A peptide derived from chaperonin 60.1, IRL201104, inhibits LPS-induced acute lung inflammation.

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1ß and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.

Mycobacterium tuberculosis Heat-Shock Protein 16.3 Induces Macrophage M2 Polarization Through CCRL2/CX3CR1.

Mycobacterium tuberculosis, the pathogen of tuberculosis (TB), can survive in host macrophages and induce macrophages to M2 phenotype might result in latent MTB infection. During the latent phase, the expression of MTB heat-shock protein 16.3 (Hsp16.3) is markedly increased among most of bacterial proteins, but the role of Hsp16.3 in macrophage M2 polarization is not clear. In this work, we found that macrophages incubated with 100 ng/ml MTB Hsp16.3 increased the production of Arg-1, IL-10, TGF-beta, and CD206. These results showed that MTB Hsp16.3 may induce macrophage M2 phenotype. And the interaction of Hsp16.3 with macrophages was found to depend on chemokine receptors CCRL2 and CX3CR1. Additionally, we used overexpression and silencing techniques to further verify the effect of CCRL2 and CX3CR1 on MTB Hsp16.3-induced M2 polarization macrophages. Furthermore, we explored the downstream signaling molecules of CCRL2 and CX3CR1 and we found MTB Hsp16.3 altered the signal transduction of the AKT/ERK/p38-MAPK. Taken together, this study provides evidence that MTB Hsp16.3 promotes macrophages to M2 phenotype and explores its underlying mechanism.

ER homeostasis and motility of NSCLC cell lines can be therapeutically targeted with combined Hsp90 and HDAC inhibitors.

BACKGROUND AND OBJECTIVE: Lung cancer remains the most common cause of cancer-related death in the world for which novel systemic treatments are urgently needed. Protein homeostasis that regulates protein levels and their fold is critical for cancer cell proliferation and survival. A complex network of cellular organelles and signaling cascades is involved in control of protein homeostasis including endoplasmic reticulum (ER). Thus, proteins in control of ER homeostasis are increasingly recognized as potential therapeutic targets. Molecular chaperone heat shock protein 90 (Hsp90) and histone deacetylase (HDAC) play an important role in ER homeostasis. Previous studies demonstrate that Hsp90 and HDAC inhibitors are individually functional against lung cancer. In this work we suggested that combined Hsp90 and HDAC inhibitors may elevate ER stress thereby enhancing the anti non small lung cancer (NSCLC) activity. METHODS AND RESULTS: Using an in vitro cell line model we demonstrated that 17-DMAG (HSP90 inhibitor) co-administration with PTACH (HDAC inhibitor) caused elevated ER stress (immunoblotting) (more than 110%↑, p < 0.05) accompanied by apoptotic cell death (Annexin V) (7-21%↑, p < 0.05). Moreover, 17-DMAG/PTACH treated cells lost the ability to migrate (scratch test) (57-85%↓ of scratch closure, p < 0.05). CONCLUSIONS: Our findings provide proof-of-concept that targeting ER homeostasis is therapeutically beneficial in lung cancer cell lines. Indeed, the elevated ER stress caused by 17-DMAG/PTACH combined treatment leads to increased cell death of NSCLC cell lines compared to the application of the drugs separately.

Tolerization with Hsp65 induces protection against adjuvant-induced arthritis by modulating the antigen-directed interferon-gamma, interleukin-17, and antibody responses.

OBJECTIVE: Pretreatment of Lewis rats with soluble mycobacterial Hsp65 affords protection against subsequent adjuvant-induced arthritis (AIA). This study was aimed at unraveling the mechanisms underlying mycobacterial Hsp65-induced protection against arthritis, using contemporary parameters of immunity. METHODS: Lewis rats were given 3 intraperitoneal injections of mycobacterial Hsp65 in solution prior to the initiation of AIA with heat-killed Mycobacterium tuberculosis. Thereafter, mycobacterial Hsp65-specific T cell proliferative, cytokine, and antibody responses were tested in tolerized rats. The roles of anergy and the indoleamine 2,3 dioxygenase (IDO)-tryptophan pathway in tolerance induction were assessed, and the frequency and suppressive function of CD4+FoxP3+ Treg cells were monitored. Also tested was the effect of mycobacterial Hsp65 tolerization on T cell responses to AIA-related mycobacterial Hsp70, mycobacterial Hsp10, and rat Hsp65. RESULTS: The AIA-protective effect of mycobacterial Hsp65-induced tolerance was associated with a significantly reduced T cell proliferative response to mycobacterial Hsp65, which was reversed by interleukin-2 (IL-2), indicating anergy induction. The production of interferon-gamma (but not IL-4/IL-10) was increased, with concurrent down-regulation of IL-17 expression by mycobacterial Hsp65-primed T cells. Neither the frequency nor the suppressive activity of CD4+FoxP3+ T cells changed following tolerization, but the serum level of anti-mycobacterial Hsp65 antibodies was increased. However, no evidence was observed for a role of IDO or cross-tolerance to mycobacterial Hsp70, mycobacterial Hsp10, or rat Hsp65. CONCLUSION: Tolerization with soluble mycobacterial Hsp65 leads to suppression of IL-17, anergy induction, and enhanced production of anti-mycobacterial Hsp65 antibodies, which play a role in protection against AIA. These results are relevant to the development of effective immunotherapeutic approaches for autoimmune arthritis.

Effects of vaccination with heat shock proteins on streptozotocin induced diabetes in histidine decarboxylase knockout mice.

RATIONALE: Type 1 diabetes mellitus (T1D) is an immune mediated disease in which heat shock proteins (hsps) may be involved in the development of the disease. Furthermore, vaccination with different hsps prevented the development of multiple low-dose streptozotocin (STZ) induced autoimmune diabetes in C57BL/KSJ mice. Histamine influences many aspects of the immune response, including Th1/Th2 balance and antibody production. Therefore, a study of diabetes-related immune processes was considered of interest in histidine decarboxylase knockout (HDC-KO) mice. AIM OF THE STUDY: The aim of our study was i) to characterize antibody production in response to vaccination with p277 or hsp65 in wild type (WT) BALB/c and HDC-KO mice, and ii) to establish a possible correlation between vaccination and the changes in the pattern of STZ diabetes. MATERIALS AND METHODS: An ELISA was employed to measure the hsp65- and p277-specific antibody levels. To induce diabetes multiple low-dose of STZ was used. RESULTS: Vaccination with p277 and hsp65 altered the pattern of STZ diabetes both in HDC-KO and WT animals, characterized by a transient increase followed by sustained reduction of blood sugar levels as compared to controls. However, vaccination with hsp65 and p277 caused a significant anti-p277 and anti-hsp65 antibody level increase only in WT animals. CONCLUSION: Multiple low-doses of STZ were able to induce diabetes in HDC-KO mice and the development of diabetes was prevented by vaccination with hsps. This protection developed in spite of the fact that vaccination caused a significant antibody level increase only in WT animals. To explain the therapeutic effect of vaccination we need further examination of the HDC KO strain.

[Prokaryotic expression, purification of HSP65-MUC1 VNTR2 fusion protein and primary research on its tumoricidal effect].

AIM: To express the HSP65-MUC1 VNTR(2) in E.coli and to evaluate its activity of inhibiting tumor growth in vivo. METHODS: HSP65 and MUC1 VNTR(2) were generated by PCR method and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR(2)-pET28a(+). E.coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell linejB16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR(2) was evaluated in mice C57BL/6. RESULTS: The gene HSP65 and MUC1 VNTR(2) confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR(2)-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR(2) protein could be above 95% after purification by Q ion-exchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR(2) fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control group. CONCLUSION: In summary, HSP65-MUC1 VNTR(2) fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.

Down-regulation of glutaminase C in human hepatocarcinoma cell by diphenylarsinic acid, a degradation product of chemical warfare agents.

In a poisonous incident in Kamisu, Japan, it is understood that diphenylarsinic acid (DPAA) was a critical contaminant of ground water. Most patients showed dysfunction of the central nervous system. To understand the overall mechanism of DPAA toxicity and to gain some insight into the application of a remedy specific for intoxication, the molecular target must be clarified. As an approach, a high throughput analysis of cell proteins in cultured human hepatocarcinoma HpG2 exposed to DPAA was performed by two-dimensional electrophoresis (2-DE). Four proteins, which were up- and down-regulated by exposure of cultured HepG2 cells to DPAA, were identified. They were chaperonin containing TCP-1 (CCT) beta subunit, aldehyde dehydrogenase 1 (ALDH1), ribosomal protein P0 and glutaminase C (GAC). Of these, GAC was the only protein that was down-regulated by DPAA exposure, and cellular expression levels were reduced by DPAA in a concentration- and time-dependent manner. Decrease in cellular GAC levels was accompanied by decreased activity of the enzyme, phosphate-activated glutaminase (PAG). Decreased expression of GAC by DPAA was also observed in human cervical carcinoma HeLa and neuroblastoma SH-SY5Y cells. By contrast, no significant changes in GAC protein expression were observed when cells were incubated with arsenite [iAs (III)] and trivalent dimethylarsinous acid [DMA (III)]. In the central nervous system, GAC plays a role in the production of the neurotransmitter glutamic acid. Selective inhibition of GAC expression by DPAA may be a cause of dysfunction of glutamatergic neuronal transmission and the resultant neurological impairments.

Chaperone-mediated inhibition of tubulin self-assembly.

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self-assembly efficiently. Chaperones tested include alpha-crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and alpha (s)-casein from milk. All of them inhibit polymerization in a dose-dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self-assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and alpha (s)-casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 xx 10(4) and 9.8 xx 10(5) M(-1) for HSP16.3 and alpha (s)-casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones-tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin-chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein-protein interaction site involved in the nucleation phase of self-assembly.

Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein.

Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed.

Immunity to heat shock protein 65--an additional determinant in intimal thickening.

Inflammation occurring consequent to vessel injury is thought to play an important role in atherosclerosis and restenosis. Autoimmunity to HSP65 has been shown to accelerate early atherogenesis in rabbits and mice, whereas in humans epidemiological data support this contention. In the current study, we explored the possibility of HSP65 influencing the extent of neointimal growth in the rat carotid injury model. Rats were either immunized with recombinant mycobacterial HSP65, heat killed preparation of Mycobacterium tuberculosis (MT), or with PBS, all emulsified in incomplete Freund's adjuvant. Animals were boosted with a similar protocol 3 weeks following the primary immunization and 2 weeks later carotid injury was applied in all animals by balloon inflation. Upon sacrifice 2 weeks later, sera were obtained for measurement of anti-HSP65 antibodies by ELISA, splenocytes were assessed for proliferative response to in vitro priming with HSP65, and carotid arteries were removed for evaluation of neointimal growth. Rats immunized with HSP65 exhibited a brisk and sustained humoral immune response to HSP65, and cellular immunity was also evident by thymidine uptake to splenocytes primed with the respective protein. Neointimal/medial ratio was significantly increased in HSP65 immunized rats, in comparison with MT injected and control animals. In conclusion, immunity to HSP65 can play a role in accelerating restenosis following arterial injury. These results should be further investigated in humans as they may provide a possible link between infections and restenosis/accelerated arteriosclerosis.

BCG promotes cord blood monocyte-derived dendritic cell maturation with nuclear Rel-B up-regulation and cytosolic I kappa B alpha and beta degradation.

Mycobacterium bovis bacillus Calmette-Guerin (BCG) is given to millions of neonates in developing countries as a vaccine against Mycobacterium tuberculosis; however, little is known about the initiation of response in neonatal dendritic cells (DCs) to BCG. To address this issue, the interaction of BCG with human cord blood monocyte-derived DCs was studied. We showed that BCG could promote cord blood monocyte-derived DC maturation by up-regulation of CD80, CD83, CD86, CD40, and MHC class II molecules and down-regulation of mannose receptor. BCG was able to induce similar levels of tumor necrosis factor-alpha and IL-10 but no bioactive IL-12p70 production from cord blood DCs as from adult blood DCs. Functionally BCG-treated cord blood DCs had higher ability to induce mixed lymphocyte reaction than non-BCG-treated cord blood DCs. Both non-BCG-treated and BCG-treated cord blood DCs efficiently induced a high level of IL-10, medium level of interferon-gamma, but little IL-4 production by cord blood naïve CD4+ T cells. Heat shock protein 65, a key component of BCG, had no effect on cord blood DC maturation in terms of CD86, MHC class II, and mannose receptor up-regulation. During the BCG-induced maturation process of cord blood DCs, nuclear transcription factor Rel-B was up-regulated and cytosolic Rel-B down-regulated with cytosolic IkappaB alpha and beta degradation. These results suggest that BCG can promote cord blood monocyte-derived DC maturation, and that the mechanism is through the up-regulation of nuclear Rel-B secondary to the degradation of cytosolic IkappaB alpha and beta.

Activity of HspE7, a novel immunotherapy, in patients with anogenital warts.

PURPOSE: Human papillomavirus causes anogenital squamous intraepithelial lesions, warts, and cancer. Treatment of squamous intraepithelial lesions to prevent cancer often requires extensive surgery. We tested a human papillomavirus-specific immunotherapy, HspE7, as a potential alternative. METHODS: HspE7 was constructed by fusing heat shock protein Hsp65 from bacille Calmette-Guerin to E7 protein from human papillomavirus-16. Improvement in pathologic diagnosis of patients with persistent high-grade squamous intraepithelial lesions was studied in an open-label trial (HspE7 500 microg monthly x3). Anogenital warts were not a trial parameter, but a retrospective review of the medical records of the first 22 patients enrolled at one site was undertaken to estimate the quality and frequency of responses of anogenital warts. Patients with warts by physical examination at baseline were scored at 24 weeks as to the percent reduction in wart size. RESULTS: Fourteen of the 22 patients had warts at baseline. At Week 24, 3 of the 14 patients had complete resolution of their warts, and 10 had warts reduced in size an estimated 70 to 95 percent. The remaining patient's warts increased in size. The reduction in size in most patients greatly diminished the procedure necessary for complete ablation. No serious or severe adverse events were related to HspE7. CONCLUSIONS: A retrospective review of patients' medical records suggests that HspE7 may be broadly active in anogenital warts. This activity crosses multiple human papillomavirus types. The warts improved substantially but usually did not totally disappear within six months. Patient follow-up continues. A new randomized, placebo-controlled trial is underway to evaluate these findings.

Amelioration of arthritis in two murine models using antibodies to oncostatin M.

OBJECTIVE: Oncostatin M (OSM) is a member of the interleukin-6 cytokine family, with well-documented effects on cell growth and differentiation. OSM also has proinflammatory and cartilage degradative properties. The aim of this study was to investigate the significance of OSM in arthritis pathology using a neutralizing antibody in arthritis models. METHODS: Collagen-induced arthritis (CIA) was established in male DBA/1 mice. Reverse transcriptase-polymerase chain reaction was used to detect OSM messenger RNA (mRNA) message levels in arthritic joints. Neutralizing anti-OSM antibody or control immunoglobulin was administered on days 1 and 3 after disease onset. Animals were assessed for clinical arthritis for 2 weeks, followed by a histologic analysis of paws. Pristane-induced arthritis (PIA) was produced in male CBA mice dosed with anti-OSM or control immunoglobulin immediately before disease onset. Mice with PIA were assessed for clinical arthritis over a period of 100 days. RESULTS: Levels of mRNA for OSM, but not GAPDH, were elevated in arthritic joints of mice with CIA compared with those of normal controls. Mice with CIA treated with anti-OSM antibody showed significant amelioration of both the clinical severity (P < 0.01) and the number of affected paws (P < 0.01) compared with control animals. Histologic analysis confirmed these clinical findings, revealing a marked reduction in cellular infiltration of synovium and cartilage damage. In the PIA model, the incidence of arthritis was 65% in the control group compared with 0% in the anti-OSM-treated animals. CONCLUSION: These results demonstrate a key role for endogenously produced OSM as a potent mediator of joint pathology, and suggest that OSM might be a potentially important, novel therapeutic target for treatment of established rheumatoid arthritis.

T cells recognize an immunodominant epitope of heat shock protein 65 in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is an acute systemic vasculitis of infancy and early childhood that is characterized by endothelial cell damage associated with T-cell activation. Lymphocytes infiltrating damaged tissues might be responsible for the disease through secretion of cytokines, such as tumor necrosis factor (TNF)-alpha, that could cause fever, as well as endothelial tissue damage. Debate is growing about the nature of antigen responsible for T-cell activation in KD. Bacillus Calmette Guerin (BCG) and purified protein derivative (PPD) hyperresponsiveness was observed in KD patients and this phenomenon was hypothetically ascribed to cross-reactivity between mycobacterial Heat Shock Protein (HSP) 65 and human homologue HSP63. MATERIALS AND METHODS: CD4+ and CD8+ T-cell clones were obtained from peripheral blood of KD patients in acute phase, or control subjects. The clones were tested for reactivity toward HSP65 and derived peptides. Both proliferation and cytokine production were analyzed. RESULTS: A significant fraction of CD4 and CD8 T-cell clones from KD patients recognized an epitope from HSP65, spanning amino acids 65-85. T-cell clones cross-reacted with the corresponding 90-110 peptide sequence of human HSP-63. CONCLUSIONS: Cross-reactivity between specific epitopes of mycobacterial and human HSP could play a role in the development of the tissue-damage characteristic of KD.

Refolding a glutamine synthetase truncation mutant in vitro: identifying superior conditions using a combination of chaperonins and osmolytes.

A new method that uses a combination of bacterial GroE chaperonins and cellular osmolytes for in vitro protein folding is described. With this method, one can form stable chaperonin-protein folding intermediate complexes to prevent deleterious protein aggregation and, using these complexes, screen a large array of osmolyte solutions to rapidly identify the superior folding conditions. As a test substrate, we used GSDelta468, a truncation mutant of bacterial glutamine synthetase (GS) that cannot be refolded to significant yields in vitro with either chaperones or osmolytes alone. When our chaperonin/osmolyte method was employed to identify and optimize GSDelta468 refolding conditions, 67% of enzyme activity was recovered, comparable with refolding yields of wild type GS. This method can potentially be applied to the refolding of a broad spectrum of proteins.

T cell responses to 60/65 kDa heat shock protein derived peptides in Turkish patients with Behçet's disease.

OBJECTIVE: Sequence homology and cross reactivity between microbial and human heat shock proteins (HSP) led to the concept that HSP might be involved in the etiopathogenesis of Behçet's disease (BD). We investigated T cell responses to 8 synthetic peptides derived from the mycobacterial 65 kDa and homologous human 60 kDa HSP in patients with BD. METHODS: T cell proliferative responses to synthetic peptides were studied in 49 patients with BD and 46 disease (DC) and 34 healthy controls (HC) with 3H-thymidine uptake test. RESULTS: Positive T cell responses to one or more of the mycobacterial peptides were observed in 52% (12/23) of patients with BD compared with 17% (3/18) of DC (p = 0.02) and to homologous human peptides in 57% (13/23) of BD and 11% (2/18) of DC (p < 0.01). Responses to the mixtures of 4 mycobacterial peptides were also significantly higher in BD compared with controls (stimulation index in BD 4.7 +/- 3.5 vs DC 2.0 +/- 1.2, HC 1.6 +/- 0.4; BD vs DC and HC, p < 0.001). Similar elevated responses to the mixture of 4 human peptides was also observed in patients with BD (BD 3.4 +/- 2.3; DC 1.9 +/- 0.8; HC 1.4 +/- 0.6; BD vs DC, p < 0.01; BD vs HC, p < 0.001). CONCLUSION: These results suggest that cellular immunity against the 65 kDa mycobacterial and 60 kDa human HSP derived peptides is significantly increased in Turkish patients with BD compared to controls, as observed in the UK and Japan.

Cytokine analysis at the single cell level and lymphoproliferative responses to mycobacterial antigens in HIV-1 patients with successful virologic response to potent antiretrovirals.

Immunologic parameters, known to be grossly abnormal in HIV-1-infected subjects, were analyzed in 22 patients with sustained viral load suppression (<200 copies/ml) following long-term highly active antiretroviral therapy (HAART). Responses were compared with those from 18 HIV-seronegative healthy controls. Persistent phenotypic alterations in patients' blood mononuclear cells were minimal, though the percentages of lymphocytes that could be activated to produce interleukin-2 (IL-2) remained severely depressed. Using lymphoproliferative assays, a striking deficit in the capacity of patients to respond to the common mycobacterial antigens and particularly to recombinant heat-shock proteins paralleled the absence of responses to virus p24 antigen. In view of the important immunoregulatory role of stress proteins, these findings reveal profound functional deficiencies and persistent immune dysregulation in HIV-1 patients, despite successful HAART and a considerable recovery of CD4+ lymphocyte numbers. Rational immunotherapeutic approaches should be aimed to correct the characterized immune abnormalities.

Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: off-pathway aggregation propensity does not determine the co-chaperonin requirement.

One of the proposed roles of the GroEL-GroES cavity is to provide an "infinite dilution" folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these "nonpermissive" folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these "low Mg2+" conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

The recombinant thermosome from the hyperthermophilic archaeon Methanopyrus kandleri: in vitro analysis of its chaperone activity.

The archaeon Methanopyrus kandleri is the most thermophilic methanogen presently known. It contains a chaperonin (thermosome) which represents a 951 kDa homo-hexadecameric protein complex with NH4+-dependent ATPase activity. Since its synthesis is not increased upon heat shock, we set out to test its chaperone function. In order to obtain the chaperonin in amounts sufficient for functional investigations, the gene encoding the 60 kDa subunit was expressed in E. coili BL21 (DE3) cells. Purification yielded soluble, high-molecular-mass double-ring complexes, indistinguishable from the natural thermosome. In order to study the functional properties of the recombinant protein complex, pig citrate synthase, yeast alcohol dehydrogenase, yeast alpha-glucosidase, bovine insulin, and Thermotoga phosphoglycerate kinase were used as model substrates. The results demonstrate that the recombinant M. kandleri thermosome possesses a chaperone-like activity in vitro, inhibiting aggregation as the major off-pathway-reaction during thermal unfolding and refolding of proteins after chemical denaturation. However, the chaperonin only forms dead-end complexes with its non-native substrates, no release is detectable at temperatures between 25 and 60 degrees C.

Abnormal proteins enhance stress-induced cell death.

The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides. Escherichia coli methionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability. The polypeptide chain was split by linker insertion and expressed as two fragments. Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity. The in vitro activities of these split mutants were enhanced by the presence of chaperonin, GroESL. However, cells containing these split polypeptides became sensitive to conditions that induce GroESL. The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.