Recombinant Heat Shock Protein Research Articles

Immune response in susceptible BALB/c mice immunized with DNA encoding Lipophosphoglycan 3 of Leishmania infantum.

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN-γ and IL-10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co-administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime-boost approach with a powerful adjuvant to elicit a robust parasite-specific protective Th1 response.

Spectroscopic and thermodynamic properties of recombinant heat shock protein A6 from Camelus dromedarius.

Heat shock protein A6, also known as HSP70B', is a member of the Hsp70 family of molecular chaperones. Under stressed conditions, the level of HSPA6 increases substantially, and the protein has been targeted as a biomarker of cellular stress in several studies. We report the spectroscopic and thermodynamic properties of Arabian camel species cHSPA6, determined by measurement of intrinsic and extrinsic fluorescence emission, and use of far-UV circular dichroism and dynamic multimode spectroscopy. Our results showed that cHSPA6 has similar binding affinity for both ATP and ADP (K D=~50nM). Binding of ATP and ADP reduced the surface hydrophobicity of the protein, and slightly altered its secondary structure, suggesting localized conformational rearrangement after ATP or ADP binding. Dynamic multimode spectroscopy revealed that cHSPA6 unfolds through three transitions with melting points (T m) of 42.3±0.2, 61.3±0.1, and 81.2±0.2°C. To the best of the author's knowledge, and literature search, this is the first report of the spectroscopic and thermodynamic properties of the Arabian camel heat shock protein.

Quaternary structure of human small heat shock protein HSPB6 (Hsp20) in crowded media modeled by trimethylamine N-oxide (TMAO): Effect of protein phosphorylation.

Effect of trimethylamine N-oxide (TMAO), well-known osmolyte, widely used to imitate crowded intracellular conditions, on the quaternary structure of recombinant human small heat shock protein HspB6 (Hsp20) was analyzed by means of size-exclusion chromatography, chemical crosslinking and analytical ultracentrifugation. Consistent with previous reports, in the absence of TMAO unphosphorylated, pseudophosphorylated (S16D mutant) and phosphorylated HspB6 form only small oligomers (presumably dimers). Addition of TMAO to unphosphorylated HspB6 leads to formation of different large oligomers being in equilibrium with dimers. Pseudophosphorylation (S16D mutation) or phosphorylation partially or completely prevent TMAO-induced oligomerization of HspB6. Pseudophosphorylation affects bis-ANS binding suggesting decreased hydrophobicity of HspB6. According to size-exclusion chromatography, TMAO-induced changes of HspB6 oligomerization result in its altered interaction with HspB1 and this effect can be reversed by HspB6 phosphorylation. It is concluded that under conditions of molecular crowding, characteristic for intracellular environment, HspB6 undergoes reversible changes of its oligomeric state which can affect its physiologically important properties and can be delicately regulated by phosphorylation.

Heat shock protein 65 promotes atherosclerosis through impairing the properties of high density lipoprotein

Aim: To explicit whether the functions of high density lipoprotein (HDL) are impaired in murine atherosclerosis by subcutaneous immunization with recombinant mycobacterial heat shock protein 65 (HSP65). Methods: C57BL/6 mice were fed a normal chow-diet with non immunization as normal group. ApoE knockout (ApoE−/−) mice on high-fat diet were randomly divided into three groups (n = 8) and immunized subcutaneously with different concentrations of HSP65 or phosphate-buffered saline (PBS). All animals were treated for 16 weeks. Reverse cholesterol efflux, the anti-oxidant and anti-inflammatory functions of HDL were assayed. Hepatocytes and peritoneal macrophages were isolated to examine the expression of cholesterol transport regulating proteins, including SR-B1, ABCA1, ABCG1, PPAR-γ and LXR-α. Results: In HSP65-immunized mice, paraoxonase1 (PON1) activity and the expression of IL-10 were reduced, while High-density lipoprotein inflammatory index (HII), myeloperoxidase (MPO) activity, and the expression of IFN-γ were elevated gradually. The MPO/PON1 ratio amount was significantly higher in HSP65-immunized group than in normal or PBS-immunized group. In addition, compared with normal or PBS-immunized group, cholesterol efflux rate and the expression of regulating proteins were markedly decreased in HSP65-immunized group. The mice immunized with HSP65 developed significantly larger aorta atherosclerotic plaques when compared with PBS-treated littermates. The high MPO/PON1 ratio was correlated with HII, cholesterol efflux rate and atherosclerotic plaques. Conclusions: This study demonstrates that subcutaneous immunization with HSP65 impairs the properties of HDL, which may contribute to its important pathogenic role of HSP65 in atherogenesis. Also, MPO/PON1 ratio may be a predictor of AS.

Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL− natural killer cells that were adoptively transferred into Rag-2−/− γ chain−/− mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

O7.06 * MAGNETIC NANOPARTICLES CONJUGATED TO A RECOMBINANT HEAT SHOCK PROTEIN HSP70 FOR TARGETING EXPERIMENTAL GLIOBLASTOMA

Superparamagnetic nanoparticles (SPIONs) due to their unique properties could be applied for theranostic approach in neuro-oncology. Accumulation and retention of the nanoparticles in the tumour can be directly imaged by MRI proving its application as contrast agents. Due to high heating capacity SPIONs could also be applied for thermotherapy of brain tumours. To further increase the biocompatibility and targeted delivery of SPIONs we have functionalized the surface of particles with recombinant heat shock protein Hsp70. Nuclear magnetic resonance (NMR) spectra and magnetic relaxation times of the synthesized conjugates T1, T2, T2* were measured with the help of an NMR spectrometer (CXP-300, Bruker) in a magnetic field of 7.1 T. Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. In series of in vitro experiments we observed enhanced internalization of the SPION-Hsp70 conjugates by C6 glioma cells in comparison to control SPIONs. The contrast-enhancing ability of SPION-Hsp70 conjugates was analyzed in the model of intracranial C6 glioma in rodents. The rat images were acquired with the help of a Bruker Avance II NMR spectrometer equipped with a microtomographic accessory at a magnetic field of 11 T. In vivo analysis of the conjugates distribution in the model of intracranial glioma revealed the retention of the functionilzed particles inside the tumour site. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. Immunofluoresence studies confirmed that retention in the glioma of nanoparticles was due to the receptor-mediated endocytosis of the SPION-Hsp70 conjugates via the CD40 receptors. The uptake of SPION-Hsp70 by tumour cells permitted MRI contrast enhancement after systemic delivery while sparring surrounding normal brain tissues.

Generation of oxyradicals ( and H2O2), mitochondrial activity and induction of apoptosis of PBMC of Cyprinus carpio carpio treated in vivo with halomethanes and with recombinant HSP60 kDa and with LPS of Klebsiella pneumoniae

Halomethanes (HM) can be immunotoxic in mammals; however, in the fish immune system HM effects are unknown. In the current study, we evaluated the mitochondrial activity (MA) by MTT, induction of apoptosis by SubG0 technique and quantified serum ROS concentration ( and H2O2) and ROS production in PBMC of Cyprinus carpio carpio treated i.p. with CH2Cl2, CHCl3 and BrCHCl2 (0.004–40.0 mg/kg) for 96 h. Positive controls were recombinant heat shock protein of 60 kDa (rHSP60 kDa) of Klebsiella pneumoniae and its LPS. In addition, for in vitro PBMC cultures, two culture media and two sources of sera were tested. Both positive controls increased the MA more than 4-fold as well as the production of (26-fold) and H2O2 (5-fold) compared to their controls. HM induced different effects on MA, ROS production and an induction of apoptosis, depending on the chlorination patterns and the dose; however, a systemic damage prevails. To fish treated with CH2Cl2, the apoptosis was related with serum ROS concentration and with MA. In contrast, in fish dosed with CHCl3 relationships were not found, deducing a systemic damage. However, in fish treated with BrCHCl2, serum concentration and in vitro ROS generation performed by PBMC were involved in the induction of apoptosis of these cells but not with MA suggesting also immunotoxic effects. The current study demonstrated that HMs are immunomodulators increasing an acute inflammatory response and that rHSP60kDA of K. pneumoniae and its LPS are appropriate antigens to assess the immune response of C. c. carpio.

The Mycoplasma hyopneumoniae recombinant heat shock protein P42 induces an immune response in pigs under field conditions.

Enzootic pneumonia (EP), resulting from Mycoplasma hyopneumoniae infection is one of the most prevalent diseases in pigs and is a major cause of economic losses to the swine industry worldwide. EP is often controlled by vaccination with inactivated, adjuvanted whole-cell bacterin. However, these bacterins provide only partial protection and do not prevent M. hyopneumoniae colonization. Attempts to develop vaccines that are more efficient have made use of the recombinant DNA technology. The objective of this study was to assess the potential of recombinant M. hyopneumoniae heat shock protein P42 in vaccine preparations against EP, using piglets housed under field conditions in a M. hyopneumoniae-positive farm. The cellular and humoral immune responses were elicited after a single intramuscular inoculation of rP42 in an oil-based adjuvant, or in conjunction with whole-cell vaccine preparation. The production of INF-γ and IL-10 cytokines was quantified in the supernatant of the cultured mononuclear cells. The rP42 emulsified in oil-based adjuvant was able to trigger a strong humoral immune response. Further, it induced a cellular immune response, accompanied by the production of antibodies that reacted with the native M. hyopneumoniae protein. The rP42 mediated induction of cellular and humoral immune response in the host suggests that rP42 emulsified in an oil-based adjuvant holds promise as an effective recombinant subunit vaccine against EP.

Pilot study of intratumoral injection of recombinant heat shock protein 70 in the treatment of malignant brain tumors in children.

Intratumoral injections of recombinant heat shock protein (Hsp)70 were explored for feasibility in patients with brain tumors. Patients aged 4.5–14 years with untreated newly diagnosed tumors (n=12) were enrolled. After tumor resection, five injections of recombinant Hsp70 (total 2.5 mg) were administered into the resection cavity through a catheter. Before administration of Hsp70 and after the last injection, specific immune responses to the autologous tumor lysate were evaluated using the delayed-type hypersensitivity test. Further, peripheral blood was monitored to identify possible changes in lymphocyte subpopulations, cytokine levels, and the cytolytic activity of natural killer cells. The follow-up period in this trial was 12 months. Intratumoral injections of Hsp70 were well tolerated by patients. One patient had a complete clinical response documented by radiologic findings and one patient had a partial response. A positive delayed-type hypersensitivity test was observed in three patients. In peripheral blood, there was a shift from cytokines provided by Th2 cells toward cytokines of a Th1-cell-mediated response. These data corresponded to changes in lymphocyte subpopulations. Immunosuppressive T-regulatory cell levels were also reduced after injection of Hsp70, as well as production of interleukin-10. The cytolytic activity of natural killer cells was unchanged. The present study demonstrates the feasibility of intratumoral delivery of recombinant Hsp70 in patients with cancer. Further randomized clinical trials are recommended to assess the optimum dose of the chaperone, the treatment schedule, and clinical efficacy.

Epitope mapping of the HSP83.1 protein of Leishmania braziliensis discloses novel targets for immunodiagnosis of tegumentary and visceral clinical forms of leishmaniasis.

Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.

Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats.

Recombinant 70 kDa heat shock protein (Hsp70) is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg). To assess Hsp70’s neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia). Rats were then kept alive for 72 hours. The ischemic region was analyzed using a high-field 11 T MRI scanner. Administration of the Hsp70 decreased the infarction zone in a dose-dependent manner with an optimal (threefold) therapeutic response at 5 mg/kg. Long-term treatment of the ischemic rats with Hsp70 formulated in alginate granules with retarded release of protein further reduced the infarct volume in the brain as well as apoptotic area (annexin V staining). Due to its high neurotherapeutic potential, prolonged delivery of Hsp70 could be useful in the management of acute ischemic stroke.

OP0019 Immunomodulatory potency of recombinant heat shock protein HSP70 in therapy of malignant brain tumours

<h3>Background</h3> HSP70 is known to stimulate anti-tumour immunity, validating its possible application in immunotherapeutic approaches. In a series of in vitro experiments we demonstrated that HSP70, when added to cancer cells, can penetrate the latter inducing the translocation of endogenous HSP70 to plasma membrane. Interchange of exogenous and intracellular HSP70 induced lymphocyte-mediated anti-tumour activity. In an intracranial C6 glioma model, we administered HSP70 intratumourally. The infusion of the chaperone caused inhibition of tumour growth, as measured by high-field (7.1T) MRI, and significantly prolonged survival. These changes correlated with induction of the tumour-specific innate response mediated by NK cells (production of Granzyme B) and adaptive immune response generated by CD8+ T-lymphocytes (proved by IFN<i>γ</i> ELISPOT assay). Immunological changes were accompanied by glioma infiltration by NK-cells (Ly-6c+) and T-cells (i.e., CD3+, CD4+, CD8+). Following the in vivo experiments, the effects of intratumoural injection of HSP70 were explored in a pilot study for feasibility, safety, and possible systemic anti-tumour immune responses in patients with brain tumours. <h3>Methods</h3> Patients with untreated newly diagnosed tumours (<i>n</i>=12) were enrolled in the study. After tumour resection, five injections of HSP70 were performed into the residual tumour cavity through a catheter. Specific immune responses were evaluated with a delayed hypersensitivity test. <h3>Findings</h3> Intratumoural injections of HSP70 were well tolerated by patients. One patient had a complete clinical response documented by radiological findings and one patient had a partial response. In three patients we observed positive delayed hypersensitivity tests. In peripheral blood there was a shift toward cytokines of the Th1-cell mediated response that corresponded to changes in lymphocyte subpopulations. <h3>Interpretation</h3> The present pilot study shows the feasibility and safety of intratumoural delivery of recombinant HSP70 in patients. On the basis of our studies we propose a novel mechanism of immunomodulatory activity of HSP70.

Sublingual Vaccine with GroEL Attenuates Atherosclerosis

Autoimmune responses to heat-shock protein 60 (HSP60) contribute to the progression of atherosclerosis, whereas immunization with HSP60 may induce atheroprotective responses. We assessed the capacity of an atheroprotective vaccine that targeted a recombinant HSP60 from Porphyromonas gingivalis (rGroEL) to induce a protective mucosal immune response. Female apolipoprotein E-deficient spontaneously hyperlipidemic (Apoeshl) mice received sublingual delivery of rGroEL prior to P. gingivalis 381 injection. The animals were euthanized 16 weeks later. Sublingual immunization with rGroEL induced significant rGroEL-specific serum IgG responses. Antigen-specific cells isolated from spleen produced significantly high levels of IL-10 and IFN-γ after antigen re-stimulation in vitro. Flow cytometric analysis indicated that the frequencies of both IL-10+ and IFN-γ+ CD4+ Foxp3+ cells increased significantly in submandibular glands (SMG). Furthermore, sublingual immunization with rGroEL significantly reduced atherosclerosis lesion formation in the aortic sinus and decreased serum CRP, MCP-1, and ox-LDL levels. These findings suggest that sublingual immunization with rGroEL is associated with the increase of IFNγ+ or IL-10+ Foxp3+ cells in SMG and a systemic humoral response, which could be an effective strategy for the prevention of naturally occurring or P. gingivalis-accelerated atherosclerosis.

Production of Recombinant Heat Shock Protein 60 (HSP60) From Salmonella enterica serovar Typhimurium ATCC 19585 and Its Evaluation as a Vaccine Candidate in BALB/c Mice

The gene for heat shock protein 60 (HSP60) was amplified from the DNA extract of Salmonella enterica serovar Typhimurium strain (LT2), ATCC 19585, using Polymerase Chain Reaction (PCR). The HSP60 gene was partially sequenced, inserted into expression vector and cloned into a competent Escherichia coli. The expressed recombinant HSP60 protein was purified by Ni-NTA affinity chromatography. Immunization of BALB/c mice with the purified recombinant HSP60 protein resulted in a significant anti-HSP60 antibody titers. The groups of the immunized and control mice were challenged with lethal doses of S. Typhimurium (LT2) ATCC 19585. Immunized mice had a higher survival rate than control mice suggesting the protective value of the recombinant HSP60 protein used for vaccination.

Evaluating the protective efficacy of antigen combinations against Photobacterium damselae ssp. piscicida infections in cobia, Rachycentron canadum L.

Cobia, Rachycentron canadum L., is a very important aquatic fish that faces the risk of infection with the bacterial pathogen Photobacterium damselae ssp. piscicida, and there are few protective approaches available that use multiple antigens. In the present study, potent bivalent antigens from P. damselae ssp. piscicida showed more efficient protection than did single antigens used in isolation. In preparations of three antigens that included recombinant heat shock protein 60 (rHSP60), recombinant α-enolase (rENOLASE) and recombinant glyceraldehyde-3-phosphate dehydrogenase (rGAPDH), we analysed the doses that elicited the best immune responses and found that this occurred at a total of 30 μg of antigen per fish. Subsequently, vaccination of fish with rHSP60, rENOLASE and rGAPDH achieved 46.9, 52 and 25% relative per cent survival (RPS), respectively. In addition, bivalent subunit vaccines--combination I (rHSP60 + rENOLASE), combination II (rENOLASE + rGAPDH) and combination III (rHSP60 + rGAPDH)--were administered and the RPS in these groups (65.6, 64.0 and 48.4%, respectively), was higher than that achieved with single-antigen administration. Finally, in combination IV, the trivalent vaccine rHSP60 + rENOLASE + rGAPDH, the RPS was 1.6%. Taken together, our results suggest that combinations of two antigens may achieve a better efficiency than monovalent or trivalent antigens, and this may provide new insights into pathogen prevention strategies.

Sperm activation by heat shock protein 70 supports the migration of sperm released from sperm storage tubules in Japanese quail (Coturnix japonica)

Systems for maintaining the viability of ejaculated sperm in the female reproductive tract are widespread among vertebrates and invertebrates. In birds, this sperm storage function is performed by specialized simple tubular invaginations called sperm storage tubules (SSTs) in the uterovaginal junction (UVJ) of the oviduct. Although the incidence and physiological reasons for sperm storage in birds have been reported extensively, the mechanisms of sperm uptake by the SSTs, sperm maintenance within the SSTs, and control of sperm release from the SSTs are poorly understood. In this study, we demonstrated that the highly conserved heat shock protein 70 (HSP70) stimulates sperm motility in vitro and also that HSP70 expressed in the UVJ may facilitate the migration of sperm released from the SSTs. Quantitative RT-PCR analysis demonstrated that the expression of HSP70 mRNA in the UVJ increases before ovulation/oviposition. Gene-specific in situ hybridization and immunohistochemical analysis with a specific antibody to HSP70 demonstrated that HSP70 is localized in the surface epithelium of the UVJ. Furthermore, injection of anti-HSP70 antibody into the vagina significantly inhibited fertilization in vivo. In addition, we found that recombinant HSP70 activates flagellar movement in the sperm and that the binding of recombinant HSP70 to the sperm surface is mediated through an interaction with voltage-dependent anion channel protein 2 (VDAC2). Our results suggest that HSP70 binds to the sperm surface by interacting with VDAC2 and activating sperm motility. This binding appears to play an important role in sperm migration within the oviduct.

A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients

BackgroundWe report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.ResultsFive cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.ConclusionThis protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

Molecular and Immunological Characterization of Heat Shock Protein 70 (HSP70) Gene from Buffalo

Heat shock protein 70 (HSP70) is a predominant member of the HSP family of proteins, which play a variety of functions in the cells and are responsible for cytoprotection under stress conditions. The present study was characterized by HSP70 (orf) in buffalo (Buablus bubalis). Genomic DNA was isolated from lymphocytes and that was used for PCR amplification of HSP70 gene. Polymerase chain reaction product (1,926 bp) was cloned in pGEM-T easy vector and sequenced. Sequence analysis revealed 1,926 bp long open reading frame of HSP70 gene encoding 641 amino acids in buffalo. The amino acid sequence showed 98 % identity with Bos taurus, Bos indicus, Yak, Capra hircus and 90–95 % identity with Camelus dromedaries, Felis catus, Canis familiaris, Sus scrofa, and Homo sapiens. The expression of this gene was in prokaryotic expression vector pPRoExHTa producing a recombinant protein of ~70 kDa, which is detected by SDS-PAGE. The tagged protein purified by Ni-NTA affinity chromatography under denaturing conditions was confirmed by western blotting using Ni-NTA HRP conjugate and 4 chloro-1-naphthol as substrate. Recombinant HSP70 was injected in mice and antiserum contained polyclonal antibody, which was detected by western blot. The recombinant HSP70 protein obtained may be used for the development of an assay for detection of thermal stress.

Identification of heat shock protein 60 as the regulator of the hypoxia-inducible factor subunit HIF-1α

The hypoxia-inducible factor (HIF) takes part in transcriptional activation of hypoxia-responsive genes such as vascular endothelial growth factor (VEGF), insulin-like growth factor, and inducible nitric oxide synthase. Since VEGF plays an important role in pathological angiogenesis such as tumor growth and ischemic diseases, the inhibition of VEGF inducer HIF is an attractive approach for the inhibition of pathological angiogenesis. Recently, we have reported that the introduction of boronic acid and a carborane moiety into phenoxyacetanilide induced a potent inhibitory effect on HIF-1α activation under hypoxic conditions. In the present study, to clarify the mechanism of action of carboranylphenoxyacetanilide GN26361 against HIF inhibition, we designed and synthesized molecular probes of GN26361 substituted with benzophenone to induce covalent binding with the target protein by UV (photoaffinity labeling) and an acetylenic moiety to conjugate with the green-fluorescent Alexa Fluor 488-azide by click reaction. In-gel fluorescent imaging of target protein bound with the probe was identified as heat shock protein 60 (HSP60). Moreover, direct binding in gel fluorescent imaging was observed by photoaffinity labeling and click reaction of the probe with recombinant HSP60. These results indicate that HSP60 is the target protein of GN26361 and might be a new molecular target for HIF inhibition.

Abstract 544: Extracellular Heat Shock Protein 27 Inhibits Foam Cell Formation by Targeting Scavenger Receptor-A in the Vessel Wall

Background: We showed that injection of recombinant heat shock protein 27 (rHSP27) into ApoE -/- mice reduces atherosclerosis lesions and total serum cholesterol levels. Extracellular HSP27 also interacts with scavenger receptor (SR)-A and inhibits acLDL uptake in macrophages, but the role of SR-A in HSP27 atheroprotection remains unclear. Purpose: To elucidate the mechanism of HSP27 atheroprotection by exploring if the presence of SR-A is required to facilitate its salutary effects. Methods/Results: rHSP27 treatment of bone-marrow derived macrophages from ApoE -/- mice reduced acLDL uptake (38%, p&lt;0.05) using flow cytometry - effects not observed (p=0.171) in cells from ApoE -/- SR-A -/- mice. rHSP27 reduced acLDL binding (38%, p&lt;0.01) and uptake (34%, p&lt;0.01) in THP-1 macrophages associated with downregulation of SR-A transcript (46%, p&lt;0.01) and total protein levels (63%, p&lt;0.001). Flow cytometry and confocal microscopy revealed reduction in SR-A expression at the cell surface. To explore the signaling involved, NF-kB activation was assessed in THP-1 cells stably transfected with an NF-kB reporter. rHSP27 increased NF-kB activity (7 fold, p&lt;0.001) and induced translocation of the NF-kB p65 subunit from the cytosol to the nucleus - effects that were inhibited (p&lt;0.05) by the NF-kB antagonist, BAY. Addition of BAY inhibited the effect of rHSP27 on SR-A gene and protein expression and acLDL uptake (p&lt;0.05). To explore the importance of SR-A in HSP27 atheroprotection, rHSP25 (mouse homologue) or PBS was administered to ApoE -/- and ApoE -/- SR-A -/- mice for 3 weeks on a high fat diet. Treatment of ApoE -/- mice revealed reductions of en face aortic lesions (39%, p&lt;0.001), SR-A expression in the intima, and total serum cholesterol levels (32%, p&lt;0.01). In the absence of SR-A, HSP27 atheroprotection was abolished (p=0.79) despite similar decreases in serum cholesterol levels (25%, p&lt;0.05). Conclusions: Extracellular HSP27 inhibits foam cells by downregulating SR-A expression via an NF-κB-dependent mechanism. HSP27 atheroprotection requires the presence of SR-A expression in vivo. In the absence of SR-A, loss of atheroprotection occurs despite reductions in total serum cholesterol levels, suggesting SR-A as a novel target for HSP27 in the vessel wall.