Binding of the maize cytosolic Hsp70 to calmodulin, and identification of calmodulin-binding site in Hsp70

Using a gel-overlay technique of biotinylated calmodulin (CaM), we showed that maize cytosolic Hsp70 protein could bind to CaM in the presence of 1 mM CaCl2. The purified maize cytosolic Hsp70 inhibited the activity of CaM-dependent NADK in a concentration-dependent manner. A synthetic peptide, which possesses the 21 amino acid sequence, PRALRRLRTACERAKRTLSST, at positions 261-281 in maize cytosolic Hsp70, could associate with CaM in the presence of 1 mM calcium. The synthetic peptide inhibited CaM-dependent NADK activity and PDE activity. This indicates that the 21-amino acid sequence at positions 261-281 is the CaM-binding site. The binding of CaM to Hsp70 inhibited the ATPase activity of Hsp70. The possible regulator function of Hsp70 in cell signaling events in response to heat stress is discussed.     

Tubocapsenolide A, a novel withanolide, inhibits proliferation and induces apoptosis in MDA-MB-231 cells by thiol oxidation of heat shock proteins

Tubocapsenolide A (TA), a novel withanolide-type steroid, exhibits potent cytotoxicity against several human cancer cell lines. In the present study, we observed that treatment of human breast cancer MDA-MB-231 cells with TA led to cell cycle arrest at G(1) phase and apoptosis. The actions of TA were correlated with proteasome-dependent degradation of Cdk4, cyclin D1, Raf-1, Akt, and mutant p53, which are heat shock protein 90 (Hsp90) client proteins. TA treatment induced a transient increase in reactive oxygen species and a decrease in the intracellular glutathione contents. Nonreducing SDS-PAGE revealed that TA rapidly and selectively induced thiol oxidation and aggregation of Hsp90 and Hsp70, both in intact cells and in cell-free systems using purified recombinant proteins. Furthermore, TA inhibited the chaperone activity of Hsp90-Hsp70 complex in the luciferase refolding assay. N-Acetylcysteine, a thiol antioxidant, prevented all of the TA-induced effects, including oxidation of heat shock proteins, degradation of Hsp90 client proteins, and apoptosis. In contrast, non-thiol antioxidants (trolox and vitamin C) were ineffective to prevent Hsp90 inhibition and cell death. Taken together, our results demonstrate that the TA inhibits the activity of Hsp90-Hsp70 chaperone complex, at least in part, by a direct thiol oxidation, which in turn leads to the destabilization and depletion of Hsp90 client proteins and thus causes cell cycle arrest and apoptosis in MDA-MB-231 cells. Therefore, TA can be considered as a new type of inhibitor of Hsp90-Hsp70 chaperone complex, which has the potential to be developed as a novel strategy for cancer treatment.     

Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins

Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper.     

Production of reagents and optimization of methods for studying calmodulin-binding proteins

Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encoding D. discoideum CaM has previously been cloned allowing production of recombinant protein. The present study describes the expression of D. discoideum CaM in Escherichia coli and its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novel D. discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

SNX-2112, a Novel Hsp90 Inhibitor,Induces G2/M Cell Cycle Arrest and Apoptosis in MCF-7 Cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.     

SNX-2112, a novel Hsp90 inhibitor, induces G2/M cell cycle arrest and apoptosis in MCF-7 cells

SNX-2112 is a heat shock protein 90 (Hsp90) inhibitor with anticancer properties currently in clinical trials. This study investigated the effects of SNX-2112 on inhibition of cell growth, the cell cycle, and apoptosis in MCF-7 human breast cancer cells, in addition to the various molecular mechanisms. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis suggest that SNX-2112 inhibits cell growth in a time- and dose-dependent manner more potently than 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), a traditional Hsp90 inhibitor, probably as a result of cell-cycle arrest at the G2/M phase and the induction of apoptosis. Downregulation of Bcl-2 and Bcl-xL, upregulation of Bax, cleavage of caspase-9 and poly (ADP-ribose) polymerase (PARP), and degradation of the breast cancer-related Hsp90 client proteins human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, and nuclear factor kappa-B kinase (IKK) were observed in SNX-2112 treated cells by Western blot assay. These findings suggest that the molecular mechanisms of cell-growth inhibition by SNX-2112 involve activation of the mitochondrial apoptotic pathway and the degradation of breast cancer-related proteins.     

Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R

Viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) is an accessory protein that plays an important role in viral pathogenesis. This pathogenic activity of Vpr is related in part to its capacity to induce cell cycle G2 arrest and apoptosis of target T cells. A screening for multicopy suppressors of these Vpr activities in fission yeast identified heat shock protein 70 (Hsp70) as a suppressor of Vpr-induced cell cycle arrest. Hsp70 is a member of a family of molecular chaperones involved in innate immunity and protection from environmental stress. In this report, we demonstrate that HIV-1 infection induces Hsp70 in target cells. Overexpression of Hsp70 reduced the Vpr-dependent G2 arrest and apoptosis and also reduced replication of the Vpr-positive, but not Vpr-deficient, HIV-1. Suppression of Hsp70 expression by RNA interference (RNAi) resulted in increased apoptosis of cells infected with a Vpr-positive, but not Vpr-defective, HIV-1. Replication of the Vpr-positive HIV-1 was also increased when Hsp70 expression was diminished. Vpr and Hsp70 coimmunoprecipitated from HIV-infected cells. Together, these results identify Hsp70 as a novel anti-HIV innate immunity factor that targets HIV-1 Vpr.     

Role of inducible heat shock protein 70 in radiation-induced cell death

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.     

Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol

Hsp70, Hsp32, and Hsp27 were induced by celastrol in rat cerebral cortical cultures at dosages that did not affect cell viability. Pronounced differences were observed in the cellular localization of these heat shock proteins in cell types of cerebral cortical cultures. Celastrol-induced Hsp70 localized to the cell body and cellular processes of neurons that were identified by neuron-specific βIII-tubulin. Hsp70 was not detected in adjacent GFAP-positive glial cells that demonstrated a strong signal for Hsp27 and Hsp32 in both glial cell bodies and cellular processes. Cells in the cerebral cortex region of the brain are selectively impacted during the progression of Alzheimer’s disease which is a “protein misfolding disorder.” Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins. Celastrol is a potential agent to counter this neurodegenerative disorder as recent evidence indicates that in vivo administration of celastrol in a transgenic model of Alzheimer’s reduces an important neuropathological hallmark of this disease, namely, amyloid beta pathology that involves protein aggregation.     

Affinity isolation of heat-shock and other calmodulin-binding proteins following hyperthermia

The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.     

Mass spectrometry identification of NASP binding partners in HeLa cells

Nuclear autoantigenic sperm protein (NASP) is a linker histone binding protein that is cell-cycle regulated. Synchronized HeLa cells are delayed in progression through the G1/S border when transiently transfected to overexpress full-length NASP, but not the histone-binding site (HBS) deletion mutant (NASP-DeltaHBS). The purpose of the current study was to identify possible NASP-associated proteins in HeLa cell nuclei that could elucidate NASP's influence on the cell cycle and chromatin remodeling. For this purpose, we employed a new approach: mass spectrometry identification of initially cross-linked proteins after their separation in a second dimension by reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Of the twelve proteins identified, three appear to be relevant to NASP's function: heat shock protein 90 (HSP90), DNA-activated protein kinase, and ATP-dependent DNA helicase II (70-kDa subunit). Individual protein-protein interactions were tested by immunoprecipitation techniques. This new method can be used for expedited identification of binding partners of different proteins in enriched fractions and as a complementary or alternative strategy to the yeast two-hybrid system and immunoprecipitation methods.     

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

Pim-1 kinase stability is regulated by heat shock proteins and the ubiquitin-proteasome pathway

Elevated expression of the serine/threonine kinase Pim-1 increases the incidence of lymphomas in Pim-1 transgenic mice and has also been found to occur in some human cancers. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. It was therefore of interest to understand to what extent maintenance and degradation of Pim-1 protein is affected by heat shock proteins (Hsp) and the ubiquitin-proteasome pathway in K562 and BV173 human leukemic cells. The half-life of Pim-1 protein in these cells was found to increase from 1.7 to 3.1 hours when induced by heat shock or by treating the cells with the proteasome inhibitor PS-341 (bortezomib). The Hsp90 inhibitor geldanamycin prevented the stabilization of Pim-1 by heat shock. Using immunoprecipitation, it was determined that Pim-1 is targeted for degradation by ubiquitin and that Hsp70 is associated with Pim-1 under these circumstances. Conversely, Hsp90 was found to protect Pim-1 from proteasomal degradation. A luminescence-based kinase assay showed that Pim-1 kinase bound to Hsp70 or Hsp90 remains active, emphasizing the importance of its overall cellular levels. This study shows how Pim-1 levels can be modulated in cells through degradation and stabilization.     

Dynamics of chaperone complex Hdj1-Hsp70-Bag1 as a response of erythroleukemia K562 cells to heat stress

Heat shock protein Hsp70 is known to play an important role in cell protection against a variety of harmful factors. This property, at least in part, is due to Hsp70 ability to restore the native conformation of newly synthetized or damaged proteins. In this activity Hsp70 is accompanied by two proteins, Hdj1 and Bag1, that enable Hsp70 to peform cycles of binding-release of target proteins. The aim of this study was to investigate interactions of Hdj1 and Bag1 co-chaperones with Hsp70 in vivo. The accumulation of Hsp70 was stimulated by heat stress, and later, at certain periods following the stress, cell probes were collected for biochemical and microscopic analysis. The data of Western blotting showed that within 24 h after heat shock amounts of Hsp70 and Hdj1 raised to remain at the elevated level for nearly 48 h. Several time points within this period were chosen for analysis of the complexes between Hsp70 and co-chaperones. The data of reciprocal immunoprecipitation/immunoblotting and confocal microscopy showed that Hsp70-Hdj1 complexes were detected primarily at early stage after heat shock, then Hsp70 was preferably bound to Bag1. The dynamics of chaperone complex formation and changes in their intracellular localization are discussed in terms of cell reaction to stress.     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

Calmodulin signaling: analysis and prediction of a disorder-dependent molecular recognition

Calmodulin (CaM) signaling involves important, wide spread eukaryotic protein-protein interactions. The solved structures of CaM associated with several of its binding targets, the distinctive binding mechanism of CaM, and the significant trypsin sensitivity of the binding targets combine to indicate that the process of association likely involves coupled binding and folding for both CaM and its binding targets. Here, we use bioinformatics approaches to test the hypothesis that CaM-binding targets are intrinsically disordered. We developed a predictor of CaM-binding regions and estimated its performance. Per residue accuracy of this predictor reached 81%, which, in combination with a high recall/precision balance at the binding region level, suggests high predictability of CaM-binding partners. An analysis of putative CaM-binding proteins in yeast and human strongly indicates that their molecular functions are related to those of intrinsically disordered proteins. These findings add to the growing list of examples in which intrinsically disordered protein regions are indicated to provide the basis for cell signaling and regulation.     

An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility

CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63 kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca²⁺-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote.