Hsp90 ATPase Activity Research Articles

Hsp90 interacts with inducible NO synthase client protein in its heme‐free state and then drives heme insertion by an ATP‐dependent process

Maturation of NOS enzymes requires that they incorporate heme to become active, but how this cellular process occurs is unclear. We investigated a role for chaperone heat shock protein 90 (hsp90) in enabling heme insertion into the cytokine-inducible mouse NOS. We used macrophage cell line RAW 264.7 and human embryonic kidney HEK293T cells and studied insertion of native heme during iNOS expression and insertion of exogenous heme into preformed apo-iNOS. Pulldown experiments showed that the hsp90-iNOS complex was present in cells, but the extent of their association was inversely related to iNOS heme content. Hsp90 was primarily associated with apo-iNOS monomer and was associated 11-fold less with heme-containing iNOS monomer or dimer in cells. Kinetic studies showed that hsp90 dissociation occurred coincident with cellular heme insertion into apo-iNOS (0.8 h(-1)). The hsp90 inhibitor radicicol or coexpression of an ATPase-defective hsp90 blocked heme insertion into apo-iNOS by 90 and 75%, respectively. The ATPase activity of hsp90 was not required for complex formation with iNOS but was essential for heme insertion to occur. We conclude that hsp90 plays a primary role in maturation of iNOS protein by interacting with the apoenzyme in cells and then driving heme insertion in an ATP-dependent manner.

SWI+], the Prion Formed by the Chromatin Remodeling Factor Swi1, Is Highly Sensitive to Alterations in Hsp70 Chaperone System Activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.

Some sulfonamide drugs inhibit ATPase activity of heat shock protein 90: investigation by docking simulation and experimental validation

Eight selected sulfonamide drugs were investigated as inhibitors of heat shock protein 90 (Hsp90). The investigation included simulated docking experiments to fit the selected compounds within the binding pocket of Hsp90. The selected molecules were found to readily fit within the ATP-binding pocket of Hsp90 in low-energy poses. The sulfonamides torsemide, sulfathiazole, and sulfadiazine were found to inhibit the ATPase activity of Hsp90 with IC50 values of 1.0, 2.6, and 1.5 μM, respectively. Our results suggest that these well-established sulfonamides can be good leads for subsequent optimization into potent Hsp90 inhibitors.

Insights into Radicicol Biosynthesis via Heterologous Synthesis of Intermediates and Analogs

Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2' ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7',8'-dehydrozearalenol.

Gambogic acid inhibits Hsp90 and deregulates TNF-α/NF-κB in HeLa cells

Gambogic acid (GB) is an important anti-cancer drug candidate, but the target protein by which it exerts its anti-cancer effects has not been identified. This study is the first to show that GB inhibits heat shock protein 90 (Hsp90) and down-regulates TNF-α/NF-κB in HeLa cells. The effects of GB on Hsp90 were studied by characterizing its physical interactions with Hsp90 upon binding, the noncompetitive inhibition of Hsp90 ATPase activity, and the degradation of Hsp90 client proteins (i.e., Akt, IKK) in HeLa cells. GB seems to bind to the N-terminal ATP-binding domain of Hsp90. Additionally, GB suppresses the activation of TNF-α/NF-κB and decreases XIAP expression levels and the ratio of Bcl-2/Bax, which in turn induces HeLa cell apoptosis. Thus, GB represents a promising therapeutic agent for cancer; it may also be useful as a probe to increase understanding of the biological functions of Hsp90.

The Pih1-Tah1 Cochaperone Complex Inhibits Hsp90 Molecular Chaperone ATPase Activity

Hsp90 (heat shock protein 90) is an ATP-dependent molecular chaperone regulated by collaborating proteins called cochaperones. This machinery is involved in the conformational activation of client proteins like signaling kinases, transcription factors, or ribonucleoproteins (RNP) such as telomerase. TPR (TetratricoPeptide Repeat)-containing protein associated with Hsp90 (Tah1) and protein interacting with Hsp90 (Pih1) have been identified in Saccharomyces cerevisiae as two Hsp90 cochaperones involved in chromatin remodeling complexes and small nucleolar RNP maturation. Tah1 possesses a minimal TPR domain and binds specifically to the Hsp90 C terminus, whereas Pih1 displays no homology to other protein motifs and has been involved in core RNP protein interaction. While Pih1 alone was unstable and was degraded from its N terminus, we showed that Pih1 and Tah1 form a stable heterodimeric complex that regulates Hsp90 ATPase activity. We used different biophysical approaches such as analytical ultracentrifugation, microcalorimetry, and noncovalent mass spectrometry to characterize the Pih1-Tah1 complex and its interaction with Hsp90. We showed that the Pih1-Tah1 heterodimer binds to Hsp90 with a similar affinity and the same stoichiometry as Tah1 alone. However, the Pih1-Tah1 complex antagonizes Tah1 activity on Hsp90 and inhibits the chaperone ATPase activity. We further identified the region within Pih1 responsible for interaction with Tah1 and inhibition of Hsp90, allowing us to suggest an interaction model for the Pih1-Tah1/Hsp90 complex. These results, together with previous reports, suggest a role for the Pih1-Tah1 cochaperone complex in the recruitment of client proteins such as core RNP proteins to Hsp90.

The L279P Mutation of Nuclear Distribution Gene C (NudC) Influences Its Chaperone Activity and Lissencephaly Protein 1 (LIS1) Stability

LIS1, a gene mutated in classical lissencephaly, plays essential roles in cytoplasmic dynein regulation, mitosis and cell migration. However, the regulation of LIS1 (lissencephaly protein 1) protein remains largely unknown. Genetic studies in Aspergillus nidulans have uncovered that the Nud (nuclear distribution) pathway is involved in the regulation of cytoplasmic dynein complex and a temperature-sensitive mutation in the nudC gene (L146P) greatly reduces the protein levels of NudF, an Aspergillus ortholog of LIS1. Here, we showed that L146 in Aspergillus NudC and its flanking region were highly conservative during evolution. The similar mutation in human NudC (L279P) obviously led to reduced LIS1 and cellular phenotypes similar to those of LIS1 down-regulation. To explore the underlying mechanism, we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90, which is also associated with LIS1. Inhibition of Hsp90 chaperone function by either geldanamycin or radicicol resulted in a decrease in LIS1 levels. Ectopic expression of Hsp90 partially reversed the degradation of LIS1 caused by overexpression of NudC-L279P. Furthermore, NudC was found to regulate the ATPase activity of Hsp90, which was repressed by the mutation of L279P. Interestingly, NudC itself was shown to possess a chaperone function, which also was suppressed by the L279P mutation. Together, these data suggest that NudC may be involved in the regulation of LIS1 stability by its chaperone function.

The Sacsin Repeating Region (SRR): A Novel Hsp90-Related Supra-Domain Associated with Neurodegeneration

Protein supra-domains are defined as recurring arrangements of two or three domains present adjacent to each other along a polypeptide chain. Such combinations have novel functions beyond those of the individual partner domains that compose them, which can exist in isolation. Here, we describe a new type of large supra-domain (∼360 residues) in which one of the component partners (∼200 residues) appears to be incapable of existing in a context other than immediately adjacent to the C-terminus of the well-characterized Hsp90-like ATPase domain. We found that this supra-domain has a broad phylogenetic distribution, with examples in Archaea, Bacteria, and Eukarya. There is strong selective pressure for this arrangement to occur as part of repeated regions of unprecedented length. We identified multiple strategies of convergent evolution to attain such configurations. In humans, this supra-domain is present in triplicate at the N-terminus of the protein sacsin (4579 residues), mutated in the neurodegenerative disorder known as spastic ataxia of Charlevoix–Saguenay, and thus, we termed it “sacsin repeating region” (SRR). Biochemical characterization demonstrated that SRRs possess ATPase activity, which appears to be a requirement for sacsin function, as a disease-causing mutation leads to an alternate conformation completely incapable of hydrolyzing ATP. We also found evidence of a convergent evolutionary strategy to place SRRs in proteins containing C-terminal J domains, which we demonstrated here to be capable of stimulating the intrinsic ATPase activity of Hsp70. Our sequence and biochemical analyses indicate that SRRs necessitate nucleotide hydrolysis for their function, provided by the common Hsp90 ATPase domain, which, when coupled to the unique adjacent sequence, may give rise to a novel activity related to protein quality control.

Binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40.

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

Inhibition of Hsp70 by Methylene Blue Affects Signaling Protein Function and Ubiquitination and Modulates Polyglutamine Protein Degradation

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

HSP90 inhibitor, celastrol, arrests human monocytic leukemia cell U937 at G0/G1 in thiol-containing agents reversible way

BackgroundBecause some of heat shock protein 90's (HSP90) clients are key cell cycle regulators, HSP90 inhibition can affect the cell cycle. Recently, celastrol is identified both as a novel inhibitor of HSP90 and as a potential anti-tumor agent. However, this agent's effects on the cell cycle are rarely investigated. In this study, we observed the effects of celastrol on the human monocytic leukemia cell line U937 cell cycle.ResultsCelastrol affected the proliferation of U937 in a dose-dependent way, arresting the cell cycle at G0/G1 with 400 nM doses and triggering cell death with doses above 1000 nM. Cell cycle arrest was accompanied by inhibition of HSP90 ATPase activity and elevation in HSP70 levels (a biochemical hallmark of HSP90 inhibition), a reduction in Cyclin D1, Cdk4 and Cdk6 levels, and a disruption of the HSP90/Cdc37/Cdk4 complex. The observed effects of celastrol on the U937 cell cycle were thiol-related, firstly because the effects could be countered by pre-loading thiol-containing agents and secondly because celastrol and thiol-containing agents could react with each other to form new compounds.ConclusionsOur results disclose a novel action of celastrol-- causing cell cycle arrest at G0/G1 phase based upon thiol-related HSP90 inhibition. Our work suggests celastrol's potential in tumor and monocyte-related disease management.

Hsp90 Cochaperone Aha1 Is a Negative Regulator of the Saccharomyces MAL Activator and Acts Early in the Chaperone Activation Pathway

Aha1 is a ubiquitous cochaperone of the Hsp90/Hsp70 chaperone machine. It binds the middle domain of Hsp90 and stimulates ATPase activity, suggesting a function late in the chaperone pathway. Saccharomyces Mal63 MAL activator is a DNA-binding transcription factor and Hsp90 client protein. This study utilizes several MAL activator mutants to investigate Aha1 function in vivo. Deletion of AHA1 enhances induced Mal63-dependent maltase activity levels 2-fold, whereas overproduction of Aha1 represses expression. Maltase expression in strains carrying constitutive and super-inducible mutant activators with alterations near the C terminus (particularly residues 433-463) is unaffected by either aha1Delta or Aha1 overproduction. However, another constitutive activator with alterations outside of this C-terminal region is sensitive to Aha1 regulation. Previously, we showed that in the absence of inducer, Mal63 forms a stable intermediate complex with Hsp70, Hsp90, and Sti1, whereas noninducible mutant activators bind only with Hsp70 in an apparent early complex. Here, we find that triple Myc-tagged Aha1/Myc3 copurifies with all noninducible Mal63 mutant activators tested. Aha1/Myc3 association with inducible Mal63 is observed only in a sti1Delta strain, in which Hsp90 binding and intermediate complex formation are defective. Constitutive and super-inducible mutant activators with C-terminal alterations do not bind Aha1 even in a sti1Delta strain. Mal63 binding to Hsp90 and Hsp70 is enhanced 3-fold by loss of Aha1. These results suggest an interaction between Aha1 and residues near the C terminus of Mal63 thereby regulating Hsp90 association. A novel mechanism for the negative regulation of the MAL activator by Aha1 cochaperone is proposed.

Characterization of plant p23-like proteins for their co-chaperone activities.

The small acidic protein p23 is best described as a co-chaperone of Hsp90, an essential molecular chaperone in eukaryotes. p23 binds to the ATP-bound form of Hsp90 and stabilizes the Hsp90-client protein complex by slowing down ATP turnover. The stabilizing activity of p23 was first characterized in studies of steroid receptor-Hsp90 complexes. Earlier studies of the Hsp90 chaperone complex in plants suggested that a p23-like stabilizing activity was absent in plant cell lysates. Here, we show that p23-like proteins are present in plants and are capable of binding Hsp90, but unlike human p23 and yeast ortholog Sba1, the plant p23-like proteins do not stabilize the steroid receptor-Hsp90 complexes formed in wheat germ lysate. Furthermore, these proteins do not inhibit the ATPase activity of plant Hsp90. While transcripts of Arabidopsis thaliana p23-1 and Atp23-2 were detected under normal growing conditions, those of the closely related Brassica napus p23-1 were present only after moderate heat stress. These observations suggest that p23-like proteins in plants are conserved in their binding to Hsp90 but have evolved mechanisms of action different from their yeast and animal counterparts.

Mutations that increase both Hsp90 ATPase activity in vitro and Hsp90 drug resistance in vivo

Hsp90 inhibitors are currently tested in clinical trials as anticancer agents. We investigated whether inhibitor resistance may arise as a result of a point mutation in Hsp90. We used yeast cells that expressed human Hsp90β to select inhibitor-resistant mutants from the randomly mutagenized library. Single amino acid substitution, I123T, in a selected mutant was sufficient to confer inhibitor resistance. Transfection of human cells with the HSP90β I123T and the corresponding HSP90α I128T yielded cell lines resistant to inhibitors of the Hsp90 ATPase. Unexpectedly, mutations did not result in diminished inhibitor binding in vitro. Similarly resistant cells were obtained after transfection with previously described A116N and T31I mutants of HSP90β that cause increase in ATPase activity in vitro. Inhibitor-resistant phenotypes of the I123T and A116N mutants depended on their increased affinity for Aha1, whereas T31I mutation did not result in increased Aha1 binding. These results show possible scenario by which resistance may arise in patients treated with Hsp90 inhibitors. Additionally, our results show that each isoform of Hsp90 can alone sustain cellular functions.

Detection of the ATPase Activity of the Molecular Chaperones Hsp90 and Hsp72 Using the Transcreener™ ADP Assay Kit

The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats.

Asymmetric Activation of the Hsp90 Dimer by Its Cochaperone Aha1

The chaperone Hsp90 is an ATP-dependent, dimeric molecular machine regulated by several cochaperones, including inhibitors and the unique ATPase activator Aha1. Here, we analyzed the mechanism of the Aha1-mediated acceleration of Hsp90 ATPase activity and identified the interaction surfaces of both proteins using multidimensional NMR techniques. For maximum activation of Hsp90, the two domains of Aha1 bind to sites in the middle and N-terminal domains of Hsp90 in a sequential manner. This binding induces the kinetically unfavored N terminally dimerized state of Hsp90, which primes for the hydrolysis-competent conformation. Surprisingly, this activation mechanism is asymmetric. The presence of one Aha1 molecule per Hsp90 dimer is sufficient to bridge the two subunits and to fully stimulate Hsp90 ATPase activity. This seems to functionalize the two subunits of the Hsp90 dimer in different ways, in that one subunit can be used for conformational ATPase regulation and the other for substrate protein processing.

Biological and Structural Basis for Aha1 Regulation of Hsp90 ATPase Activity in Maintaining Proteostasis in the Human Disease Cystic Fibrosis

The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

Facilitating Akt Clearance via Manipulation of Hsp70 Activity and Levels

Members of the 70-kDa heat shock family can control and manipulate a host of oncogenic client proteins. This role of Hsp70 in both the folding and degradation of these client proteins makes it a potential drug target for certain forms of cancer. The phenothiazine family of compounds, as well as the flavonoid myricetin, was recently shown to inhibit Hsp70-ATPase activity, whereas members of the dihydropyrimidine family stimulated ATPase function. Akt, a major survival kinase, was found to be under the regulation of Hsp70, and when the ATPase activity of Hsp70 was increased or decreased by these compounds, Akt levels were also increased or decreased. Also, increasing Hsp70 levels concurrent with inhibition of its ATPase function synergistically reduced Akt levels to a greater extent than either manipulation alone, providing new insights about client fate decisions. Akt reductions mediated by Hsp70 inhibitors were prevented when Hsp70 expression was silenced with small interfering RNA. Inhibiting Hsp70 ATPase function produced cytotoxic events only in breast cancer cell lines where Akt dysfunction was previously shown, suggesting therapeutic specificity depending on the Hsp70 client profile. Thus, increasing Hsp70 levels combined with inhibiting its ATPase function may serve to dramatically reduce Akt levels and facilitate cell death in certain types of cancer.

Cwc23, an Essential J Protein Critical for Pre-mRNA Splicing with a Dispensable J Domain

J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.

Characterization of a Novel Mammalian Co-chaperone, DnaJB7, an HSP40 Family Member

Molecular chaperones of HSP70 family members usually work together with HSP40/DNAJ family members in protein biogenesis and protein homeostasis. In mammals, approximately 50 members of the HSP40/DNAJ family are identified so far. Here, we isolated a cDNA clone of DnaJB7, one of the HSP40/DNAJ family members, and examined the properties of DnaJB7 protein in vivo and in vitro . Since the DnaJB7 gene in the human genome has no obvious heat shock element (HSE), DnaJB7 could not be induced by heat shock in HeLa cells. While overexpressed DnaJB7 in HeLa cells was distributed to both the cytoplasm and nucleolus at normal temperature, upon heat shock it accumulated in the nucleolus, where it co-localized with Hsp70. This localization pattern was very similar to that of Hsp40 (DnaJB1). Direct interaction of Hsp70 with DnaJB7 in HeLa cells was demonstrated by immunoprecipitation. Also, DnaJB7 could stimulate the ATPase activity of Hsp70. Hsp70 had molecular chaperone activity in suppressing aggregation of heat denatured luciferase, and DnaJB7 could facilitate the chaperone activity of Hsp70. In murine tissues, DnaJB7 was highly expressed in the stomach and faintly in pancreas, uterus and ovary. These results suggest that DnaJB7 is a stomach-enriched co-chaperone of Hsp70.