RESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Chaperonina 60/farmacología , Chaperoninas/farmacología , Infiltración Neutrófila/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Neumonía/prevención & control , Animales , Anexina A1/genética , Líquido del Lavado Bronquioalveolar/química , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Citocinas/análisis , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrinas/biosíntesis , Interleucina-1beta/biosíntesis , Lipopolisacáridos/toxicidad , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , Neutrófilos/inmunología , Receptor Toll-Like 4/genéticaRESUMEN
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.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Proteínas de Ciclo Celular/farmacología , Chaperoninas/farmacología , Retículo Endoplásmico/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Pulmonares/fisiopatología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Homeostasis , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Polaridad Celular/efectos de los fármacos , Chaperoninas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptores CCR/metabolismo , Animales , Proteínas Bacterianas/farmacología , Polaridad Celular/fisiología , Células Cultivadas , Chaperoninas/farmacología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
Asunto(s)
Microtúbulos/fisiología , Chaperonas Moleculares/farmacología , Tubulina (Proteína)/química , Proteínas Bacterianas/farmacología , Calorimetría , Caseínas/farmacología , Chaperoninas/farmacología , Proteínas HSP70 de Choque Térmico/farmacología , Microtúbulos/ultraestructura , Polímeros/metabolismo , Termodinámica , Tubulina (Proteína)/efectos de los fármacos , alfa-Cristalinas/farmacologíaRESUMEN
Glyoxysomal (gMDH) and mitochondrial malate dehydrogenase (mMDH) from watermelon are synthesized as higher molecular weight precursor proteins. By overexpressing the precursor forms as well as the mature subunits with a histidine arm at the carboxy-terminus, it has been possible to purify relatively large amounts especially of the glyoxysomal precursor protein for studies of their refolding capacities after denaturation with guanidinium hydrochloride, heat or low pH. Glyoxysomal MDH and its precursor is capable of its spontaneous folding over a wide range of temperature conditions. Refolding can be enhanced by inclusion of BSA and ATP as stabilisers in the folding buffer. The N-terminal transit peptide of gMDH facilitates folding, but does not function as an intramolecular chaperon. Chemically denatured mitochondrial MDH requires chaperones for refolding. GroEL/GroES/ATP increase the yield and rate of watermelon mMDH folding dramatically while GroEL and Mg-ATP alone are not sufficient to provide folding assistance similar to the results with hydrophobic mammalian mMDH. The watermelon glyoxysomal MDH interacts with GroEL-like hydrophilic mammalian cytoplasmic MDH, a binding which has to be released by Mg-ATP before spontaneous folding can ensue. Interestingly, watermelon mMDH exhibited a much higher heat stability than gMDH or mammalian mMDH in the presence of BSA/ATP as well as GroEL/GroES/ATP. The differences between glyoxysomal and chaperone-assisted mitochondrial folding patterns are discussed.
Asunto(s)
Chaperoninas/farmacología , Malato Deshidrogenasa/química , Mitocondrias/enzimología , Plantas/enzimología , Pliegue de Proteína , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chaperonina 10/farmacología , Chaperonina 60/farmacología , Malato Deshidrogenasa/aislamiento & purificación , Datos de Secuencia Molecular , PorcinosRESUMEN
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.
Asunto(s)
Acil-CoA Deshidrogenasa/química , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/deficiencia , Acil-CoA Deshidrogenasa/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Chaperoninas/farmacología , ADN/genética , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Fenotipo , Mutación Puntual , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EspectrofotometríaRESUMEN
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.
Asunto(s)
Chaperoninas/farmacología , Glutamato-Amoníaco Ligasa/química , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Chaperonina 10/farmacología , Chaperonina 10/fisiología , Chaperonina 60/farmacología , Chaperonina 60/fisiología , Chaperoninas/fisiología , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dimerización , Escherichia coli/química , Glutamato-Amoníaco Ligasa/efectos de los fármacos , Glutamatos/farmacología , Cinética , Magnesio/farmacología , Metilaminas/farmacología , Pliegue de ProteínaRESUMEN
It has previously been demonstrated that the unnatural amino acid p-Cl-phenylalanine can be attached to tRNA(Phe) by a modified phenylalanyl-tRNA synthetase with relaxed amino acid substrate specificity. We show that this modification to the translational machinery of Escherichia coli is the only requirement for the incorporation of either p-Cl- or p-Br-phenylalanine into full-length luciferase in vitro. The incorporation of p-Cl-phenylalanine was also demonstrated in vivo using a suitably modified host strain. These results represent the first description of the incorporation into a protein in vivo of an unnatural amino acid which is normally rejected by the cellular translational machinery.
Asunto(s)
Fenilalanina-ARNt Ligasa/metabolismo , Fenilalanina/análogos & derivados , Chaperoninas/farmacología , Escherichia coli/genética , Fenclonina/metabolismo , Técnicas de Inmunoadsorción , Luciferasas/biosíntesis , Luciferasas/química , Fenilalanina/metabolismo , Fenilalanina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/genética , Biosíntesis de Proteínas , Salmonella typhimurium , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Arteria Carótida Común/inmunología , Arteria Carótida Común/patología , Chaperoninas/inmunología , Inmunidad/inmunología , Túnica Íntima/inmunología , Túnica Íntima/patología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/farmacología , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/microbiología , Arteria Carótida Común/efectos de los fármacos , División Celular/inmunología , Chaperonina 60 , Chaperoninas/farmacología , Colesterol/metabolismo , Modelos Animales de Enfermedad , Adyuvante de Freund/administración & dosificación , Adyuvante de Freund/inmunología , Humanos , Inmunidad/efectos de los fármacos , Inmunización , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inyecciones Intraarteriales , Lípidos/administración & dosificación , Lípidos/inmunología , Modelos Cardiovasculares , Mycobacterium tuberculosis/inmunología , Ratas , Ratas Wistar , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/inmunología , Túnica Íntima/efectos de los fármacosRESUMEN
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.
Asunto(s)
Chaperoninas/farmacología , Glutamato-Amoníaco Ligasa/química , Pliegue de Proteína , Chaperonina 10/farmacología , Chaperonina 60/farmacología , Glicerol/farmacología , MutaciónAsunto(s)
Proteínas Bacterianas , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Tuberculosis Pulmonar/inmunología , Adulto , Chaperonina 60 , Chaperoninas/farmacología , Femenino , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Tuberculina/farmacología , Prueba de Tuberculina , Tuberculosis Pulmonar/prevención & controlRESUMEN
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.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Proteínas Bacterianas/farmacología , Chaperoninas/farmacología , Tolerancia Inmunológica/inmunología , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Animales , Autoanticuerpos/inmunología , Proteínas Bacterianas/inmunología , Antígenos CD4/metabolismo , Chaperonina 10/inmunología , Chaperonina 10/farmacología , Chaperonina 60 , Chaperoninas/inmunología , Reacciones Cruzadas/inmunología , Regulación hacia Abajo/inmunología , Factores de Transcripción Forkhead/metabolismo , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/farmacología , Tolerancia Inmunológica/efectos de los fármacos , Inyecciones Intraperitoneales , Interleucina-2/metabolismo , Masculino , Ratas , Ratas Endogámicas Lew , Solubilidad , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/uso terapéutico , Chaperoninas/uso terapéutico , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Proteínas de Choque Térmico/uso terapéutico , Histidina Descarboxilasa/metabolismo , Fragmentos de Péptidos/uso terapéutico , Vacunación , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Antiidiotipos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/farmacología , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Chaperonina 60 , Chaperoninas/inmunología , Chaperoninas/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Proteínas de Choque Térmico/inmunología , Proteínas de Choque Térmico/farmacología , Histamina/metabolismo , Histidina Descarboxilasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , EstreptozocinaRESUMEN
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.
Asunto(s)
Arsenicales/farmacología , Regulación hacia Abajo/efectos de los fármacos , Glutaminasa/metabolismo , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Arsenicales/metabolismo , Arsenitos/farmacología , Ácido Cacodílico/análogos & derivados , Ácido Cacodílico/farmacología , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Chaperonina con TCP-1 , Chaperoninas/farmacología , Sustancias para la Guerra Química/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Immunoblotting , Isoenzimas/metabolismo , Retinal-Deshidrogenasa , Proteínas Ribosómicas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de TiempoRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Chaperoninas/genética , Chaperoninas/farmacología , Repeticiones de Minisatélite/genética , Mucina-1/genética , Mucina-1/farmacología , Mycobacterium bovis , Animales , Antineoplásicos/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chaperonina 60 , Chaperoninas/biosíntesis , Chaperoninas/aislamiento & purificación , Escherichia coli/genética , Humanos , Ratones , Mucina-1/biosíntesis , Mucina-1/aislamiento & purificación , Células Procariotas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The 65-kDa heat shock protein (Hsp65) is supposed to play a role in host defense against infections with various microbial pathogens and in autoimmune inflammatory disorders. These effects are thought to result mainly from an Hsp65-specific T-lymphocyte-mediated immune response that recognizes conserved epitopes. The aim of the present study was to assess whether mycobacterial Hsp65 has a direct effect on resident murine peritoneal macrophages, independent of Hsp65-sensitized T lymphocytes. Exposure of peritoneal macrophages from naive C57BL/6 mice to the mycobacterial Hsp65 in vitro induced an enhanced release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6. These cells also produced large amounts of reactive nitrogen intermediates (RNI) and inhibited the intracellular proliferation of Toxoplasma gondii. Small amounts of gamma interferon acted synergistically with Hsp65. Thus, exposure of murine macrophages to Hsp65 results in activation of these cells. The acquisition of these characteristics by peritoneal macrophages occurred in the absence of sensitized T lymphocytes. Addition of anti-TNF-alpha antiserum resulted in an attenuation of the Hsp65-induced release of RNI and toxoplasmastatic activity, indicating that endogenous TNF-alpha is involved in the Hsp65-induced macrophage activation. The conclusion of this study is that in vitro exposure of peritoneal macrophages to the mycobacterial Hsp65 induces the release of proinflammatory cytokines and RNI and results in inhibition of the intracellular proliferation of T. gondii. These effects on murine macrophages occur independently of Hsp65-specific T lymphocytes. The proinflammatory effect of Hsp65 demonstrated in this study suggests that this heat shock protein may play a role in the initiation of inflammation that adds to a non-species-specific resistance in the early stages of infections.
Asunto(s)
Proteínas Bacterianas/farmacología , Chaperoninas/farmacología , Interleucina-6/biosíntesis , Activación de Macrófagos/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Toxoplasma/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Células CHO , Chaperonina 60 , Cricetinae , Femenino , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos C57BL , Nitritos/metabolismoRESUMEN
Carbamylation of lens proteins may contribute to cataract formation in populations with high levels of blood urea. Urea comes to equilibrium with cyanate. Changes induced by cyanate binding to lens crystallin have been described but little is known about the carbamylation of the enzymes. The present study investigated the in vitro carbamylation of 6-phospho-D-gluconate dehydrogenase (E.C.1.1.1.44) and its effect on the enzymic activity, as well as a possible way to prevent the cyanate binding to the enzyme. The covalent cyanate binding to protein inactivated the enzyme in a concentration-dependent fashion. Aspirin and paracetamol did not protect the enzyme against inactivation by carbamylation, while alpha-crystallin was specifically protective as compared with other control proteins, consistent with its suggested role as a molecular chaperone.
Asunto(s)
Chaperoninas/farmacología , Cristalinas/farmacología , Cianatos/metabolismo , Fosfogluconato Deshidrogenasa/sangre , Acetaminofén/farmacología , Animales , Aspirina/farmacología , Radioisótopos de Carbono , Bovinos , Electroforesis en Gel de Poliacrilamida , Eritrocitos/enzimología , Glicosilación , Humanos , Cinética , Muramidasa/farmacología , Fosfogluconato Deshidrogenasa/antagonistas & inhibidores , Fosfogluconato Deshidrogenasa/aislamiento & purificación , Unión Proteica , Albúmina Sérica Bovina/farmacologíaRESUMEN
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.
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Proteínas Bacterianas , Chaperoninas/inmunología , Epítopos Inmunodominantes/inmunología , Síndrome Mucocutáneo Linfonodular/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Líquido Cefalorraquídeo/citología , Chaperonina 60 , Chaperoninas/genética , Chaperoninas/farmacología , Humanos , Interleucina-2/biosíntesis , Interleucina-2/metabolismo , Activación de Linfocitos , Datos de Secuencia Molecular , Síndrome Mucocutáneo Linfonodular/etiología , Mycobacterium/genética , Mycobacterium/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Linfocitos T/metabolismo , Tuberculina/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Síndrome de Behçet/sangre , Chaperonina 60/farmacología , Chaperoninas/farmacología , Fragmentos de Péptidos/farmacología , Linfocitos T/efectos de los fármacos , Adulto , Secuencia de Aminoácidos/genética , Proteínas Bacterianas/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Chaperonina 60/genética , Chaperoninas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mycobacterium , Valores de Referencia , Linfocitos T/patologíaRESUMEN
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.