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The existing literature points towards the presence of robust mitochondrial mechanisms aimed at mitigating protein dyshomeostasis within the organelle. However, the precise molecular composition of these mechanisms remains unclear. Our data show that inorganic polyphosphate (polyP), a polymer well-conserved throughout evolution, is a component of these mechanisms. In mammals, mitochondria exhibit a significant abundance of polyP, and both our research and that of others have already highlighted its potent regulatory effect on bioenergetics. Given the intimate connection between energy metabolism and protein homeostasis, the involvement of polyP in proteostasis has also been demonstrated in several organisms. For example, polyP is a bacterial primordial chaperone, and its role in amyloidogenesis has already been established. Here, using mammalian models, our study reveals that the depletion of mitochondrial polyP leads to increased protein aggregation within the organelle, following stress exposure. Furthermore, mitochondrial polyP is able to bind to proteins, and these proteins differ under control and stress conditions. The depletion of mitochondrial polyP significantly affects the proteome under both control and stress conditions, while also exerting regulatory control over gene expression. Our findings suggest that mitochondrial polyP is a previously unrecognized, and potent component of mitochondrial proteostasis.
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OBJECTIVES: Prior studies noted that chondrocyte SIRT6 activity is repressed in older chondrocytes rendering cells susceptible to catabolic signalling events implicated in osteoarthritis (OA). This study aimed to define the effect of Sirt6 deficiency on the development of post-traumatic and age-associated OA in mice. METHODS: Male cartilage-specific Sirt6-deficient mice and Sirt6 intact controls underwent destabilisation of the medial meniscus (DMM) or sham surgery at 16 weeks of age and OA severity was analysed at 6 and 10 weeks postsurgery. Age-associated OA was assessed in mice aged 12 and 18 months of age. OA severity was analysed by micro-CT, histomorphometry and scoring of articular cartilage structure, toluidine blue staining and osteophyte formation. SIRT6-regulated pathways were analysed in human chondrocytes by RNA-sequencing, qRT-PCR and immunoblotting. RESULTS: Sirt6-deficient mice displayed enhanced DMM-induced OA severity and accelerated age-associated OA when compared with controls, characterised by increased cartilage damage, osteophyte formation and subchondral bone sclerosis. In chondrocytes, RNA-sequencing revealed that SIRT6 depletion significantly repressed cartilage extracellular matrix (eg, COL2A1) and anabolic growth factor (eg, insulin-like growth factor-1 (IGF-1)) gene expression. Gain-of-function and loss-of-function studies in chondrocytes demonstrated that SIRT6 depletion attenuated, whereas adenoviral overexpression or MDL-800-induced SIRT6 activation promoted IGF-1 signalling by increasing Aktser473 phosphorylation. CONCLUSIONS: SIRT6 deficiency increases post-traumatic and age-associated OA severity in vivo. SIRT6 profoundly regulated the pro-anabolic and pro-survival IGF-1/Akt signalling pathway and suggests that preserving the SIRT6/IGF-1/Akt axis may be necessary to protect cartilage from injury-associated or age-associated OA. Targeted therapies aimed at increasing SIRT6 function could represent a novel strategy to slow or stop OA.
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Cartilagem Articular , Osteoartrite , Osteófito , Sirtuínas , Masculino , Animais , Camundongos , Humanos , Idoso , Fator de Crescimento Insulin-Like I/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Condrócitos/metabolismo , Cartilagem Articular/metabolismo , RNA/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Modelos Animais de DoençasRESUMO
Background: Dysferlinopathies represent a group of limb girdle or distal muscular dystrophies with an autosomal-recessive inheritance pattern resulting from the presence of pathogenic variants in the dysferlin gene (DYSF). Objective: In this work, we describe a population from a small city in Brazil carrying the c.5979dupA pathogenic variant of DYSF responsible for limb girdle muscular dystrophy type 2R and distal muscular dystrophy. Methods: Genotyping analyses were performed by qPCR using customized probe complementary to the region with the duplication under analysis in the DYSF. Results: A total of 104 individuals were examined. c.5979dupA was identified in 48 (46.15%) individuals. Twenty-three (22%) were homozygotes, among whom 13 (56.5%) were female. A total of 91.3% (21) of homozygous individuals had a positive family history, and seven (30.4%) reported consanguineous marriages. Twenty-five (24%) individuals were heterozygous (25.8±16 years) for the same variant, among whom 15 (60%) were female. The mean CK level was 697 IU for homozygotes, 140.5 IU for heterozygotes and 176 IU for wild-type homo-zygotes. The weakness distribution pattern showed 17.3% of individuals with a proximal pattern, 13% with a distal pattern and 69.6% with a mixed pattern. Fatigue was present in 15 homozygotes and one heterozygote. Conclusion: The high prevalence of this variant in individuals from this small community can be explained by a possible founder effect associated with historical, geographical and cultural aspects.
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Introduction: Inorganic polyphosphate (polyP) is an ancient polymer which is extremely well-conserved throughout evolution, and found in every studied organism. PolyP is composed of orthophosphates linked together by high-energy bonds, similar to those found in ATP. The metabolism and the functions of polyP in prokaryotes and simple eukaryotes are well understood. However, little is known about its physiological roles in mammalian cells, mostly due to its unknown metabolism and lack of systematic methods and effective models for the study of polyP in these organisms. Methods: Here, we present a comprehensive set of genetically modified cellular models to study mammalian polyP. Specifically, we focus our studies on mitochondrial polyP, as previous studies have shown the potent regulatory role of mammalian polyP in the organelle, including bioenergetics, via mechanisms that are not yet fully understood. Results: Using SH-SY5Y cells, our results show that the enzymatic depletion of mitochondrial polyP affects the expression of genes involved in the maintenance of mitochondrial physiology, as well as the structure of the organelle. Furthermore, this depletion has deleterious effects on mitochondrial respiration, an effect that is dependent on the length of polyP. Our results also show that the depletion of mammalian polyP in other subcellular locations induces significant changes in gene expression and bioenergetics; as well as that SH-SY5Y cells are not viable when the amount and/or the length of polyP are increased in mitochondria. Discussion: Our findings expand on the crucial role of polyP in mammalian mitochondrial physiology and place our cell lines as a valid model to increase our knowledge of both mammalian polyP and mitochondrial physiology.
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In tumor cells, higher expression of glucose transporter proteins (GLUT) and carbonic anhydrases (CAIX) genes is influenced by hypoxia-induced factors (HIF).Thus, we aimed to study the expression profile of these markers in sequential peripheral blood collections performed in breast cancer patients in order to verify their predictive potential in liquid biopsies. Gene expressions were analyzed by qPCR in tumor and blood samples from 125 patients and 25 healthy women. Differential expression was determined by the 2(-ΔCq) method. Expression of HIF-1α and GLUT1 in the blood of breast cancer patients is significantly higher (90-91 and 160-161 fold increased expression, respectively; p < 0.0001) than that found in healthy women. Their diagnostic power was confirmed by ROC curve. CAIX is also more expressed in breast cancer women blood, but its expression was detected only in a few samples. But none of these genes could be considered predictive markers. Therefore, evaluation of the expression of HIF-1α and GLUT1 in blood may be a useful laboratory tool to complement the diagnosis of breast cancer, in addition to being useful for follow-up of patients and of women with a family history of breast cancer.
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Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Anidrase Carbônica IX/genética , Hipóxia Celular/genética , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Biópsia Líquida/métodos , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: McArdle disease presents clinical and genetic heterogeneity. There is no obvious association between genotype and phenotype. PYGM (muscle glycogen phosphorylase gene) mRNA expression and its association with clinical, morphological, and genetic aspects of the disease as a set have not been studied previously. METHODS: We investigated genetic variation in PYGM considering the number of PTCs (premature termination codon) per sample and compared mRNA expression in skeletal muscle samples from 15 patients with McArdle disease and 16 controls to PTCs number and different aspects of the disease. RESULTS: The main variant found was c.148C>T (PTC-premature termination codon). Patients with two PTCs showed 42% mRNA expression compared to the control group. Most cases showed an inversely proportional relation among PTCs and mRNA expression. Association between mRNA expression and other aspects of the disease showed no statistically significant difference (p> 0.05). DISCUSSION: mRNA expression is not useful as a predictor factor for the prognosis and severity of the disease. Different mechanisms as post-transcriptional events, epigenetics factors or protein function may be involved.
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Demografia , Regulação Enzimológica da Expressão Gênica , Glicogênio Fosforilase Muscular/genética , Doença de Depósito de Glicogênio Tipo V/genética , Adulto , Códon sem Sentido/genética , Estudos Transversais , Feminino , Doença de Depósito de Glicogênio Tipo V/epidemiologia , Doença de Depósito de Glicogênio Tipo V/patologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Adulto JovemRESUMO
BACKGROUND: Patients with breast cancer-the deadliest cancer among women-are at constant risk of developing metastasis. Oxidative stress and hypoxia are common feature of tumor cells that can proliferate even in a resultant metabolic acidosis. Despite the low extracellular pH, intracellular pH of tumor cells remains relatively normal, or even more alkaline due to the action of a membrane protein family known as monocarboxylate transporters (MCTs). The objective of this study was to verify the diagnostic and prognostic value of MCT1, MCT4 and CD147 in tumor and peripheral blood samples of patients with breast cancer undergoing chemotherapic treatment. METHODS: Differential expression of MCT1, MCT4 and CD147 obtained by qPCR was determined by 2-ΔΔCq method between biological samples (tumor and serial samples of peripheral) of patients (n = 125) and healthy women (n = 25). RESULTS: tumor samples with higher histological grades have shown higher expression of these markers; this higher expression was also observed in blood samples obtained at diagnosis of patients when compared to healthy women and in patients with positive progression of the disease (metastasis development). CONCLUSION: markers studied here could be a promising strategy in routine laboratory evaluations as breast cancer diagnosis and prognosis.