Hsp90 Cofactors Research Articles

Coordinated targeting of CK2 and KIT in gastrointestinal stromal tumours

BackgroundMost gastrointestinal stromal tumours (GIST) are driven by activating oncogenic mutations of KIT/PDGFRA, which provide a compelling therapeutic target. Our previous studies showed that CDC37, regulated by casein kinase 2 (CK2), is a crucial HSP90 cofactor for KIT oncogenic function and a promising and more selective therapeutic target in GIST.MethodsBiologic mechanisms of CK2-mediated CDC37 regulation were assessed in GISTs by immunoblotting, immunoprecipitations, knockdown and inactivation assays. The effects of a combination of KIT and CK2 inhibition were assessed by immunoblotting, cell viability, colony growth, cell cycle analysis, apoptosis, migration and invasiveness.ResultsCK2 overexpression was demonstrated by immunoblotting in GIST cell lines and patient biopsies. Treatment with a specific CK2 inhibitor, CX4945, leads to CDC37 dephosphorylation and inhibits KIT signalling in imatinib-sensitive and in imatinib-resistant GIST cell lines. Immunoprecipitation demonstrated that CK2 inhibition blocks KIT:HSP90:CDC37 interaction in GIST cells. Coordinated inhibition of CK2 and KIT by CX4945 (or CK2 shRNA) and imatinib, respectively, leads to increased apoptosis, anti-proliferative effects and cell cycle arrest and decreased p-AKT and p-S6 expression, migration and invasiveness in all GIST cell lines compared with either intervention alone, indicating additive effects of inhibiting these two important regulators of GIST biology.ConclusionOur findings suggest that combinatorial inhibition of CK2 and KIT warrants evaluation as a novel therapeutic strategy in GIST, especially in imatinib-resistant GIST.

Hsp90-downregulation influences the heat-shock response, innate immune response and onset of oocyte development in nematodes.

Hsp90 is a molecular chaperone involved in the regulation and maturation of kinases and transcription factors. In Caenorhabditis elegans, it contributes to the development of fertility, maintenance of muscle structure, the regulation of heat-shock response and dauer state. To understand the consequences of Hsp90-depletion, we studied Hsp90 RNAi-treated nematodes by DNA microarrays and mass spectrometry. We find that upon development of phenotypes the levels of chaperones and Hsp90 cofactors are increased, while specific proteins related to the innate immune response are depleted. In microarrays, we further find many differentially expressed genes related to gonad and larval development. These genes form an expression cluster that is regulated independently from the immune response implying separate pathways of Hsp90-involvement. Using fluorescent reporter strains for the differentially expressed immune response genes skr-5, dod-24 and clec-60 we observe that their activity in intestinal tissues is influenced by Hsp90-depletion. Instead, effects on the development are evident in both gonad arms. After Hsp90-depletion, changes can be observed in early embryos and adults containing fluorescence-tagged versions of SEPA-1, CAV-1 or PUD-1, all of which are downregulated after Hsp90-depletion. Our observations identify molecular events for Hsp90-RNAi induced phenotypes during development and immune responses, which may help to separately investigate independent Hsp90-influenced processes that are relevant during the nematode’s life and development.

Nematode Sgt1-Homologue D1054.3 Binds Open and Closed Conformations of Hsp90 via Distinct Binding Sites

Heat shock protein 90 (Hsp90) is a highly conserved ATP-driven machine involved in client protein maturation, folding, and activation. The chaperone is supported by a set of cochaperones that confer client specificities. One of those proteins is the suppressor of G2 allele of skp1 (Sgt1), which participates together with Hsp90 in the immune responses of plants. Sgt1 consists of three domains: a TPR-, CS-, and SGS-domain, conserved in plants, yeast, and humans. The TPR-domain though is lacking in nematodes and insects. We observe that the Caenorhabditis elegans Sgt1 homologue D1054.3 binds to Hsp90 in the absence of nucleotides but much stronger in the presence of ATP and ATPγS. The latter binding mode is similar to p23, another CS-domain containing Hsp90 cofactor, even though binding is not observable for p23 in the absence of nucleotides. We use point mutations in Hsp90, which accumulate different conformations in the ATPase cycle, to differentiate between binding to open and closed Hsp90 conformations. These data support a strong contribution of the Hsp90 conformation to Sgt1 binding and highlight the ability of this cofactor to interact with all known Hsp90 conformations albeit with different affinities.

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.

The promise of heat shock protein inhibitors in the treatment of castration resistant prostate cancer

To present the recent advances in novel agents that target heat shock proteins (Hsps) to treat or delay the development of castration resistant prostate cancer (CRPC). Multiple preclinical studies have shown that silencing Hsp27, Hsp90, or clusterin sensitizes prostate cancer cells to modern chemotherapy and radiation treatments; and overexpression of these chaperones confers resistance to these therapies. Antisense oligonucleotides targeting Hsp27 and clusterin have shown good biological activity in human phase II trials and phase III studies are ongoing. Despite promising preclinical efficacy, a number of phase I/II human trials with various Hsp90 inhibitors have been disappointing with negligible anticancer activity and dose-limiting toxicity profiles. Newer Hsp90 inhibitors with better toxicity profiles, and inhibitors that target Hsp90 cofactors, such as FKBP52, are currently being investigated in human studies. Many Hsp chaperone client proteins are key components of alternative growth factor pathways upregulated in CRPC and are involved in key resistance pathways to current chemotherapy and radiotherapy regimes. New treatments that inhibit Hsps are attractive anticancer strategies as they have the ability to simultaneously target multiple pathways involved in CRPC.

Genome-wide functional screening identifies CDC37 as a crucial HSP90-cofactor for KIT oncogenic expression in gastrointestinal stromal tumors

Most gastrointestinal stromal tumors (GISTs) contain KIT or PDGFRA kinase gain-of-function mutations, and therefore respond clinically to imatinib and other tyrosine kinase inhibitor (TKI) therapies. However, clinical progression subsequently results from selection of TKI-resistant clones, typically containing secondary mutations in the KIT kinase domain, which can be heterogeneous between and within GIST metastases in a given patient. TKI-resistant KIT oncoproteins require HSP90 chaperoning and are potently inactivated by HSP90 inhibitors, but clinical applications in GIST patients are constrained by the toxicity resulting from concomitant inactivation of various other HSP90 client proteins, beyond KIT and PDGFRA. To identify novel targets responsible for KIT oncoprotein function, we performed parallel genome-scale short hairpin RNA (shRNA)-mediated gene knockdowns in KIT-mutant GIST-T1 and GIST882. GIST cells were infected with a lentiviral shRNA pooled library targeting 11 194 human genes, and allowed to proliferate for 5-7 weeks, at which point assessment of relative hairpin abundance identified the HSP90 cofactor, CDC37, as one of the top six GIST-specific essential genes. Validations in treatment-naive (GIST-T1, GIST882) vs imatinib-resistant GISTs (GIST48, GIST430) demonstrated that: (1) CDC37 interacts with oncogenic KIT; (2) CDC37 regulates expression and activation of KIT and downstream signaling intermediates in GIST; and (3) unlike direct HSP90 inhibition, CDC37 knockdown accomplishes prolonged KIT inhibition (>20 days) in GIST. These studies highlight CDC37 as a key biologic vulnerability in both imatinib-sensitive and imatinib-resistant GIST. CDC37 targeting is expected to be selective for KIT/PDGFRA and a subset of other HSP90 clients, and thereby represents a promising strategy for inactivating the myriad KIT/PDGFRA oncoproteins in TKI-resistant GIST patients.

The 26S proteasome binds and degrades Pih1 via the C‐terminal degron elements in yeast Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae, R2TP complex is required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. This complex is composed of Rvb1 and Rvb2, two ATP‐dependent DNA helicases, Tah1, an Hsp90 cofactor, and Pih1, a subunit that acts as a scaffold and likely regulates the Rvb1/Rvb2 helicase activities. We have previously shown that Rvb1 and Rvb2 bind to the N‐terminal fragment of Pih1, while Tah1 binds to the Pih1 C‐terminal fragment. Additionally, the C‐terminus of Pih1 acts as a degron that regulates Pih1 degradation in vivo and controls R2TP complex assembly. To understand the mechanism of Pih1 degradation in vivo, we purified Pih1 protein complex from yeast cells and immunoblotting analysis indicated that Pih1 is able to pull down some 26S proteasome subunits. In vitro binding assays indicated that Pih1 C‐terminal degron elements are likely to interact with 26S proteasome directly. Based on in vitro and in vivo degradation assay, we propose a model of proteasome dependent, but ubiquitin independent degradation of Pih1 in yeast cells. (This work is supported by a Connaught New Staff Matching Fund and an NSERC Discovery Grant to RZ)

The Stability of the Small Nucleolar Ribonucleoprotein (snoRNP) Assembly Protein Pih1 in Saccharomyces cerevisiae Is Modulated by Its C Terminus

Pih1 is an unstable protein and a subunit of the R2TP complex that, in yeast Saccharomyces cerevisiae, also contains the helicases Rvb1, Rvb2, and the Hsp90 cofactor Tah1. Pih1 and the R2TP complex are required for the box C/D small nucleolar ribonucleoprotein (snoRNP) assembly and ribosomal RNA processing. Purified Pih1 tends to aggregate in vitro. Molecular chaperone Hsp90 and its cochaperone Tah1 are required for the stability of Pih1 in vivo. We had shown earlier that the C terminus of Pih1 destabilizes the protein and that the C terminus of Tah1 binds to the Pih1 C terminus to form a stable complex. Here, we analyzed the secondary structure of the Pih1 C terminus and identified two intrinsically disordered regions and five hydrophobic clusters. Site-directed mutagenesis indicated that one predicted intrinsically disordered region IDR2 is involved in Tah1 binding, and that the C terminus of Pih1 contains multiple destabilization or degron elements. Additionally, the Pih1 N-terminal domain, Pih1(1-230), was found to be able to complement the physiological role of full-length Pih1 at 37 °C. Pih1(1-230) as well as a shorter Pih1 N-terminal fragment Pih1(1-195) is able to bind Rvb1/Rvb2 heterocomplex. However, the sequence between the two disordered regions in Pih1 significantly enhances the Pih1 N-terminal domain binding to Rvb1/Rvb2. Based on these data, a model of protein-protein interactions within the R2TP complex is proposed.

The function of HSP90 in MHC class II presentation (106.31)

Abstract A role for heat shock proteins (HSP) in promoting antigen presentation via the MHC class I pathway is well established and appears to play a key role in host recognition of tumors and transformed cells. Studies suggest that some HSP may promote the presentation of cytoplasmic antigens via the class I pathway, as well as selecting cytoplasmic antigens for presentation in the context of MHC class II molecules. A role for intracellular HSP90 as well as HSC70 in regulating the presentation of a cytoplasmic autoantigen, glutamic acid decarboxylase (GAD) has been demonstrated in B lymphoblasts. Modulating intracellular expression of HSP90 alpha in human B lymphoblasts altered class II presentation of GAD epitopes. Yet, diminished cellular HSP90 alpha expression did not alter the steady state levels of GAD antigen, or disrupt the trafficking of this native antigen from the cytoplasm to membraneous intracellular organelles. This may suggest HSP90 acts after antigen processing to promote epitope transit into endosomal/lysosomal compartments. Prolonged knockdown of HSP90 alpha in B cells, lead to an increase in the expression of HSP90 beta subunit. Studies are currently underway to examine whether the activation state of B cells influences cytoplasmic antigen presentation as well as the role of HSP90 co-factors in regulating this pathway for class II presentation.

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways (1,2). Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid (3). Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored (4) . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [(3)H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 (5). A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source (6). In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K(+), which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity (7). This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding (8-11).

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Hsp90: a chaperone for protein folding and gene regulation

Molecular chaperones are essential components of a quality control machinery present in the cell. They can either aid in the folding and maintenance of newly translated proteins, or they can lead to the degradation of misfolded and destabilized proteins. Hsp90 is a key member of this machinery. It is a ubiquitous molecular chaperone that is found in eubacteria and all branches of eukarya. It plays a central role in cellular signaling since it is essential for maintaining the activity of several signaling proteins, including steroid hormone receptors and protein kinases. Hsp90 is currently a novel anticancer drug target since it is overexpressed in some cancer cells. The chaperone typically functions as part of large complexes, which include other chaperones and essential cofactors that regulate its function. It is thought that different cofactors target Hsp90 to different sets of substrates. However, the mechanism of Hsp90 function remains poorly understood. As part of an effort to elucidate the Hsp90 chaperone network, we carried out a large-scale proteomics study to identify physical and genetic interactors of the chaperone. We identified 2 highly conserved novel Hsp90 cofactors, termed Tah1 and Pih1, that bind to the chaperone and that also associate physically and functionally with the essential DNA helicases Rvb1 and Rvb2. These helicases are key components of the chromatin remodeling complexes Ino80 and SWR-C. Tah1 and Pih1 seem to represent a novel class of Hsp90 cofactors that allow the chaperone to indirectly affect gene regulation in the cell in addition to its ability to directly promote protein folding. In this review, we provide an overview of Hsp90 structure and function, and we discuss the literature that links the chaperone activity to gene regulation.

Navigating the Chaperone Network: An Integrative Map of Physical and Genetic Interactions Mediated by the Hsp90 Chaperone

Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.

Specific Binding of Tetratricopeptide Repeat Proteins to the C-terminal 12-kDa Domain of hsp90

The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90alpha (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the approximately 25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains.