RESUMO
This study identified tumorigenic processes most dependent on murine heat shock protein 72 (HSP72) in the mouse mammary tumor virus-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72-/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the tumor growth factor-ß-suppressor of mothers against decapentaplegic-3 signaling pathway and evidence of suppressor of mothers against decapentaplegic-3 and HSP72 coprecipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+ breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+ breast tumor gene expression data using a gene set comprising ECM-related gene and protein folding-related gene as an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between the ECM and protein-folding genes may contribute to tumor progression.
Assuntos
Matriz Extracelular , Proteínas de Choque Térmico HSP72 , Animais , Humanos , Matriz Extracelular/metabolismo , Feminino , Camundongos , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP72/genética , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Regulação Neoplásica da Expressão Gênica , Camundongos Knockout , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Transdução de Sinais , Metástase NeoplásicaRESUMO
NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), a cell cycle regulatory gene, was found to regulate cardiac hypertrophy. However, its role in diabetes-induced cardiomyopathy has not been fully elucidated. This research was designed to illustrate the effect of NEK6 involved in diabetic cardiomyopathy. Here we used a streptozotocin (STZ)-induced mice diabetic cardiomyopathy model and NEK6 knockout mice to explore the role and mechanism of NEK6 in diabetic-induced cardiomyopathy. NEK6 knockout mice and wild-type littermates were subjected to STZ injection (50 mg/kg/day for 5 days) to induce a diabetic cardiomyopathy model. As a result, 4 months after final STZ injection, DCM mice revealed cardiac hypertrophy, fibrosis, and systolic and diastolic dysfunction. NEK6 deficiency causes deteriorated cardiac hypertrophy, fibrosis, and cardiac dysfunction. Furthermore, we observed inflammation and oxidative stress in the hearts of NEK6 deficiency mice under diabetic cardiomyopathy pathology. Adenovirus was used to upregulate NEK6 in neonatal rat cardiomyocytes, and it was found that NEK6 ameliorated high glucose-induced inflammation and oxidative stress. Our findings revealed that NEK6 increased the phosphorylation of heat shock protein 72 (HSP72) and increased the protein level of PGC-1α and NRF2. Co-IP assay experiment confirmed that NEK6 interacted with HSP72. When HSP72 was silenced, the anti-inflammation and anti-oxidative stress effects of NEK6 were blurred. In summary, NEK6 may protect diabetic-induced cardiomyopathy by interacting with HSP72 and promoting the HSP72/PGC-1α/NRF2 signaling. KEY MESSAGES: NEK6 knockout deteriorated cardiac dysfunction, cardiac hypertrophy, fibrosis as well as inflammation response, and oxidative stress. NEK6 overexpression attenuated high glucose induced inflammation and oxidative stress. The underlying mechanisms of the protective role of NEK6 in the development of diabetic cardiomyopathy seem to involve the regulation of HSP72-NRF2- PGC-1α pathway. NEK6 may become a new therapeutic target for diabetic cardiomyopathy.
Assuntos
Diabetes Mellitus , Cardiomiopatias Diabéticas , Ratos , Camundongos , Animais , Cardiomiopatias Diabéticas/metabolismo , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismo , Modelos Animais de Doenças , Mitose , Glucose , Cardiomegalia/metabolismo , Fibrose , Camundongos KnockoutRESUMO
Passive hyperthermia induces a range of physiological responses including augmenting skeletal muscle mRNA expression. This experiment aimed to examine gene and protein responses to prolonged passive leg hyperthermia. Seven young participants underwent 3 h of resting unilateral leg heating (HEAT) followed by a further 3 h of rest, with the contralateral leg serving as an unheated control (CONT). Muscle biopsies were taken at baseline (0 h), and at 1.5, 3, 4, and 6 h in HEAT and 0 and 6 h in CONT to assess changes in selected mRNA expression via qRT-PCR, and HSP72 and VEGFα concentration via ELISA. Muscle temperature (Tm) increased in HEAT plateauing from 1.5 to 3 h (+3.5 ± 1.5°C from 34.2 ± 1.2°C baseline value; P < 0.001), returning to baseline at 6 h. No change occurred in CONT. Endothelial nitric oxide synthase (eNOS), Forkhead box O1 (FOXO-1), Hsp72, and VEGFα mRNA increased in HEAT (P < 0.05); however, post hoc analysis identified that only Hsp72 mRNA statistically increased (at 4 h vs. baseline). When peak change during HEAT was calculated angiopoietin 2 (ANGPT-2) decreased (-0.4 ± 0.2-fold), and C-C motif chemokine ligand 2 (CCL2) (+2.9 ± 1.6-fold), FOXO-1 (+6.2 ± 4.4-fold), Hsp27 (+2.9 ± 1.7-fold), Hsp72 (+8.5 ± 3.5-fold), Hsp90α (+4.6 ± 3.7-fold), and VEGFα (+5.9 ± 3.1-fold) increased from baseline (all P < 0.05). At 6 h Tm were not different between limbs (P = 0.582; CONT = 32.5 ± 1.6°C, HEAT = 34.3 ± 1.2°C), and only ANGPT-2 (P = 0.031; -1.3 ± 1.4-fold) and VEGFα (P = 0.030; 1.1 ± 1.2-fold) differed between HEAT and CONT. No change in VEGFα or HSP72 protein concentration were observed over time; however, peak change in VEGFα did increase (P < 0.05) in HEAT (+140 ± 184 pg·mL-1) versus CONT (+7 ± 86 pg·mL-1). Passive hyperthermia transiently augmented ANGPT-2, CCL2, eNOS, FOXO-1, Hsp27, Hsp72, Hsp90α and VEGFα mRNA, and VEGFα protein.
Assuntos
Proteínas de Choque Térmico HSP72 , Hipertermia Induzida , Músculo Esquelético , Neovascularização Fisiológica , Humanos , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Extremidade Inferior/irrigação sanguínea , Extremidade Inferior/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Impaired endothelial insulin signaling and consequent blunting of insulin-induced vasodilation is a feature of type 2 diabetes (T2D) that contributes to vascular disease and glycemic dysregulation. However, the molecular mechanisms underlying endothelial insulin resistance remain poorly known. Herein, we tested the hypothesis that endothelial insulin resistance in T2D is attributed to reduced expression of heat shock protein 72 (HSP72). HSP72 is a cytoprotective chaperone protein that can be upregulated with heating and is reported to promote insulin sensitivity in metabolically active tissues, in part via inhibition of JNK activity. Accordingly, we further hypothesized that, in individuals with T2D, 7 days of passive heat treatment via hot water immersion to waist level would improve leg blood flow responses to an oral glucose load (i.e., endogenous insulin stimulation) via induction of endothelial HSP72. In contrast, we found that: 1) endothelial insulin resistance in T2D mice and humans was not associated with reduced HSP72 in aortas and venous endothelial cells, respectively; 2) after passive heat treatment, improved leg blood flow responses to an oral glucose load did not parallel with increased endothelial HSP72; and 3) downregulation of HSP72 (via small-interfering RNA) or upregulation of HSP72 (via heating) in cultured endothelial cells did not impair or enhance insulin signaling, respectively, nor was JNK activity altered. Collectively, these findings do not support the hypothesis that reduced HSP72 is a key driver of endothelial insulin resistance in T2D but provide novel evidence that lower-body heating may be an effective strategy for improving leg blood flow responses to glucose ingestion-induced hyperinsulinemia.
Assuntos
Diabetes Mellitus Tipo 2 , Proteínas de Choque Térmico HSP72 , Resistência à Insulina , Animais , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliais/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Insulina/metabolismo , CamundongosRESUMO
Histone deacetylase inhibitors (HDACis) are one of the therapeutic options for cutaneous T-cell lymphoma (CTCL), but they have limited effects. We previously demonstrated that HSP72 overexpression is associated with chemoresistance to HDACis in lymphoma cells. The purpose of this study was to investigate whether the functional depletion of HSP72 enhances the effect of the HDACi vorinostat. First, we established a stable HSP72-knockdown CTCL cell line and confirmed the influence of HSP72 reduction on the antitumor effects of vorinostat. Next, we studied the effect of quercetin, an inhibitor of HSP72, on the antineoplastic effects of vorinostat. In five CTCL cell lines examined, HSP72 expression was highest in Hut78 cells, and HSP72 knockdown enhanced vorinostat-induced apoptosis in these cells. Low-dose quercetin reduced HSP72 expression, increased HDAC activity, and enhanced vorinostat-induced suppression of Hut78 cell proliferation. A single low dose of quercetin induced G2 arrest and only slightly increased the sub-G1 cell fraction. Quercetin also significantly enhanced vorinostat-induced apoptosis, caspase-3, caspase-8, and caspase-9 activity, and the loss of mitochondrial membrane potential. HSP72 knockdown enhanced vorinostat-induced apoptosis in an HSP72-overexpressing CTCL cell line, and thus, quercetin may be a suitable candidate for combination therapy with vorinostat in clinical settings.
Assuntos
Proteínas de Choque Térmico HSP72/genética , Linfoma Cutâneo de Células T/genética , Quercetina/farmacologia , RNA Interferente Pequeno/farmacologia , Neoplasias Cutâneas/genética , Vorinostat/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP72/antagonistas & inibidores , Histona Desacetilases/metabolismo , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológicoRESUMO
Growth arrest and DNA damage-inducible 45ß (GADD45ß) belongs to the GADD45 family which is small acidic proteins in response to cellular stress. GADD45ß has already been reported to have excellent capabilities against cancer, innate immunity and neurological diseases. However, there is little information regard GADD45ß and non-alcoholic fatty liver disease (NAFLD). In the current work, we found that the expression of GADD45ß was markedly decreased in the livers of NAFLD patients via analyzing Gene Expression Omnibus (GEO) dataset and in mouse model through detecting its mRNA in high-fat-high-fructose diet (HFHFr)-fed mice. Moreover, the results from in vivo experiment demonstrated that overexpression of GADD45ß by AAV8-mediated gene transfer in HFHFr-fed mouse model could reduce the level of serum and hepatic triglyceride (TG), and alleviate insulin resistance. Subsequently, by combining immunoprecipitation (IP) and mass spectrometry, we identified that HSP72 directly interacted with GADD45ß to prevent GADD45ß from being degraded by the proteasome pathway. Finally, the benefits of GADD45ß in regulating key factors of TG synthesis and insulin signaling pathway were abolished after HSP72 knockdown. In conclusion, GADD45ß stabilized by the interaction with HSP72 could alleviate the NAFLD-related pathologies, suggested it might be a potential target for the treatment of NAFLD.
Assuntos
Antígenos de Diferenciação/genética , Proteínas de Choque Térmico HSP72/genética , Resistência à Insulina , Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Regulação para Baixo , Células HEK293 , Proteínas de Choque Térmico HSP72/metabolismo , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Hsp70 proteins are among the most ubiquitous chaperones and play important roles in maintaining proteostasis and resisting environmental stress. Multiple copies of Hsp70s are widely present in eukaryotic cells with redundant and divergent functions, but they have been less well investigated in prokaryotes. Myxococcus xanthus DK1622 is annotated as having many hsp70 genes. In this study, we performed a bioinformatic analysis of Hsp70 proteins and investigated the functions of six hsp70 genes in DK1622, including two genes that encode proteins with the conserved PRK00290 domain (MXAN_3192 and MXAN_6671) and four genes that encode proteins with the cl35085 or cd10170 domain. We found that only MXAN_3192 is essential for cell survival and heat shock induction. MXAN_3192, compared with the other hsp70 genes, has a high transcriptional level, far exceeding that of any other hsp70 gene, which, however, is not the reason for its essentiality. Deletion of MXAN_6671 (sglK) led to multiple deficiencies in development, social motility, and oxidative resistance, while deletion of each of the other four hsp70 genes decreased sporulation and oxidative resistance. MXAN_3192 or sglK, but not the other genes, restored the growth deficiency of the E. colidnaK mutant. Our results demonstrated that the PRK00290 proteins play a central role in the complex cellular functions of M. xanthus, while the other diverse Hsp70 superfamily homologues probably evolved as helpers with some unknown specific functions.IMPORTANCE Hsp70 proteins are highly conserved chaperones that occur in all kingdoms of life. Multiple copies of Hsp70s are often present in genome-sequenced prokaryotes, especially taxa with complex life cycles, such as myxobacteria. We investigated the functions of six hsp70 genes in Myxococcus xanthus DK1622 and demonstrated that the two Hsp70 proteins with the PRK00290 domain play a central role in complex cellular functions in M. xanthus, while other Hsp70 proteins probably evolved as helpers with some unknown specific functions.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional/métodos , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Myxococcus xanthus/química , Myxococcus xanthus/genética , Proteínas de Choque Térmico HSP72/classificação , Myxococcus xanthus/metabolismo , Filogenia , Estresse Fisiológico , Transcrição GênicaRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by airway obstruction and progressive lung inflammation. As the primary ingredient of a traditional Chinese medical herb, Baicalin has been previously shown to possess anti-inflammatory abilities. Thus, the current study aimed to elucidate the mechanism by which baicalin alleviates COPD. METHODS: Baicalin was adopted to treat cigarette smoke in extract-exposed MLE-12 cells after which cell viability and apoptosis were determined. The production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8 were determined by enzyme-linked immunoassay. A COPD mouse model was constructed via exposure to cigarette smoke and lipopolysaccharide, baicalin treatment. Lung function and inflammatory cell infiltration were determined and the production of Muc5AC, TNF-α, IL-6, IL-8 in the bronchoalveolar lavage fluid (BALF) was assayed by ELISA. The effect of HSP72 and JNK on COPD following treatment with baicalin was assessed both in vivo and in vitro by conducting loss- and gain- function experiments. RESULTS: Baicalin improved lung function evidenced by reduction in inflammatory cell infiltration and Muc5AC, TNF-α, IL-6 and IL-8 levels observed in BALF in mice. Baicalin was further observed to elevate cell viability while inhibited apoptosis and TNF-α, IL-6 and IL-8 levels in MLE-12 cells. Baicalin treatment increased HSP72 expression, while its depletion reversed the effect of baicalin on COPD. HSP72 inhibited the activation of JNK, while JNK activation was found to inhibit the effect of baicalin on COPD. CONCLUSIONS: Baicalin upregulated the expression of HSP72, resulting in the inhibition of JNK signaling activation, which ultimately alleviates COPD.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Flavonoides/farmacologia , Proteínas de Choque Térmico HSP72/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Biomarcadores , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Flavonoides/uso terapêutico , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP72/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/efeitos adversos , Camundongos , Modelos Biológicos , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/etiologia , Testes de Função Respiratória , Resultado do TratamentoRESUMO
Plasmodium falciparum expresses two essential cytosol localised chaperones; PfHsp70-1 and PfHsp70-z. PfHsp70-z (Hsp110 homologue) is thought to facilitate nucleotide exchange function of PfHsp70-1. PfHsp70-1 is a refoldase, while PfHsp70-z is restricted to holdase chaperone function. The structural features delineating functional specialisation of these chaperones remain unknown. Notably, PfHsp70-z possesses a unique linker segment which could account for its distinct functions. Using recombinant forms of PfHsp70-1, PfHsp70-z and E. coli Hsp70 (DnaK) as well as their linker switch mutant forms, we explored the effects of the linker mutations by conducting several assays such as circular dichroism, intrinsic and extrinsic fluorescence coupled to biochemical and in cellular analyses. Our findings demonstrate that the linker of PfHsp70-z modulates global conformation of the chaperone, regulating several functions such as client protein binding, chaperone- and ATPase activities. In addition, as opposed to the flexible linker of PfHsp70-1, the PfHsp70-z linker is rigid, thus regulating its notable thermal stability, making it an effective stress buffer. Our findings suggest a crucial role for the linker in streamlining the functions of these two chaperones. The findings further explain how these distinct chaperones cooperate to ensure survival of P. falciparum particularly under the stressful human host environment.
Assuntos
Citosol/metabolismo , Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP110/metabolismo , Proteínas de Choque Térmico HSP72/química , Proteínas de Choque Térmico HSP72/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Adenosina Trifosfatases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72/genética , Ligação de Hidrogênio , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.
Assuntos
Sistemas de Liberação de Medicamentos , Técnicas de Transferência de Genes , Proteínas de Choque Térmico HSP72/genética , Ondas Ultrassônicas , Sobrevivência Celular/efeitos da radiação , Expressão Gênica/efeitos da radiação , Células HL-60 , Proteínas de Choque Térmico HSP72/química , Proteínas de Choque Térmico HSP72/farmacologia , Humanos , Lipídeos/química , Lipídeos/farmacologia , Microbolhas/uso terapêuticoRESUMO
Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.
Assuntos
Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Mutação , Plasmodium falciparum , Proteínas de Protozoários/genética , Dicroísmo Circular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP72/química , Proteínas de Choque Térmico/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Estabilidade Proteica , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Espectrometria de FluorescênciaRESUMO
Visceral leishmaniasis is spreading in Brazil where the main vector of its agent, Leishmania infantum Nicolle, 1908, is the Lutzomyia longipalpis (Lutz & Neiva, 1912) species complex (Diptera: Psychodidae: Phlebotominae), on which many of the activities of the visceral leishmaniasis surveillance program are based. However, there are areas where canine, and/or human cases have been occurring without the presence of this species complex as in the western part of the Greater São Paulo Metropolitan region, where Embu das Artes municipality is situated. In this area, Pintomyia fischeri (Pinto, 1926) has been implicated as potential vector of Le. infantum but so far its natural infection with this parasite has not yet been ascertained. Therefore, the present study sought to investigate the natural infection in sand flies of a CVL focus in Embu das Artes. The sand fly collections were undertaken with Shannon and CDC traps, monthly, between 1800 and 2100 hours from November 2018 to October 2019, inclusive. A total of 951 sand flies (457 males and 494 females), belonging to 10 species, were captured. Pintomyia fischeri was the predominant species (89.5%); of which 426 females were dissected and one of them (0.23%) was found to be harboring flagellates in its midgut. A sample of these flagellates was isolated in culture and characterized by a 234 base pair fragment of Leishmania heat-shock protein 70 gene (hsp70) and restriction fragment length polymorphism with Hae III restriction enzyme as Le. infantum. This finding reinforces previous evidence of Pi. fischeri as a vector of Le. infantum in foci of visceral leishmaniasis and highlights the importance of vector surveillance in areas where this species occurs.
Assuntos
Leishmania infantum/isolamento & purificação , Leishmaniose Visceral , Psychodidae/parasitologia , Animais , Brasil/epidemiologia , Vetores de Doenças/classificação , Doenças do Cão/prevenção & controle , Doenças do Cão/transmissão , Cães , Monitoramento Epidemiológico , Genes de Protozoários , Proteínas de Choque Térmico HSP72/genética , Humanos , Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania infantum/genética , Leishmaniose Visceral/prevenção & controle , Leishmaniose Visceral/transmissão , Psychodidae/classificação , Doenças Transmitidas por Vetores/prevenção & controle , Doenças Transmitidas por Vetores/transmissão , Doenças Transmitidas por Vetores/veterinária , Zoonoses/prevenção & controle , Zoonoses/transmissãoRESUMO
Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite's chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite the structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition, oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate the folding of client-substrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain the substrate in folding-competent state to minimize its misfolding. Structural investigation through small angle x-ray scattering gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggest that the parasite has evolved functionally diverged and efficient chaperone machinery which allows the human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for the exploration of small-molecule based antimalarial interventions.
Assuntos
Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP72/química , Plasmodium falciparum/química , Dobramento de Proteína , Proteínas de Protozoários/química , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Temperature elevations constitute a major threat to plant performance. In recent years, much was learned about the general molecular mode of heat stress reaction of plants. The current research focuses on the integration of the knowledge into more global networks, including the reactions of cellular compartments. For instance, chloroplast function is central for plant growth and survival, and the performance of chloroplasts is tightly linked to the general status of the cell and vice versa. We examined the changes in photosynthesis, chloroplast morphology and proteomic composition posed in Arabidopsisthaliana chloroplasts after a single or repetitive heat stress treatment over a period of two weeks. We observed that the acclimation is potent in the case of repetitive application of heat stress, while a single stress results in lasting alterations. Moreover, the physiological capacity and its adjustment are dependent on the efficiency of the protein translocation process as judged from the analysis of mutants of the two receptor units of the chloroplast translocon, TOC64, and TOC33. In response to repetitive heat stress, plants without TOC33 accumulate Hsp70 proteins and plants without TOC64 have a higher content of proteins involved in thylakoid structure determination when compared to wild-type plants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Resposta ao Choque Térmico/genética , Proteínas de Membrana/genética , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/genética , Proteínas de Choque Térmico HSP72/genética , Fotossíntese/genética , ProteômicaRESUMO
BACKGROUND: Heat stroke-induced mortality is rising across the globe. So, the design of prophylactic and/or therapeutic modalities for heat stroke is pressing need. The common plant derived flavonoid exhibits strong anti-oxidant and anti-inflammatory activities; however, its effects in heat stroke remain unknown. The study aimed to investigate the cardioprotective effects of myricetin on heat stroke induced acute myocardial injury as well as lethality in rats and to explore the underlying mechanisms. METHODS: Myocardial injury was induced by subjecting the anesthetized rats to a high ambient temperature of 43 °C for 70 min. An intragastrical dose of myricetin (5-25 mg/kg body weight) was given to rats once per day for one week prior to the start of heat stress. Heat shock protein 72 antibodies was given intraperitoneally to rats 24 h before the start of heat stress. Myocardial injury severity was estimated by determing myocardial damage scores, myocardial injury indicators, myocardial oxidative and inflammatory factors. Western blot analysis was used for cardiac expression of heat shock protein (HSP)72. RESULTS: Significant (P < 0.05) up-regulation of HSP-72 after chronic administration of myricetin coincided with significant (P < 0.05) reduction in hyperthermia, hypotension, cardiac inflammatory and oxidative damage and lethality. Inhibition of HSP-72 showed a significant (P < 0.05) reversal in the cardiaprotection as well as survival. CONCLUSIONS: Our results indicate that myricetin diminishes myocardial injury as well as lethality in heat stroke by up-regulating HSP-72 and show promise as a novel prevention therapeutic for heat stroke.
Assuntos
Flavonoides/farmacologia , Proteínas de Choque Térmico HSP72/genética , Traumatismos Cardíacos/prevenção & controle , Golpe de Calor/tratamento farmacológico , Animais , Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Flavonoides/administração & dosagem , Traumatismos Cardíacos/etiologia , Golpe de Calor/complicações , Masculino , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Trypsinogen activation is the hallmark of acute pancreatitis (AP) independent of intra-acinar NF-κB activation and inflammation. We previously found that dopamine (DA) receptor 2 (DRD2) activation controls inflammation during AP via PP2A-dependent NF-κB activation. In this study, we sought to examine whether DRD2 signaling mediates trypsinogen activation and the underlying mechanisms. Pancreatic acinar cells were stimulated with cholecystokinin-8 in vitro. AP was induced by intraperitoneal injections of caerulein and LPS or l-arginine. Pancreatitis severity was assessed biochemically and histologically. We found that activation of DRD2 by quinpirole, a potent DRD2 agonist, resulted in the reduction of trypsinogen activation and the upregulation of HSP70 in vitro and in vivo. Mechanistically, we found that quinpirole induced dephosphorylation of heat shock factor 1 (HSF1), a master transcription factor of HSP70, leading to increased nuclear translocation of HSF1 in a PP2A-dependent pathway. Furthermore, DRD2 activation restored lysosomal pH and, therefore, maintained lysosomal cathepsin B activity in a HSP70-dependent manner. VER155008, a potent HSP70 antagonist, abolished the protective effects observed with DRD2 activation in vitro and in two experimental models of AP. Our data showed that besides controlling NF-κB activation, DRD2 activation prevented trypsinogen activation during acute pancreatitis via PP2A-dependent upregulation of HSP70 and further support that DRD2 agonist could be a promising therapeutic strategy for treating AP.NEW & NOTEWORTHY The current study demonstrated that activation of DRD2 by quinpirole protects against trypsinogen activation in the in vitro and in vivo setting of acute pancreatitis by upregulating HSP70 and restoring lysosomal degradation via a PP2A-dependent manner, therefore leading to reduced pancreatic injury. These findings provide a new mechanistic insight on the protective effect of DRD2 activation in acute pancreatitis.
Assuntos
Proteínas de Choque Térmico HSP72/metabolismo , Pancreatite/tratamento farmacológico , Quimpirol/farmacologia , Receptores de Dopamina D2/agonistas , Tripsinogênio/metabolismo , Animais , Ceruletídeo/farmacologia , Agonistas de Dopamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Lisossomos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/citologia , Pancreatite/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Regulação para CimaRESUMO
Adaptive correction of structural and metabolic disturbances in the lungs caused by longterm exposure to coal-rock dust were studied in experiments on rats. It was shown that the complex antioxidant preparation containing dihydroquercetin compensated disturbances in the redox balance in the lung tissue, prevented the formation of dust granulomas, and reduced the severity of degenerative changes in the bronchopulmonary system.
Assuntos
Antioxidantes/farmacologia , Carvão Mineral/efeitos adversos , Radicais Livres/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Granuloma/prevenção & controle , Quercetina/análogos & derivados , Administração Oral , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Animais , Animais não Endogâmicos , Aspartato Aminotransferases/genética , Aspartato Aminotransferases/metabolismo , Catalase/genética , Catalase/metabolismo , Esquema de Medicação , Poeira , Radicais Livres/metabolismo , Granuloma/etiologia , Granuloma/genética , Granuloma/patologia , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Hidroxibutirato Desidrogenase/genética , Hidroxibutirato Desidrogenase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Exposição por Inalação/efeitos adversos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Oxirredução , Tamanho da Partícula , Quercetina/farmacologia , Ratos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
The proliferation of fibroblasts creates an environment favoring post-operative tendon adhesion, but targeted therapy of this pathology remains in its infancy. In this study, we explored the effect of heat shock protein 72 (HSP72), a major inducible member of the heat shock protein family that can protect cells against many cellular stresses including heat shock, on fibroblast proliferation in tendon adhesion, with its underlying mechanisms investigated. HSP72 expression was examined in an established rat model of tendon injury using RT-qPCR and immunoblot analysis. After conducting ectopic expression and depletion experiments in fibroblast NIH3T3 cells, we determined the effects of HSP72 on the expression of α-SMA and STAT3 signaling pathway-related genes, fibroblast proliferation, as well as collagen production. The mRNA (65.46%) and protein (63.65%) expression of HSP72 was downregulated in the rat model of tendon injury. The in vitro experiments revealed that overexpression of HSP72 inhibited fibroblast proliferation (42.57%) and collagen production (45.60%), as well as reducing α-SMA expression (42.49%) and the extent of STAT3 phosphorylation (55.46%). Moreover, we observed that HSP72 overexpression reduced inflammation as well as the number of inflammatory cell infiltration and fibroblasts in vivo. Furthermore, the inhibited extent of STAT3 phosphorylation contributed to the impaired fibroblast proliferation and collagen production evoked by upregulated HSP72. In summary, the present study unveils an inhibitory role of HSP72 in tendon adhesion via inactivation of the STAT3 signaling pathway. This finding may enable the development of new therapeutic strategies for the prevention against tendon adhesion.
Assuntos
Colágeno/biossíntese , Fibroblastos/patologia , Proteínas de Choque Térmico HSP72/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Tendões/patologia , Aderências Teciduais/patologia , Regulação para Cima , Animais , Adesão Celular , Proliferação de Células , Proteínas de Choque Térmico HSP72/metabolismo , Masculino , Camundongos , Células NIH 3T3 , Fosforilação , Ratos Sprague-DawleyRESUMO
HSP90 (heat shock protein 90) is an ATP-dependent molecular chaperone involved in a proper folding and maturation of hundreds of proteins. HSP90 is abundantly expressed in cancer, including melanoma. HSP90 client proteins are the key oncoproteins of several signaling pathways controlling melanoma development, progression and response to therapy. A number of natural and synthetic compounds of different chemical structures and binding sites within HSP90 have been identified as selective HSP90 inhibitors. The majority of HSP90-targeting agents affect N-terminal ATPase activity of HSP90. In contrast to N-terminal inhibitors, agents interacting with the middle and C-terminal domains of HSP90 do not induce HSP70-dependent cytoprotective response. Several inhibitors of HSP90 were tested against melanoma in pre-clinical studies and clinical trials, providing evidence that these agents can be considered either as single or complementary therapeutic strategy. This review summarizes current knowledge on the role of HSP90 protein in cancer with focus on melanoma, and provides an overview of structurally different HSP90 inhibitors that are considered as potential therapeutics for melanoma treatment.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Melanoma/metabolismo , Animais , Antineoplásicos/química , Proteínas de Choque Térmico HSP72/genética , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Domínios ProteicosRESUMO
Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.