RESUMEN
A class of chaperones dubbed heat shock protein 70 (Hsp70) possesses high relevance in cancer diseases due to its cooperative activity with the well-established anticancer target Hsp90. However, Hsp70 is closely connected with a smaller heat shock protein, Hsp40, forming a formidable Hsp70-Hsp40 axis in various cancers, which serves as a suitable target for anticancer drug design. This review summarizes the current state and the recent developments in the field of (semi-)synthetic small molecule inhibitors directed against Hsp70 and Hsp40. The medicinal chemistry and anticancer potential of pertinent inhibitors are discussed. Since Hsp90 inhibitors have entered clinical trials but have exhibited severe adverse effects and drug resistance formation, potent Hsp70 and Hsp40 inhibitors may play a significant role in overcoming the drawbacks of Hsp90 inhibitors and other approved anticancer drugs.
Asunto(s)
Antineoplásicos , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico , Chaperonas Moleculares , Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Humanos , Diseño de FármacosRESUMEN
Whitefly, Bemisia tabaci (Gennadius) is an important pest of cotton causing direct damage as sap feeder and vector of Cotton leaf curl virus (CLCuV). Previous few studies suggest that female whiteflies are more efficient vector of begomovirusthan males, however the sex-biased transmission efficiency is still not clearly understood. Present studies with B. tabaci AsiaII-1 haplotype showed higher virus transmission efficiency of females compared to males. This variable begomovirus transmission efficiency has been related to previously identifiedkey factors associated with B. tabaci. The higher density of endosymbiont Arsenophonus and variable expression of some midgut proteins genes i.e. Cyclophilin, Knottin, Hsp40, Hsp70 may be possibly imparting higher vector competency to the females compared to males. The present studies suggest low abundance of Arsenophonus spp. as well as lower expressionof Cyclophilin genein males as compared to females. This is further supplemented by overexpression of Knottin, Hsp40, and Hsp70 genes in males compared to females and thus collectively all these factors might be playing a key role in low virus transmission efficiency of males. The relative density of Arsenophonus spp. and expression of midgut proteins genes in male and female whitefly first time enriches our understanding about sex-biased transmission efficiency of begomovirus.
Asunto(s)
Begomovirus/fisiología , Sistema Digestivo/metabolismo , Gammaproteobacteria/crecimiento & desarrollo , Hemípteros/virología , Proteínas de Insectos/metabolismo , Animales , Begomovirus/crecimiento & desarrollo , Ciclofilinas/antagonistas & inhibidores , Ciclofilinas/genética , Ciclofilinas/metabolismo , Femenino , Gammaproteobacteria/aislamiento & purificación , Gammaproteobacteria/fisiología , Regulación de la Expresión Génica , Silenciador del Gen , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Haplotipos , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Insectos Vectores/virología , Masculino , Enfermedades de las Plantas/virología , ARN Bicatenario/metabolismo , Factores Sexuales , Simbiosis , Carga ViralRESUMEN
Heat shock protein 70 (Hsp70) is an important molecular chaperone that regulates oncoprotein stability and tumorigenesis. However, attempts to develop anti-chaperone drugs targeting molecules such as Hsp70 have been hampered by toxicity issues. Hsp70 is regulated by a suite of co-chaperone molecules that bring "clients" to the primary chaperone for efficient folding. Rather than targeting Hsp70 itself, here we have examined the feasibility of inhibiting the Hsp70 co-chaperone DNAJA1 as a novel anticancer strategy. We found DNAJA1 to be upregulated in a variety of cancers, suggesting a role in malignancy. To confirm this role, we screened the NIH Approved Oncology collection for chemical-genetic interactions with loss of DNAJA1 in cancer. 41 compounds showed strong synergy with DNAJA1 loss, whereas 18 dramatically lost potency. Several hits were validated using a DNAJA1 inhibitor (116-9e) in castration-resistant prostate cancer cell (CRPC) and spheroid models. Taken together, these results confirm that DNAJA1 is a hub for anticancer drug resistance and that DNAJA1 inhibition is a potent strategy to sensitize cancer cells to current and future therapeutics. The large change in drug efficacy linked to DNAJA1 suggests a personalized medicine approach where tumor DNAJA1 status may be used to optimize therapeutic strategy.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico , Chaperonas Moleculares , Neoplasias/genética , Neoplasias/patología , Carcinogénesis/genética , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas Oncogénicas/metabolismo , Medicina de Precisión , Células Tumorales CultivadasRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate ß-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.
Asunto(s)
Ácidos Grasos/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Hígado/patología , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Adulto , Anciano , Animales , Conjuntos de Datos como Asunto , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Nanopartículas/administración & dosificación , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción/efectos de los fármacos , Cultivo Primario de Células , ARN Interferente Pequeño/administración & dosificación , RNA-SeqRESUMEN
Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.
Asunto(s)
Mutación con Ganancia de Función , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Fuerza Muscular/genética , Distrofia Muscular de Cinturas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Humanos , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Distrofia Muscular de Cinturas/tratamiento farmacológico , Distrofia Muscular de Cinturas/genética , Distrofia Muscular de Cinturas/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Saccharomyces cerevisiaeRESUMEN
The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.
Asunto(s)
Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Proteínas del Choque Térmico HSP40 , Proteínas de Fusión Oncogénica , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/antagonistas & inhibidores , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genéticaRESUMEN
DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-ß) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-ß signaling pathway. Poly(Iâ C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-ß signaling pathway.IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-ß signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-ß signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-ß signaling pathway.
Asunto(s)
Proteínas de la Cápside/metabolismo , Virus de la Fiebre Aftosa/efectos de los fármacos , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/metabolismo , Interferón beta/metabolismo , Lisosomas/metabolismo , Transducción de Señal/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Antivirales/metabolismo , Antivirales/farmacología , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Células HEK293 , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Interacciones Huésped-Patógeno , Humanos , Factor 3 Regulador del Interferón , Fosforilación , Complejo de la Endopetidasa Proteasomal , Dominios y Motivos de Interacción de Proteínas , Proteínas Virales/metabolismoRESUMEN
Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a devastating progressive disease caused by mutations in the iduronate 2-sulfatase (IDS) gene. IDS is one of the sulfatase enzymes required for lysosomal degradation of glycosaminoglycans. Mutant proteins linked to diseases are often prone to misfolding. These misfolded proteins accumulate in the endoplasmic reticulum (ER) and are degraded by the ubiquitin-proteasome pathway (ER-associated degradation (ERAD)). The decreased enzyme activities of IDS mutants may be due to accelerated degradation by ERAD. However, intracellular dynamics including degradation of IDS mutants is unexplored. In this report, we examined biochemical and biological characteristics of wild-type (WT) IDS and IDS mutants expressed in HeLa cells. IDS was shown to be glycosylated in the ER and Golgi apparatus and proteolytically cleaved to generate the mature forms in the Golgi apparatus. The mature WT IDS was translocated to the lysosome. In contrast, all IDS mutants we examined were found to accumulate in the ER and could not efficiently translocate to the lysosome. Accumulated IDS mutants in the ER were ubiquitinated by ERAD-related ubiquitin E3 ligase HRD1 followed by degradation via ERAD. Suppressed degradation of 'attenuated' mutant A85T IDS (the late-onset form of MPS II) by inhibiting ERAD components improved translocation to the lysosome and its activities. Our novel findings provide alternative targets to current principal therapies for MPS II. These perspectives provide a potenti al framework to develop fundamental therapeutic strategies and agents.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/fisiología , Retículo Endoplásmico/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/genética , Aparato de Golgi/metabolismo , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Mucopolisacaridosis II/metabolismo , Mucopolisacaridosis II/patología , Mutagénesis Sitio-Dirigida , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The tazarotene-induced gene 1 (TIG1) protein is a retinoid-inducible growth regulator and is considered a tumor suppressor. Here, we show that DnaJ heat shock protein family member C8 (DNAJC8) is a TIG1 target that regulates glycolysis. Ectopic DNAJC8 expression induced the translocation of pyruvate kinase M2 (PKM2) into the nucleus, subsequently inducing glucose transporter 1 (GLUT1) expression to promote glucose uptake. Silencing either DNAJC8 or PKM2 alleviated the upregulation of GLUT1 expression and glucose uptake induced by ectopic DNAJC8 expression. TIG1 interacted with DNAJC8 in the cytosol, and this interaction completely blocked DNAJC8-mediated PKM2 translocation and inhibited glucose uptake. Furthermore, increased glycose uptake was observed in cells in which TIG1 was silenced. In conclusion, TIG1 acts as a pivotal repressor of DNAJC8 to enhance glucose uptake by partially regulating PKM2 translocation.
Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proliferación Celular/fisiología , Citosol/metabolismo , Femenino , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/biosíntesis , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/biosíntesis , Proteínas del Choque Térmico HSP40/genética , Células HeLa , Humanos , Ácido Láctico/biosíntesis , Proteínas de la Membrana/genética , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Transfección , Neoplasias del Cuello Uterino/genética , Proteínas de Unión a Hormona TiroideRESUMEN
Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes (e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Descubrimiento de Drogas , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Preparaciones Farmacéuticas/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Unión ProteicaRESUMEN
Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Proteínas del Choque Térmico HSP40/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Adolescente , Adulto , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Modelos Animales de Enfermedad , Sobredosis de Droga/complicaciones , Sobredosis de Droga/etiología , Complejo I de Transporte de Electrón/metabolismo , Femenino , Técnicas de Inactivación de Genes , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Hepatocitos , Humanos , Hígado/citología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Cultivo Primario de Células , ARN Interferente Pequeño/metabolismo , Rotenona/farmacología , Rotenona/uso terapéutico , Desacopladores/farmacología , Desacopladores/uso terapéutico , Adulto JovenRESUMEN
DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/metabolismo , Estrés del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sustitución de Aminoácidos , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Células COS , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Chlorocebus aethiops , Resistencia a Antineoplásicos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células HEK293 , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Mutación , Proteínas de Neoplasias/antagonistas & inhibidores , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Interferencia de ARN/efectos de los fármacos , Receptores del Factor Autocrino de Motilidad/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Tiazoles/farmacologíaRESUMEN
A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins.
Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Virus de la Influenza A/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Compuestos de Bencidrilo/farmacología , Línea Celular , Núcleo Celular/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/química , Proteínas del Choque Térmico HSP40/genética , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/fisiología , Gripe Humana/metabolismo , Gripe Humana/virología , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Pirrolidinonas/farmacología , ARN Interferente Pequeño/genética , Ribonucleoproteínas/química , Proteínas Virales/químicaRESUMEN
Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110-C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.
Asunto(s)
Proteínas del Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Neuronas/metabolismo , Rodopsina/genética , Línea Celular Tumoral , Disulfuros/química , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Mutación , Neuronas/citología , Plásmidos/química , Plásmidos/metabolismo , Agregado de Proteínas , Pliegue de Proteína , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/metabolismo , Transducción de Señal , TransfecciónRESUMEN
Ischemic stroke is the second leading cause of death worldwide. One of the main risk factors of the ischemic stroke is atherosclerosis which is a chronic inflammatory and immune-mediated disease. Bacterial infections generate specific human antibodies against various antigens, including Hsps. It has been demonstrated that Hsps are selectively overexpressed in the atherosclerotic lesions. The amino acid sequence homology between human and bacterial Hsps may lead to an autoimmune response by immunological cross-reaction. Such immune response against Hsps overexpressed in the blood vessels under stressful conditions may contribute to inflammatory processes and subsequent development of atherosclerosis. In this study we determined the antibody levels against bacterial and human Hsp by ELISA in blood plasma obtained from stroke patients. Using ANOVA we analyzed levels of Hsp-antibodies in control and patient groups and correlate them with several stroke risk factors. The group of stroke patients had elevated levels of anti-Hsp antibodies compared to the control group. We also discovered an antibody level increase in patients that previously underwent another stroke. Our data provide evidence that autoimmunity could underlie formation of atherosclerosis plaque leading to stroke.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Aterosclerosis/inmunología , Autoanticuerpos/sangre , Isquemia Encefálica/inmunología , Accidente Cerebrovascular/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/biosíntesis , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/patología , Autoanticuerpos/biosíntesis , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/sangre , Proteínas Bacterianas/inmunología , Isquemia Encefálica/sangre , Isquemia Encefálica/complicaciones , Isquemia Encefálica/patología , Estudios de Casos y Controles , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/sangre , Chaperonina 60/inmunología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/sangre , Proteínas de Escherichia coli/inmunología , Femenino , Expresión Génica/inmunología , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/sangre , Proteínas del Choque Térmico HSP40/inmunología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/sangre , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas del Choque Térmico HSP72/antagonistas & inhibidores , Proteínas del Choque Térmico HSP72/sangre , Proteínas del Choque Térmico HSP72/inmunología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/sangre , Proteínas HSP90 de Choque Térmico/inmunología , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/patologíaRESUMEN
Insulin stimulates glucose transporter 4 (GLUT4) vesicle recruitment from its intracellular storage site to the plasma membrane. Cysteine string protein 1 (CSP1) is a SNARE-binding protein involved in the vesicular trafficking of neurotransmitters and other exocytic processes. In this study, we investigated the involvement of CSP1 in insulin-dependent GLUT4 recruitment in 3T3-L1 adipocytes. Over-expression of wild-type CSP1 led to attenuated insulin-stimulated glucose uptake without any change in GLUT4 content in the plasma membrane, rather it inhibits docking by blocking the association of VAMP2 with syntaxin 4. In contrast, knockdown of CSP1 enhanced insulin-stimulated glucose uptake. The mRNA and protein expression of CSP1 was elevated in 3T3-L1 adipocytes in insulin resistant states caused by high levels of palmitate and chronic insulin exposure. Taken together, the results of this study suggest that CSP1 is involved in insulin resistance by interrupting GLUT4 vesicle docking with the plasma membrane.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Resistencia a la Insulina/fisiología , Proteínas de la Membrana/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Proteínas Qa-SNARE/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos MolecularesRESUMEN
The heat shock proteins are essential players in the development of cancer and they are prime therapeutic targets. Targeting multiple hsps in dual therapies decreases the likelihood of drug resistance compared to utilizing mono-therapies. Further, employing an hsp inhibitor in combination with another therapy has proven clinically successful. Examples of efficacious strategies include the inhibition of hsp27, which prevents protein aggregation, controlling hsp40's role as an ATPase modulator, and inhibiting hsp70 from acting as a molecular chaperone. While hsp40 therapies are just in the beginning stages, hsp27 and hsp70 therapies have been successfully used in dual inhibition treatments with hsp90 inhibitors and in combinational therapy with antineoplastic drugs. Both dual and combinatorial therapies show encouraging results when used in treating chemotherapeutically resistant diseases.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Choque Térmico HSP27/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/química , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Relación Estructura-ActividadRESUMEN
It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue-derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress-induced metabolic dysfunctions.
Asunto(s)
Adipoquinas/metabolismo , Estrés del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Grasa Intraabdominal/metabolismo , Proteínas de Neoplasias/metabolismo , Obesidad/metabolismo , Serpinas/metabolismo , Adipoquinas/química , Adipoquinas/genética , Adipoquinas/aislamiento & purificación , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas del Choque Térmico HSP40/genética , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Ligandos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Obesidad/patología , Transporte de Proteínas , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serpinas/química , Serpinas/genética , Serpinas/aislamiento & purificación , Transducción de Señal , Regulación hacia ArribaRESUMEN
Data obtained for the last 12 years and modern hypotheses on key function of sleep and the role of Heat Shock Protein 70 kDa (HSP70) molecular chaperones family in sleep modulation are insufficient to determine assotiation of sleep quantity to the level of chaperones in the basic "center" of sleep in the ventrolateral preoptic area (VLPA) of the hypothalamus. In the present study, to reduce the content of Hdj1 major co-chaperone of Hsp70 in the VLPA we employed a novel approach based on lentiviral construction containing specific Hdj1-shRNA. The immunoblotting data showed that in 6 weeks after infection the level of Hdj1 in VLPA was reduced by 80% that was accompanied by a considerable increase in the quantity of slow-wave sleep and a marked decrease in the level of anxiety; earlier we found that elevation of Hsp70 level in the rat brain resulted in similar changes. It is suggested that the increase in quantity of slow wave sleep and the decrease in the level of anxiety can be related to a sustained disorder in the integration between molecular systems based on chaperones Hdj1 and Hsp70 and to a compensatory increase in the Hsp70 chaperone activity/level in VLPA.