Hsp90 Mutants Research Articles

Controlling oligomerization through protein engineering: in vivo analysis of Hsp90

Many homo-oligomeric proteins are vital for biology including ion-channels, the p53 tumor suppressor, and the essential kinase-associated chaperone Hsp90. Mutational analyses of these homo-oligomeric systems in vivo is complicated by cross-oligomerization between wild-type and mutant subunits. We have devised a generalizable thermodynamic strategy to prevent cross-dimerization. Appending an oligomerization domain to the mutant subunits reduces the free energy of homocomplexes relative to wild-type/mutant heterocomplexes. We have used this strategy to engineer super-stabilized Hsp90 dimers that do not cross-oligomerize with wild-type Hsp90. Super-stabilized Hsp90 supports yeast viability and is fully active in the maturation of v-src kinase. Thus, our stabilization strategy does not disturb the biochemical function of Hsp90.We have used superstabilized Hsp90 to address a fundamental and long-unanswered question regarding Hsp90: what clients or substrate proteins depend on Hsp90 ATPase activity in vivo. The identification of ATP dependent Hsp90 substrates has been a major challenge both in vitro and in vivo. In vitro studies are complicated by the large number of co-chaperones required for Hsp90 to function efficiently. In vivo studies are complicated both because ATPase deficient Hsp90 mutants do not support viability and because when different Hsp90 variants are co-expressed they form a mixture of different dimer species. We have used our engineered super-stabilized Hsp90 to developed a yeast system to identify clients that rely on Hsp90 ATPase activity. Using this approach, we find that Hsp90 mutants deficient for ATP binding or hydrolysis have differential impacts on the activation of kinase and hormone receptor clients in vivo. These results provide a rationale for understanding anti-cancer drugs that competitively bind to the ATPase site of Hsp90.

A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes

Plus-stranded RNA viruses coopt host proteins to promote their robust replication in infected hosts. Tomato bushy stunt tombusvirus (TBSV) is a model virus that can replicate a small replicon RNA in Saccharomyces cerevisiae and in plants. The tombusvirus replicase complex contains heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, which is required for TBSV replication. To dissect the function of Hsp70 in TBSV replication, in this paper we use an Hsp70 mutant (ssa1 ssa2) yeast strain that supports a low level of TBSV replication. Using confocal laser microscopy and cellular fractionation experiments, we find that the localization of the viral replication proteins changes to the cytosol in the mutant cells from the peroxisomal membranes in wild-type cells. An in vitro membrane insertion assay shows that Hsp70 promotes the integration of the viral replication proteins into subcellular membranes. This step seems to be critical for the assembly of the viral replicase complex. Using a gene-silencing approach and quercetin as a chemical inhibitor to downregulate Hsp70 levels, we also confirm the significance of cytosolic Hsp70 in the replication of TBSV and other plant viruses in a plant host. Taken together, our results suggest that cytosolic Hsp70 plays multiple roles in TBSV replication, such as affecting the subcellular localization and membrane insertion of the viral replication proteins as well as the assembly of the viral replicase.

Sgt1, a co-chaperone of Hsp90 stabilizes Polo and is required for centrosome organization

Sgt1 was described previously in yeast and humans to be a Hsp90 co-chaperone and required for kinetochore assembly. We have identified a mutant allele of Sgt1 in Drosophila and characterized its function. Mutations in sgt1 do not affect overall kinetochore assembly or spindle assembly checkpoint. sgt1 mutant cells enter less frequently into mitosis and arrest in a prometaphase-like state. Mutations in sgt1 severely compromise the organization and function of the mitotic apparatus. In these cells, centrioles replicate but centrosomes fail to mature, and pericentriolar material components do not localize normally resulting in highly abnormal spindles. Interestingly, a similar phenotype was described previously in Hsp90 mutant cells and correlated with a decrease in Polo protein levels. In sgt1 mutant neuroblasts, we also observe a decrease in overall levels of Polo. Overexpression of the kinase results in a substantial rescue of the centrosome defects; most cells form normal bipolar spindles and progress through mitosis normally. Taken together, these findings suggest that Sgt1 is involved in the stabilization of Polo allowing normal centrosome maturation, entry and progression though mitosis.

Cryptic genetic variation

Cryptic genetic variation

The Hsp110 protein chaperone Sse1 is required for yeast cell wall integrity and morphogenesis

Molecular chaperones direct refolding and triage decisions and support signal transduction responses to cytotoxic stress. The eukaryotic chaperone Hsp110 is represented by the SSE1/2 genes in Saccharomyces cerevisiae, which act as nucleotide exchange factors (NEFs) for cognate cytosolic Hsp70 chaperones. In this report, we present evidence that Sse1 is required for signaling through the cell integrity pathway via partnership with Hsp90 and the terminal MAP kinase Slt2. We found that sse1Delta and sti1Delta mutant cells share the typical cell integrity mutant phenotypes of osmoremediated temperature-sensitive growth and sensitivity to cell wall-damaging agents. Sse1 binds to Slt2 in vivo and similar to Hsp90 mutants, Slt2 stability and phosphorylation is not compromised in sse1Delta cells, whereas activation of the downstream transcription factor Rlm1 is abolished. In addition to Rlm1, Slt2 activates the Swi4/Swi6 heterodimer SBF in response to cell wall damage. SSE1 displayed dramatic synthetic phenotypes when disrupted in combination with mutations in SBF and the related Mbp1/Swi6 heterodimer MBF, characterized by severe growth and morphological defects. These defects were reversed by restoration of Hsp70 NEF activity, providing a mechanistic model wherein Sse1 functionally partners with Hsp90 as an Hsp70 NEF to promote client protein maturation and interaction with downstream effectors.

SITE SPECIFIC PHOSPHORYLATION OF HEAT SHOCK PROTEIN 27 (HSP27) REGULATES CELL SURVIVAL AND DEATH

Hsp27 is constitutively expressed and increases in response to stress. Pretreatment of LLC‐PK1 cells with 11‐deoxy‐16,16‐dimethyl PGE2 (DDM‐PGE2) selectively increases constitutive Hsp27 expression and contributes to protection against 2,3,5‐tris(glutathion‐S‐yl)hydroquinone (TGHQ) induced cytotoxicity. We have now identified specific changes in Hsp27 phosphorylation during DDM‐PGE2 mediated cytoprotection. 2D gel electrophoresis revealed that in addition to constitutive pS15‐Hsp27, TGHQ induces the formation of 2 additional Hsp27‐positive proteins. 2D western analysis subsequently identified TGHQ induced pS84‐ and pS88‐Hsp27, both of which are translocated to the nucleus. Pretreatment of LLC‐PK1 cells with DDM‐PGE2 increases the constitutive expression of pS15‐Hsp27 in the cytosol and the nucleus, and specifically decreased TGHQ‐mediated induction of pS84‐Hsp27. These findings were confirmed by mass spectrometric analysis. Interestingly, although the inhibition of p38 MAPK prevented TGHQ‐induced pS84‐Hsp27 phosphorylation and cytotoxicity, transfection of cells with mutant Hsp27 (S15,84,88→ A15,84,88) or p‐Hsp27 (S15,84,88→D15,84,88) had no effect on TGHQ‐induced cytotoxicity. The data suggest that the specific phosphorylation of Hsp27 at Ser84 negatively regulates cell survival, and that pS15‐Hsp27 may be positively associated with cell survival. (GM56321, ES06694).

Histone Deacetylase 6 Regulates Growth Factor-Induced Actin Remodeling and Endocytosis

Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes alpha-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.

WT1 Interaction with the Chaperone Protein HSP90 Regulates Its Expression in Myeloid Leukemia.

Introduction: Wilms tumor protein 1 (WT1) is a transcription factor that is universally expressed in AML, and clinical studies have demonstrated a significant correlation between WT1 expression and disease progression. Heat shock proteins (HSP) are ubiquitous molecular chaperones that are involved in the folding, activation and assembly of many proteins. HSP90 may facilitate the maintenance of altered oncogenic client proteins such as flt-3 and bcr-abl in myeloid malignancies. It has been also shown that WT1 is a client protein of Hsp70, and this interaction may be important for the function of WT1. Since HSP70 and HSP90 have similar functions and often complement each other in engaging with client cellular proteins we wanted to determine if WT1 was chaperoned by HSP90 and if so could this HSP90-WT1 interaction be modulated.Materials and methods: We first looked for a potential interaction between HSP90 and WT1 by confocal microscopy colocalization and then by immunoprecipitation (IP) in the myeloid cell line K562. For confocal studies, cells were grown on coverslips, fixed and permeabilized and stained with the rabbit anti-WT1 and anti- HSP90 antibodies. Samples were examined by using a laser confocal/Zeiss axiovert microscope imaging system. This interaction was further confirmed by co-IP/Western and then confirmed using a GST-pulldown assay. A glutathione S-transferase (GST)-WT1 fusion protein was expressed in E. coli strain BL21(DE3) and immobilized onto glutathione sepharose beads. The full length Hsp90 and different deletion mutant proteins were in vitro translated and [35S] methionine-labeled; the bound proteins were eluted and then separated by SDS-PAGE. The gels were fixed, dried and subjected to fluorography.Results: Confocal microscopy revealed colocalization of HSP90 and WT1 mainly in the nucleus. Immunoprecipitation of K562 lysates with either anti-WT1 or anti-HSP90 antibody followed by Western analysis for WT1 and HSP90 demonstrated that WT1 and HSP90 could be co-immunoprecipitated. The GST pull down assay confirmed this interaction. Additional mutants of HSP90 from the C to N terminus were prepared to localize the WT1 interacting domain. These studies localized the WT1 interacting domain to the HSP90 region close to the binding pocket of the HSP inhibitor geldanamycin. Treatment of K562 with the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in downregulation of WT1 protein expression with an IC50 of 3μM.Conclusions: Understanding the mechanisms that regulate WT1 expression thus may lead to novel therapeutic strategies to inhibit this expression and result directly in myeloid blast cell killing. We have shown elsewhere that downmodulating WT1 gene expression with interferon or shRNA potentiates myeloid leukemic blast killing by chemotherapeutic agents. We demonstrate here that the WT1 protein interacts with the co-chaperone heat shock protein HSP90. Taken together, our findings support the hypothesis that the regulation of WT1 occurs at the protein level through an interaction with WT1-protein-HSP chaperone levels, and that pharmacologically targeting this interaction with the HSP inhibitor 17AAG results in a significant reduction in WT1 protein expression. Ongoing studies in primary AML cells will confirm these findings and define novel therapeutic strategies to manipulate WT1 protein expression levels and modulate leukemic blast survival.

Definition of the minimal fragments of Sti1 required for dimerization, interaction with Hsp70 and Hsp90 and in vivo functions

The molecular chaperone Hsp (heat-shock protein) 90 is critical for the activity of diverse cellular client proteins. In a current model, client proteins are transferred from Hsp70 to Hsp90 in a process mediated by the co-chaperone Sti1/Hop, which may simultaneously interact with Hsp70 and Hsp90 via separate TPR (tetratricopeptide repeat) domains, but the mechanism and in vivo importance of this function is unclear. In the present study, we used truncated forms of Sti1 to determine the minimal regions required for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. We found that both TPR1 and TPR2B contribute to the Hsp70 interaction in vivo and that mutations in both TPR1 and TPR2B were required to disrupt the in vitro interaction of Sti1 with the C-terminus of the Hsp70 Ssa1. The TPR2A domain was required for the Hsp90 interaction in vivo, but the isolated TPR2A domain was not sufficient for the Hsp90 interaction unless combined with the TPR2B domain. However, isolated TPR2A was both necessary and sufficient for purified Sti1 to migrate as a dimer in solution. The DP2 domain, which is essential for in vivo function, was dispensable for the Hsp70 and Hsp90 interaction, as well as Sti1 dimerization. As evidence for the role of Sti1 in mediating the interaction between Hsp70 and Hsp90 in vivo, we identified Sti1 mutants that result in reduced recovery of Hsp70 in Hsp90 complexes. We also identified two Hsp90 mutants that exhibit a reduced Hsp70 interaction, which may help clarify the mechanism of client transfer between the two molecular chaperones.

Importance of the Hsp70 ATPase Domain in Yeast Prion Propagation

The Saccharomyces cerevisiae non-Mendelian genetic element [PSI+] is the prion form of the translation termination factor Sup35p. The ability of [PSI+] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PSI+]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70-Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PSI+] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties.

An Acetylation Site in the Middle Domain of Hsp90 Regulates Chaperone Function

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.

Defining the Requirements for Hsp40 and Hsp70 in the Hsp90 Chaperone Pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.

Inhibition of histone deacetylase (HDAC) 6 sensitizes human leukemia and breast cancer cells to antagonists of heat shock protein (hsp) 90 and/or bortezomib (BZ)

13039 Background: HDAC6 is a cytosolic α-tubulin deacetylase, which shuttles misfolded polyubiquitylated proteins into aggresomes. Our studies demonstrated that the hydroxamic acid (HA) analogue pan-HDAC inhibitors (HDIs), e.g., LAQ824 and LBH589, induce α-tubulin and hsp90 acetylation, thereby inhibiting ATP-binding and chaperone function of hsp90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival hsp90 client proteins, e.g., Bcr-Abl, mutant FLT-3, c-Raf and AKT in leukemia and Her-2 in breast cancer cells. Methods: Human CML K562 and breast cancer SKBR-3 and BT-474 cells were exposed to LAQ824 (100 nM) or LBH589 (20 to 50 nM) and/or 17-AAG (1 to 5 μM) and/or BZ (5 to 100 nM). K562 and BT-574 cells were also transfected with siRNA to HDAC6. Immunoprecipitation and/or immunoblot analysis were performed to determine the expression or binding of HDAC6 with hsp90, and of hsp90 acetylation and binding with its co-chaperones and client proteins. Immunofluorescent microscopy was utilized to estimate aggresome formation. Cellular cytotoxicity was determined by % inhibition of colony growth. Results: HDAC6 knockdown by its siRNA induced the acetylation of hsp90 and α-tubulin but not of the co-chaperones p23 and hsp40. HDAC6 binds to hsp90. Hsp90 acetylation increased its binding to biotinylated-geldanamycin (GM) and its analogue 17-AAG. Depletion of HDAC6 levels (∼70%) or its activity by treatment with HA-HDI also inhibited the binding of hsp90 to ATP, p23 and hsp90 client proteins, which was augmented by co-treatment with 17-AAG. Knockdown of HDAC6 sensitized the cells to loss of clonogenic survival induced by HA-HDI and/or BZ and/or 17-AAG. Depletion of HDAC6 led to attenuation of Her-2 levels and loss of survival of Her-2 amplified SKBR-3 and BT-474 cells. Co-localization of HDAC6 and polyubiquitylated proteins into aggresomes was enhanced by co-treatment with BZ and/or 17-AAG. This was inhibited by depletion of HDAC6 activity. Conclusions: These findings support the in vivo testing of HDAC6 inhibitors, which through inhibition of hsp90 and aggresome formation sensitizes cancer cells to 17-AAG and/or BZ induced cellular stress due to misfolded and polyubiquitylated proteins. [Table: see text]

Genistein Inhibits Matrix Metalloproteinase Type 2 Activation and Prostate Cancer Cell Invasion by Blocking the Transforming Growth Factor β-Mediated Activation of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2-27-kDa Heat Shock Protein Pathway

Genistein is a candidate cancer chemopreventive drug being tested in clinical trials. We have shown that genistein blocks prostate cancer (PCa) cell invasion, that p38 mitogen-activated protein (MAP) kinase regulates activation of matrix metalloproteinase type 2 (MMP-2) and cell invasion, and that genistein prevents transforming growth factor beta (TGFbeta) from activating p38 MAP kinase. More recently, we identified MAP kinase-activated protein kinase 2 (MAPKAPK2) and the 27-kDa heat shock protein (HSP27) as downstream regulators of p38 MAP kinase. However, MAPKAPK2 and HSP27 can be regulated by factors other than p38 MAP kinase, and HSP27 is up-regulated during PCa progression. The current study was undertaken to examine the role of MAPKAPK2 and HSP27 in modulating genistein-mediated regulation of PCa cell invasion. Genistein inhibited TGFbeta-mediated phosphorylation of MAPKAPK2 and HSP27. Inhibitory effects by genistein upon cell signaling, inhibition of MMP-2, and inhibition of invasion were retained when both PC3 and PC3-M cells were transfected with either wild-type MAPKAPK2 or HSP27. However, transfection with dominant-negative MAPKAPK2 or nonphosphorylatable mutant HSP27 led to decreases in cell invasion and to abrogation of responsiveness to either TGFbeta-mediated increases or genistein-mediated decreases in MMP-2 and cell invasion. It is noteworthy that, after transfection with constitutive active MAPKAPK2 or with pseudophosphorylated HSP27, levels of MMP-2 activation and cell invasion were high and overcame any inhibitory effect of genistein. These findings demonstrate that genistein-mediated inhibition of cell invasion rests upon blocking activation of the MAPKAPK2-HSP27 pathway, and that its activation during cancer progression has the potential to mitigate therapeutic efficacy.

Hsp70 inhibits lipopolysaccharide-induced NF-κB activation by interacting with TRAF6 and inhibiting its ubiquitination

Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-κB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-κB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-κB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70ΔC(1–428aa) with N-terminal ATPase domain and Hsp70C(428–642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-κB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-κB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.

Generation of anti-tumor immunity using mammalian heat shock protein 70 DNA vaccines for cancer immunotherapy

In this study, we explored the protective anti-tumor potency of mouse (self) Hsp70 or Hsp110-based DNA vaccination approach targeting a tumor-associated antigen, human papilloma virus (HPV) type 16 E7 protein. Linkage of E7 to the N-terminus of the mouse Hsp70 not only elicits an E7-specific cytotoxic T cell (CTL) response, but also protects mice against challenge with E7 expressing tumors. CD8 + T-cells are crucial in both priming and effector phases for the induction of tumor immunity, whereas CD4 + T-cells and NK cells do not appear to play a major role. Furthermore, the ATP-binding domain deletion mutant Hsp70 382–641, when fused to E7, was immunologically effective, suggesting that the peptide-binding region, not the ATPase domain of Hsp70, is required for the vaccine activity of the E7-Hsp70 DNA. This study demonstrates that autologous Hsp70 is highly potent in enhancing antigen-specific immune responses. Functional domain mapping and orientation of the E7 and Hsp70 in the fusion gene may have clinical implications for the design and optimization of Hsp70-based DNA vaccines.

Distinct hsp70 Domains Mediate Apoptosis-inducing Factor Release and Nuclear Accumulation

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-deltaEEVD) or the ATP hydrolytic domain (hsp70-deltaATPase) failed to prevent mitochondrial AIF release. Although hsp70-deltaEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-deltaATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.

Depletion of GAK/auxilin 2 inhibits receptor-mediated endocytosis and recruitment of both clathrin and clathrin adaptors

Cyclin G-associated kinase (GAK/auxilin 2), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for the Hsc70-dependent uncoating of clathrin-coated vesicles. We have now investigated the effect of knocking down GAK in HeLa cells by vector-based small hairpin RNA. Functionally, depletion of GAK caused a marked decrease in internalization of both transferrin and epidermal growth factor and altered mannose 6-phosphate receptor trafficking, but had little effect on the recycling of transferrin receptor back to the plasma membrane. Structurally, depletion of GAK caused a marked reduction in perinuclear clathrin associated with the trans-Golgi network and in the number of clathrin-coated pits on the plasma membrane, and reduced clathrin exchange on the few clathrin-coated pits that remained. Surprisingly, while clathrin depletion does not prevent adaptors from assembling on the membrane, depletion of GAK caused a dramatic reduction in AP2 and epsin on the plasma membrane and AP1 and GGA at the trans-Golgi network. A similar effect was caused by expression of a dominant negative Hsp70 mutant. These results suggest that GAK, in conjunction with Hsc70, not only uncoats clathrin-coated vesicles and induces clathrin exchange on clathrin-coated pits, but also mediates binding of clathrin and adaptors to the plasma membrane and the trans-Golgi network.

Heat Shock Protein 27 Is the Major Differentially Phosphorylated Protein Involved in Renal Epithelial Cellular Stress Response and Controls Focal Adhesion Organization and Apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Roles of the C-terminal peptide binding domain of human inducible HSP70 in focal ischemic brain injury

The inducible heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed primarily in response to stress, including heat shock, ischemia and glucose deprivation. Hsp70 consists of a 44 kDa amino terminal ATPase domain, an 18 kDa peptide or substrate binding domain, and a 10 kDa carboxy terminal domain. While a large number of proteins interact with Hsp70, only in some cases has the domain with which they interact been identified. Although it is accepted that Hsp70 can protect cells from ischemic injury, the mechanism is not known. Possible protective effects include prevention of protein aggregation, refolding denatured proteins and reduction of apoptosis. The relative importance of the ATP-binding domain compared to the peptide binding domain of HSP70 in ischemic protection is unknown. To explore this question we tested whether two Hsp70 mutant proteins could protect against ischemia-like injury of primary cultured murine astrocytes and reduce focal ischemic injury induced by transient (2 h) middle cerebral artery occlusion (MCAO). The two mutants studied were 1) Hsp70 K71E, a point mutation that abrogates ATP binding and renders the protein deficient in folding ability and 2) Hsp70 381-640 a deletion mutant lacking the ATP binding domain. Hsp70 wild type (WT), -K71E, -381-640 and vector plasmid LXSN were expressed in primary murine astrocyte cultures using retroviral mediated transfection. After G418 selection, more than 95% of cells expressed these proteins by immunostaining. Cultures were then subjected to combined oxygen-glucose deprivation (OGD) and cell survival was assessed by MTT assay or PI staining and cell counting. Astrocytes overexpressing Hsp70 -WT, -K71E or -381-640 were all significantly protected from 4 h OGD (cell viability was increased about 50%, p < 0.05). We then tested the ability of these mutant proteins to protect rats from transient focal ischemic injury. Plasmids encoding Hsp70 -K71E, -381-640, or the control backbone plasmid LXSN were stereotactically injected into the left lateral ventricle 24 h prior to MCAO. These mutant proteins were expressed in both astrocytes and neurons as determined by double immunostaining. Animals that overexpressed either of these two mutant proteins had significantly better neurological scores and smaller infarct volumes (30-35%) assessed at 24 hr (infarction volume (mm3): 233.538.6 in control group; 152.2543.95 in Hsp70 K71E group; and 162.7132.5 in Hsp70 381-640 group, P<0.05 compared to control, n=8 in each group). Protection by both mutants was associated with reduced protein aggregation and apoptosis, as assessed by ubiquitin immunohistochemistry and Klenow staining. Both the point mutant and the deletion mutant were found to reduce ischemic injury. Since the carboxy terminal half of the protein is sufficient for protection, interaction with protein partners that bind the aminoterminal half of the protein are not essential to ischemic protection. Also, since neither mutant can facilitate folding, their efficacy against ischemia suggests that inhibiting protein aggregation is sufficient to reduce ischemic injury and apoptotic cell death.