BAG1, MGMT, FOXO1, and DNAJA1 as potential drug targets for radiosensitizing cancer cell lines.

BACKGROUND AND PURPOSE: Activation of some signaling pathways can promote cell survival and have a negative impact on tumor response to radiotherapy. Here, the role of differences in expression levels of genes related to the poly(ADP-ribose) polymerase-1 (PARP-1), heat shock protein 90 (Hsp90), B-cell lymphoma 2 (Bcl-2), and phosphoinositide 3-kinase (PI3K) pathways in the survival or death of cells following X-ray exposure was investigated. METHODS: Eight human cell cultures (MCF-7 and MDA-MB-231: breast cancers; MCF-12A: apparently normal breast; A549: lung cancer; L132: normal lung; G28, G44 and G112: glial cancers) were irradiated with X-rays. The colony-forming and real-time PCR based on a custom human pathway RT2 Profiler PCR Array assays were used to evaluate cell survival and gene expression, respectively. RESULTS: The surviving fractions at 2 Gy for the cell lines, in order of increasing radioresistance, were found to be as follows: MCF-7 (0.200 ± 0.011), G44 (0.277 ± 0.065), L132 (0.367 ± 0.023), MDA-MB-231 (0.391 ± 0.057), G112 (0.397 ± 0.113), A549 (0.490 ± 0.048), MCF-12A (0.526 ± 0.004), and G28 (0.633 ± 0.094). The rank order of radioresistance at 6 Gy was: MCF-7 < L132 < G44 < MDA-MB-231 < A549 < G28 < G112 < MCF-12A. PCR array data analysis revealed that several genes were differentially expressed between irradiated and unirradiated cell cultures. The following genes, with fold changes: BCL2A1 (21.91), TP53 (8743.75), RAD51 (11.66), FOX1 (65.86), TCP1 (141.32), DNAJB1 (3283.64), RAD51 (51.52), and HSPE1 (12887.29) were highly overexpressed, and BAX (-127.21), FOX1 (-81.79), PDPK1 (-1241.78), BRCA1 (-8.70), MLH1 (-12143.95), BCL2 (-18.69), CCND1 (-46475.98), and GJA1 (-2832.70) were highly underexpressed in the MDA-MB-231, MCF-7, MCF-12A, A549, L132, G28, G44, and G112 cell lines, respectively. The radioresistance in the malignant A549 and G28 cells was linked to upregulation in the apoptotic, DNA repair, PI3K, and Hsp90 pathway genes BAG1, MGMT, FOXO1, and DNAJA1, respectively, and inhibition of these genes resulted in significant radiosensitization. CONCLUSIONS: Targeting BAG1, MGMT, FOXO1, and DNAJA1 with specific inhibitors might effectively sensitize radioresistant tumors to radiotherapy.

DNAJB9 Inhibits p53-Dependent Oncogene-Induced Senescence and Induces Cell Transformation.

DNAJB9 is known to be a member of the molecular chaperone gene family, whose cellular function has not yet been fully characterized. Here, we investigated the cellular function of DNAJB9 under strong mitogenic signals. We found that DNAJB9 inhibits p53-dependent oncogene-induced senescence (OIS) and induces neoplastic transformation under oncogenic RAS activation in mouse primary fibroblasts. In addition, we observed that DNAJB9 interacts physically with p53 under oncogenic RAS activation and that the p53-interacting region of DNAJB9 is critical for the inhibition of p53-dependent OIS and induction of neoplastic transformation by DNAJB9. These results suggest that DNAJB9 induces cell transformation under strong mitogenic signals, which is attributable to the inhibition of p53-dependent OIS by physical interactions with p53. This study might contribute to our understanding of the cellular function of DNAJB9 and the molecular basis of cell transformation.

HLJ1 is an endogenous Src inhibitor suppressing cancer progression through dual mechanisms.

HLJ1 (DNAJB4), a DNAJ/Hsp40 chaperone, has emerged as a novel prognostic marker in lung cancers; however, the molecular contribution and functionality in neoplastic diseases remain to be established. This study demonstrated that HLJ1 inhibits epithelial-mesenchymal transition in vitro and reduces lung cancer metastasis in vivo. Using shRNA silencing and ectopic expression of HLJ1, we found that HLJ1 not only suppresses catalytic activity of Src but also downregulates the formation of oncogenic complexes associated with the EGFR, FAK and STAT3 signaling pathways. A screen of specimens from HLJ1-knockout mice and lung cancer patients validated that HLJ1 expression is inversely correlated with Src activity. Mechanistically, HLJ1 protein directly bound to catalytic and protein-binding domains of Src through its amino acid Y172 and the P301/P304 motif. Following Src-induced HLJ1 phosphorylation at Y172, HLJ1-Src interaction was elevated, resulting in Src inhibition and malignancy suppression. Interestingly, both Src-binding regions also occurred in other DNAJB family members and contributed to anti-invasive activities of DNAJB proteins. We conclude that HLJ1 is an endogenous Src inhibitor that can suppress cancer metastasis through complex interacting mechanisms. This HLJ1-Src complex might provide a promising molecular model for developing new anticancer strategies.

The J-domain of heat shock protein 40 can enhance the transduction efficiency of arginine-rich cell-penetrating peptides.

Sense and antisense oligonucleotide pairs encoding cell-penetrating peptides PTD (Tat47-57), DPV3A, E162, pVEC, R11, and TP13 were used to construct two sets of pET22b-CPP-DsRed and pET22b-CPP-J-DsRed vectors for CPP-DsRed and CPP-J-DsRed recombinant proteins expression. PTD-DsRed, DPV3A-DsRed, PTD-J-DsRed, and DPV3A-J-DsRed recombinant proteins were expressed in a soluble form. PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins were able to escape from E. coli host cells into the culture medium. The membrane-penetrating activity of PTD-J-DsRed and DPV3A-J-DsRed recombinant proteins to mammalian cells was more effective than that of PTD-DsRed and DPV3A-DsRed. The route of the cellular membrane translocation of these recombinant proteins is suggested via macropinocytosis followed by an endosomal escape pathway.

Cysteine string protein limits expression of the large conductance, calcium-activated K⁺ (BK) channel.

Large-conductance, calcium-activated K(+) (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKß4 and BKß1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

Novel role of HSP40/DNAJ in the regulation of HIV-1 replication.

OBJECTIVES: DNAJ/HSP40 is an evolutionarily conserved family of proteins bearing various functions. Historically, it has been emphasized that HSP40/DNAJ family proteins play a positive role in infection of various viruses. We identified DNAJ/HSP40B6 as a potential negative regulator of HIV-1 replication in our genetic screens. In this study, we investigated the functional interactions between HIV-1 and HSP40 family members. DESIGN: We took genetic and comparative virology approaches to expand the primary observation. METHODS: Multiple HSP40/DNAJ proteins were tested for their ability to inhibit replication of adenovirus, herpes simplex virus type 1, HIV-1, and vaccinia virus. The mechanism of inhibition was investigated by using HSP40/DNAJ mutants and measuring the efficiencies of each viral replication steps. RESULTS: HSP40A1, B1, B6, and C5, but not C3, were found to be able to limit HIV-1 production. This effect was specific to HIV-1 for such effects were not detected in adenovirus, herpes simplex virus type 1, and vaccinia virus. Genetic analyses suggested that the conserved DNAJ domain was responsible for the inhibition of HIV-1 production through which HSP40 regulates HSP70 ATPase activity. Interestingly, HSP40s lowered the levels of steady-state viral messenger RNA. This was not attributed to the inhibition of Tat/long terminal repeat-driven transcription but the downregulation of Rev expression. CONCLUSIONS: This is the first report providing evidence that HSP70-HSP40 complex confers an innate resistance specific to HIV-1. For their interferon-inducible nature, HSP40 family members should account for the anti-HIV-1 function of interferon.

Hsp40 proteins modulate humoral and cellular immune response in rheumatoid arthritis patients.

Recent research on the heat shock proteins (Hsps) in chronic inflammatory diseases indicates that Hsps may have disease-suppressive activities. Our aim was to characterize immune response directed to bacterial (DnaJ) and human Hsp40s in patients with rheumatoid arthritis (RA). We found elevated levels of anti-DnaJ, anti-Hdj2, and anti-Hdj3 (but not ant-Hdj1) serum antibodies in the RA patients (P < or = 0.001) compared to healthy controls. In peripheral blood mononuclear cells (PBMCs) culture, all tested Hsp40 proteins significantly inhibited the divisions of CD4+ and CD8+ T cells of the RA patients but not those of the controls. Both DnaJ and Hdj2 stimulated secretion of the main anti-inflammatory cytokine IL-10 by PBMCs of the RA patients (P < 0.05), and of IL-6 by PBMCs of the RA (P < 0.001) and control (P < 0.01) groups. DnaJ reduced TNFalpha secretion (P < 0.05) by both groups of PBMCs. Our results show for the first time that the RA patients have an increased humoral response to human Hsp40 proteins Hdj2 and Hdj3. This is also the first description of immunomodulatory effect of human Hsp40s on T cells and cytokine secretion in RA, suggesting that Hsp40s act as natural anti-inflammatory agents in RA.

Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer.

The p53 tumor suppressor protein induces apoptosis in response to genotoxic and environmental stresses. Recent studies have revealed the existence of a transcription-independent mitochondrial p53 apoptotic pathway; however, the mechanism that regulates its translocation to the mitochondria has been unknown. In this study, we show that the tumor suppressor Tid1 forms a complex with p53 under hypoxic conditions that directs p53 translocation to the mitochondria and the subsequent initiation of the mitochondrial apoptosis pathway. Loss of Tid1 expression abrogated p53 translocation to the mitochondria and inhibited apoptosis, whereas the over-expression of Tid1 promoted p53 mitochondrial localization and apoptosis. Tid1's mitochondrial signal sequence and DnaJ domain were both required for the movement of the p53-Tid1 complex from the cytosol to the mitochondria. When Tid is over-expressed in cancer cell lines expressing mutant p53 isoforms defective in transcriptional activity, mitochondrial localization and pro-apoptotic activities of the mutant p53 proteins was restored. Our results establish Tid1 as a novel regulator of p53-mediated apoptosis, and suggest that therapies designed to enhance Tid1's function in promoting mitochondrial localization of p53 and apoptosis could be an effective therapy in many cancers.

Immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae against lethal infection in mice.

The present study was carried out to evaluate the immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae, by cloning the full-length DnaJ of S. pneumoniae and expressing in heterologous host E. coli BL-21 (DE3). PCR amplified DnaJ was ligated in pQE-30 expression vector and subsequently transformed in E. coli DH5alpha strain. Cloning of DnaJ was confirmed by double digestion and PCR, followed by DNA sequencing. The His-tag containing recombinant protein was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of DnaJ, the mice (10 mice/group) were immunized by injecting 40 microg DnaJ protein/mouse i.p. There was a significant increase in IgG titres (2 x 10(5)) in mice immunized with DnaJ protein. Isotyping studies revealed that antibodies produced are predominantly IgG2a type indicating the predominance of Th1 response. A significant increase in lymphocyte proliferation was observed in mice immunized with DnaJ protein as compared to the control mice. Further, there was a significant increase in IL-2 and gamma-IFN levels in culture supernatants of splenocytes isolated from immunized mice. To determine the efficacy of DnaJ vaccination in eliciting protection, the mice were challenged with 1 x 10(5)cells of S. pneumoniae A66 type 3 capsular strain intra-nasally after 7 days of last immunization. All the control mice died within 2 days of post-infection, while 70% of animals immunized with DnaJ survived the lethal challenge by S. pneumoniae. The study reveals that immunization of mice with DnaJ elicits protective immunity against S. pneumoniae infection.

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2.

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.