mediated growth arrest.

In Leishmania donovani, the HSP90 chaperone complex plays an essential role in the control of the parasite's life cycle, general viability and infectivity. Several of the associated co-chaperones were also shown to be essential for viability and/or infectivity to mammalian cells. Here, we identify and describe the co-chaperone P23 and distinguish its function from that of the structurally related small heat shock protein HSP23. P23 is expressed constitutively and associates itself with members of the HSP90 complex, i.e. HSP90 and Sti1. Viable P23 gene replacement mutants could be raised and confirmed as null mutants without deleterious effects on viability under a variety of physiological growth conditions. The null mutant also displays near-wild-type infectivity, arguing against a decisive role played by P23 in laboratory settings. However, the P23 null mutant displays a marked hypersensitivity against HSP90 inhibitors geldanamycin and radicicol. P23 also appears to affect the radicicol resistance of a HSP90 Leu33-Ile mutant described previously. Therefore, the annotation of L. donovani P23 as HSP90-interacting co-chaperone is confirmed.

KU675, a Concomitant Heat-Shock Protein Inhibitor of Hsp90 and Hsc70 that Manifests Isoform Selectivity for Hsp90α in Prostate Cancer Cells.

The 90-kDa heat-shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Inhibiting Hsp90 consequently is an attractive strategy for cancer therapy as the concomitant degradation of multiple oncoproteins may lead to effective antineoplastic agents. Here we report a novel C-terminal Hsp90 inhibitor, designated KU675, that exhibits potent antiproliferative and cytotoxic activity along with client protein degradation without induction of the heat-shock response in both androgen-dependent and -independent prostate cancer cell lines. In addition, KU675 demonstrates direct inhibition of Hsp90 complexes as measured by the inhibition of luciferase refolding in prostate cancer cells. In direct binding studies, the internal fluorescence signal of KU675 was used to determine the binding affinity of KU675 to recombinant Hsp90α, Hsp90β, and Hsc70 proteins. The binding affinity (Kd) for Hsp90α was determined to be 191 μM, whereas the Kd for Hsp90β was 726 μM, demonstrating a preference for Hsp90α. Western blot experiments with four different prostate cancer cell lines treated with KU675 supported this selectivity by inducing the degradation of Hsp90α -: dependent client proteins. KU675 also displayed binding to Hsc70 with a Kd value at 76.3 μM, which was supported in cellular by lower levels of Hsc70-specific client proteins on Western blot analyses. Overall, these findings suggest that KU675 is an Hsp90 C-terminal inhibitor, as well as a dual inhibitor of Hsc70, and may have potential use for the treatment of cancer.

Antiproliferative effect of HSP90 inhibitor Y306zh against pancreatic cancer is mediated by interruption of AKT and MAPK signaling pathways.

Inhibition of HSP90 is a potential strategy to treat pancreatic cancer (PC), since many client proteins of HSP90 are found to be over-expressed and/or genetically mutated in PC. These client proteins directly participate in the modulation of proliferation and aggravation of PC. Our previous works led to Y306zh as a novel and potent HSP90 inhibitor. In this study we further investigated the mode of actions of Y306zh as an anti-PC agent. The binding affinity of Y306zh towards HSP90 was analyzed by fluorescence polarization assay. The anti-proliferative activities and molecular mechanism of Y306zh were investigated in human PC cell lines and Miapaca2 xenograft model. Y306zh binds tightly to the NH2-terminus of HSP90 with an IC50 of 85 nM, and this causes ATP incapable to attach to the same binding site, and disrupts HSP90-p23 association. Y306zh also induces the expression of HSP70, recruits the complex of CHIP, HSP70 and HSP90, and eventually results in the degradation of HSP90 client proteins (EGFR, AKT, C-RAF and CDK4) via proteasomal pathway. Amazingly, coincident with the molecular mechanism, Y306zh also inhibits the proliferation of PC cells through G2/M phase arrest, but appears to be totally insensitive to normal mammalian cells. Furthermore, Y306zh effectively inhibits tumor growth of Mia-paca2 xenograft mice without any obvious effect on body weight. Y306zh, a novel HSP90 inhibitor, interrupts ATP binding to HSP90 and disrupts HSP90-p23 interaction, and eventually inhibits the growth of PC cells.

HINT1 peptide/Hsp70 complex induces NK-cell-dependent immunoregulation in a model of autoimmune demyelination.

Heat shock proteins (Hsps) interact with the immune system and have been shown to contribute to immunoregulation. As efficient chaperones, Hsps bind many peptides and these complexes have many yet-to-be-clarified functions. We have shown that Hsp70 is complexed within the mouse CNS with peptide CLAFHDISPQAPTHFLVIPK derived from histidine triad nucleotide-binding protein-1 (HINT1₃₈₋₅₇/Hsp70). Only this complex, in contrast to other peptides complexed with Hsp70, was able to prevent experimental autoimmune encephalomyelitis (EAE) by induction of immunoregulatory mechanisms dependent on NK cells. Pretreatment of proteolipid protein peptide ₁₃₉₋₁₅₁(PLP₁₃₉₋₁₅₁) sensitized SJL/J mice with HINT1₃₈₋₅₇/Hsp70 prevented the development of EAE, suppressed PLP₁₃₉₋₁₅₁-induced T-cell proliferation, and blocked secretion of IL-17. HINT1₃₈₋₅₇ /Hsp70 stimulation of NK cells depended on synergistic activation of two NK-cell receptors, CD94 and NKG2D. NK cells with depleted CD94 or with blocked NKG2D did not inhibit PLP₁₃₉₋₁₅₁-induced spleen cell (SC) proliferation. The HINT1₃₈₋₅₇/Hsp70 complex enhanced surface expression of the NKG2D ligand-H60. Downstream signaling of CD94 and NKG2D converged at the adaptor proteins DAP10 and DAP12, and in response to HINT1₃₈₋₅₇ /Hsp70 stimulation, expression of DAP10 and DAP12 was significantly increased in NK cells. Thus, we have shown that the HINT1₃₈₋₅₇ /Hsp70 complex affects NK-cell function by enhancing NK-cell-dependent immunoregulation in the EAE model of autoimmune demyelination.

Clathrin complexes with the inhibitor kappa B kinase signalosome: imaging the interactome.

Many receptors involved with innate immunity activate the inhibitor kappa B kinase signalosome (IKK). The active complex appears to be assembled from the two kinase units, IKKα and IKKβ with the regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNFα‐induced signaling and phosphorylation of inhibitor kappa B (IκB), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKKα and NEMO interactomes. Analysis of the affinity interactomes by mass spectrometry detected clathrin with both baits with high confidence. Using the same antibodies for indirect digital immunofluorescence microscopy and FRET, the CHC–IKK complexes were visualized together with NEMO or HSP90. The natural variability of protein amounts in unsynchronized A549 cells was used to obtain statistical correlation for several complexes, at natural levels and without invasive labeling. Analyses of voxel numbers indicated that: (i) CHC–IKK complexes are not part of the IKK signalosome itself but, likely, precursors of IKK–NEMO complexes. (ii) CHC–IKKβ complexes may arise from IKKβ–HSP90 complexes.

Affinity purification probes of potential use to investigate the endogenous Hsp70 interactome in cancer.

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.

Cruentaren A Binds F1F0 ATP Synthase To Modulate the Hsp90 Protein Folding Machinery

The molecular chaperone Hsp90 requires the assistance of immunophilins, co-chaperones, and partner proteins for the conformational maturation of client proteins. Hsp90 inhibition represents a promising anticancer strategy due to the dependence of numerous oncogenic signaling pathways upon Hsp90 function. Historically, small molecules have been designed to inhibit ATPase activity at the Hsp90 N-terminus; however, these molecules also induce the pro-survival heat shock response (HSR). Therefore, inhibitors that exhibit alternative mechanisms of action that do not elicit the HSR are actively sought. Small molecules that disrupt Hsp90-co-chaperone interactions can destabilize the Hsp90 complex without induction of the HSR, which leads to inhibition of cell proliferation. In this article, selective inhibition of F1F0 ATP synthase by cruentaren A was shown to disrupt the Hsp90-F1F0 ATP synthase interaction and result in client protein degradation without induction of the HSR.

A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

Export of newly synthesized G protein-coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D2 (PGD2) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1-Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD2 synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1-ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD2) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.

Hsp90-Tau Complex Reveals Molecular Basis for Specificity in Chaperone Action

Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is poorly understood. Here, we obtained a structural model of Hsp90 in complex with its natural disease-associated substrate, the intrinsically disordered Tau protein. Hsp90 binds to a broad region in Tau that includes the aggregation-pronerepeats. Complementarily, a 106-Å-long substrate-binding interface in Hsp90 enables many low-affinity contacts. This allows recognition of scattered hydrophobic residues in late folding intermediates that remain after early burial of the Hsp70 sites. Our model resolves the paradox of how Hsp90 specifically selects for late folding intermediates but also for some intrinsically disordered proteins-through the eyes of Hsp90 they look the same.

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

IP‐FCM platform detects the existence and regulator‐caused dissociation of components in naturally assembled HSP90 complex

Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors.

Differential Modulation of Functional Dynamics and Allosteric Interactions in the Hsp90-Cochaperone Complexes with p23 and Aha1: A Computational Study

Allosteric interactions of the molecular chaperone Hsp90 with a large cohort of cochaperones and client proteins allow for molecular communication and event coupling in signal transduction networks. The integration of cochaperones into the Hsp90 system is driven by the regulatory mechanisms that modulate the progression of the ATPase cycle and control the recruitment of the Hsp90 clientele. In this work, we report the results of computational modeling of allosteric regulation in the Hsp90 complexes with the cochaperones p23 and Aha1. By integrating protein docking, biophysical simulations, modeling of allosteric communications, protein structure network analysis and the energy landscape theory we have investigated dynamics and stability of the Hsp90-p23 and Hsp90-Aha1 interactions in direct comparison with the extensive body of structural and functional experiments. The results have revealed that functional dynamics and allosteric interactions of Hsp90 can be selectively modulated by these cochaperones via specific targeting of the regulatory hinge regions that could restrict collective motions and stabilize specific chaperone conformations. The protein structure network parameters have quantified the effects of cochaperones on conformational stability of the Hsp90 complexes and identified dynamically stable communities of residues that can contribute to the strengthening of allosteric interactions. According to our results, p23-mediated changes in the Hsp90 interactions may provide “molecular brakes” that could slow down an efficient transmission of the inter-domain allosteric signals, consistent with the functional role of p23 in partially inhibiting the ATPase cycle. Unlike p23, Aha1-mediated acceleration of the Hsp90-ATPase cycle may be achieved via modulation of the equilibrium motions that facilitate allosteric changes favoring a closed dimerized form of Hsp90. The results of our study have shown that Aha1 and p23 can modulate the Hsp90-ATPase activity and direct the chaperone cycle by exerting the precise control over structural stability, global movements and allosteric communications in Hsp90.

A complex of Cox4 and mitochondrial Hsp70 plays an important role in the assembly of the cytochrome c oxidase

The formation of the mature cytochrome c oxidase (complex IV) involves the association of nuclear- and mitochondria-encoded subunits. The assembly of nuclear-encoded subunits like cytochrome c oxidase subunit 4 (Cox4) into the mature complex is poorly understood. Cox4 is crucial for the stability of complex IV. To find specific biogenesis factors, we analyze interaction partners of Cox4 by affinity purification and mass spectroscopy. Surprisingly, we identify a complex of Cox4, the mitochondrial Hsp70 (mtHsp70), and its nucleotide-exchange factor mitochondrial GrpE (Mge1). We generate a yeast mutant of mtHsp70 specifically impaired in the formation of this novel mtHsp70-Mge1-Cox4 complex. Strikingly, the assembly of Cox4 is strongly decreased in these mutant mitochondria. Because Cox4 is a key factor for the biogenesis of complex IV, we conclude that the mtHsp70-Mge1-Cox4 complex plays an important role in the formation of cytochrome c oxidase. Cox4 arrests at this chaperone complex in the absence of mature complex IV. Thus the mtHsp70-Cox4 complex likely serves as a novel delivery system to channel Cox4 into the assembly line when needed.

Hsp90 cochaperones p23 and FKBP4 physically interact with hAgo2 and activate RNA interference–mediated silencing in mammalian cells

Argonaute proteins and small RNAs together form the RNA-induced silencing complex (RISC), the central effector of RNA interference (RNAi). The molecular chaperone Hsp90 is required for the critical step of loading small RNAs onto Argonaute proteins. Here we show that the Hsp90 cochaperones Cdc37, Aha1, FKBP4, and p23 are required for efficient RNAi. Whereas FKBP4 and p23 form a stable complex with hAgo2, the function of Cdc37 in RNAi appears to be indirect and may indicate that two or more Hsp90 complexes are involved. Our data also suggest that p23 and FKBP4 interact with hAgo2 before small RNA loading and that RISC loading takes place in the cytoplasm rather than in association with RNA granules. Given the requirement for p23 and FKBP4 for efficient RNAi and that these cochaperones bind to hAgo2, we predict that loading of hAgo2 is analogous to Hsp90-mediated steroid hormone receptor activation. To this end, we outline a model in which FKBP4, p23, and Aha1 cooperatively regulate the progression of hAgo2 through the chaperone cycle. Finally, we propose that hAgo2 and RNAi can serve as a robust model system for continued investigation into the Hsp90 chaperone cycle.