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1.
Nucleic Acids Res ; 49(2): 1114-1132, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398331

RESUMO

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.


Assuntos
Proteínas de Arabidopsis/fisiologia , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Biogênese de Organelas , Fatores de Terminação de Peptídeos/fisiologia , RNA de Plantas/metabolismo , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
2.
Phytomedicine ; 80: 153369, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33070082

RESUMO

BACKGROUND: Impairment of mitochondrial biogenesis is associated with the pathological progression of Parkinson's disease (PD). Parkin-interacting substrate (PARIS) can be ubiquitinated by parkin and prevents the repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α). PURPOSE: This study investigated whether the neuroprotective mechanism of carnosic acid (CA) from rosemary is mediated via the regulation of PARIS and PGC-1α by parkin. METHODS: The Western blotting and RT-PCR were used to determine protein and mRNA, respectively. To investigate the protein-protein interaction of between PARIS and ubiquitin, the immunoprecipitation assay (IP assay) was utilized. Silencing of endogenous parkin or PGC-1α was performed by using transient transfection of small interfering RNA (siRNA). RESULTS: SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) increased PARIS protein, decreased PGC-1α protein, and reduced protein and mRNA of mitochondrial biogenesis-related genes. CA pretreatment reversed the effects of 6-OHDA. By IP assay, the interaction of PARIS with ubiquitin protein caused by CA was stronger than that caused by 6-OHDA. Moreover, knockdown of parkin attenuated the ability of CA to reverse the 6-OHDA-induced increase in PARIS and decrease in PGC-1α expression. PGC-1α siRNA was used to investigate how CA influenced the effect of 6-OHDA on the modulation of mitochondrial biogenesis and apoptosis. In the presence of PGC-1α siRNA, CA could no longer significantly reverse the reduction of mitochondrial biogenesis or the induction of cleavage of apoptotic-related proteins by 6-OHDA. CONCLUSION: The cytoprotective of CA is related to the enhancement of mitochondrial biogenesis by inhibiting PARIS and inducing PGC-1α by parkin. The activation of PGC-1α-mediated mitochondrial biogenesis by CA prevents the degeneration of dopaminergic neurons, CA may have therapeutic application in PD.


Assuntos
Abietanos/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Repressoras/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Biogênese de Organelas , Oxidopamina/toxicidade , Doença de Parkinson/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
Chin J Physiol ; 63(6): 286-293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33380613

RESUMO

Glucocorticoid (GC)-induced bone loss is the most prevalent form of secondary osteoporosis. Previous studies demonstrated that long-term incubation of dexamethasone (DEX) induced oxidative stress and mitochondrial dysfunctions, consequently leading to apoptosis of differentiated osteoblasts. This DEX-induced cell death might be the main causes of bone loss. We previously described that DEX induced biphasic mitochondrial alternations. As GC affects mitochondrial physiology through several different possible routes, the short-term and long-term effects of GC treatment on mitochondria in the osteoblast have not been carefully characterized. Here, we examined the expression levels of genes that are associated with mitochondrial functions at several different time points after incubation with DEX. Mitochondrial biogenesis-mediated genes nuclear respiratory factor 1 (Nrf1) and Nrf2 were upregulated after 4-h incubation, and then declined after 24-h incubation, suggesting that mitochondrial biogenesis were transiently upregulated by DEX. In contrast, mitochondrial fusion gene optic atrophy 1 (Opa1) and mitofusin 2 (Mfn2) started to be elevated as the biogenesis started to decrease. Finally, the mitochondrial fission increased and apoptosis becomes prominent. Agree with the mitochondrial biphasic alterations hypothesis, the results suggested an early increase of mitochondrial activities and biogenesis upon DEX stimulation to the osteoblasts. The oxidative phosphorylation and inducible nitric oxide synthase levels increased results in oxidative stress accumulation, leading to mitochondrial fusion, and subsequently fission and triggering the apoptosis. Our results indicated that the primary effects of GC on mitochondria are promoting their functions and biogenesis. Mitochondrial breakdown and the activation of the apoptotic pathways appeared to be the secondary effect after long-term treatment.


Assuntos
Biogênese de Organelas , Osteoblastos , Apoptose , Dexametasona/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mitocôndrias
4.
Nat Commun ; 11(1): 5816, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199730

RESUMO

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.


Assuntos
Cílios/metabolismo , Microcefalia/genética , Neurogênese , Biogênese de Organelas , Proteínas de Ligação a RNA/genética , Ribossomos/metabolismo , Adulto , Animais , Sequência de Bases , Encéfalo/embriologia , Encéfalo/patologia , Ciclo Celular , Nucléolo Celular/metabolismo , Centrossomo/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Camundongos , Mutação/genética , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Paquistão , Linhagem , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/embriologia
5.
Artigo em Inglês | MEDLINE | ID: mdl-32751208

RESUMO

This study examines how the high-fat diet (HFD) affects mitochondrial dynamics and biogenesis, and also whether combining it with low-intensity endurance exercise adds to these effects. Six 8-week-old male Sprague-Dawley (SD) rats were put on control (CON; standard chow diet), HF (HFD intake), and HFEx (HFD + low-intensity treadmill exercise) for 6 weeks. As a result, no change in body weight was observed among the groups. However, epididymal fat mass increased significantly in the two groups that had been given HFD. Blood free fatty acid (FFA) also increased significantly in the HF group. While HFD increased insulin resistance (IR), this was improved significantly in the HFEx group. HFD also significantly increased mitochondrial biogenesis-related factors (PPARδ, PGC-1α, and mtTFA) and mitochondrial electron transport chain proteins; however, no additional effect from exercise was observed. Mitochondrial dynamic-related factors were also affected: Mfn2 increased significantly in the HFEx group, while Drp1 and Fis-1 increased significantly in both the HF and HFEx groups. The number of mitochondria in the subsarcolemmal region, and their size in the subsarcolemmal and intermyofibrillar regions, also increased significantly in the HFEx group. Taken overall, these results show that HFD in combination with low-intensity endurance exercise has no additive effect on mitochondrial biogenesis, although it does have such an effect on mitochondrial dynamics by improving IR.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Resistência à Insulina , Dinâmica Mitocondrial , Condicionamento Físico Animal/fisiologia , Animais , Dieta Hiperlipídica/estatística & dados numéricos , Masculino , Músculo Esquelético , Biogênese de Organelas , Ratos , Ratos Sprague-Dawley
6.
Nat Cell Biol ; 22(8): 947-959, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32753669

RESUMO

The plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether the membrane tension of organelles regulates their functions, notably intracellular traffic. The endosomal sorting complexes required for transport (ESCRT)-III complex is the major membrane remodelling complex that drives intra-lumenal-vesicle (ILV) formation on endosomal membranes. Here we used a fluorescent membrane-tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when the membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro and correlates with increased ILV formation in ESCRT-III-decorated endosomes in vivo. Finally, we find that the endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and endosome trafficking, leading us to conclude that membrane tension can control organelle functions.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Biogênese de Organelas , Endossomos/fisiologia , Corantes Fluorescentes , Células HeLa , Humanos , Soluções Hipertônicas , Tensão Superficial
7.
Biochim Biophys Acta Rev Cancer ; 1874(1): 188392, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32735964

RESUMO

Although the eukaryotic genome is mainly comprised of linear chromosomal DNA, genes can also be found outside of chromosomes. The unconventional presence of extrachromosomal genes is usually found to be circular, and these structures are named extrachromosomal circular DNA (eccDNA), which are often observed in cancer cells. Various types of eccDNA including small polydispersed DNA (spcDNA), telomeric cirlces, microDNA, etc. have been discovered. Among these eccDNA, extrachromosomal DNA (ecDNA), which encompasses the full spectrum of large, gene-containing extrachromosomal particles, has regained great research interest due to recent technological advances such as next-generation sequencing and super-resolution microscopy. In this review, we summarize the different types of eccDNA and discuss the role of eccDNA, especially ecDNA in tumor heterogeneity and progression. Additionally, we discuss some possible future investigative directions related to ecDNA biogenesis and its clinical application.


Assuntos
DNA Circular/classificação , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Heterogeneidade Genética , Neoplasias/genética , DNA Circular/genética , DNA Circular/metabolismo , DNA de Neoplasias/química , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Amplificação de Genes , Humanos , Micronúcleos com Defeito Cromossômico , Neoplasias/patologia , Neoplasias/terapia , Biogênese de Organelas
8.
Anim Sci J ; 91(1): e13385, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32515535

RESUMO

Both oocytes and extracellular vesicles (EV) have emerged as critical regulators of mammalian follicular development; however, the possible interaction between the oocyte-derived paracrine factor (ODPF) and EV signals has never been examined. Therefore, to explore the possibility of an interaction between oocyte and EV signals, the effects of ODPFs on the biogenesis of EVs as well as the expression levels of transcripts related to EV biogenesis in mural granulosa cells (MGCs) were examined using mice. The results showed that, while oocyte coculture has some effects on the expression levels of transcripts related to EV biogenesis, the number of EV particles present in the conditioned medium were not significantly different between ODPF-treated and non-treated MGCs. Therefore, oocytes have no effects on the EV biogenesis by MGCs, at least with respect to the numbers of EV particles.


Assuntos
Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Oócitos/fisiologia , Comunicação Parácrina/fisiologia , Animais , Células Cultivadas , Feminino , Camundongos Endogâmicos C57BL , Biogênese de Organelas
9.
Proc Natl Acad Sci U S A ; 117(24): 13719-13729, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32482878

RESUMO

The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.


Assuntos
Apicoplastos/enzimologia , Endopeptidase Clp/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Animais , Apicoplastos/genética , Endopeptidase Clp/genética , Humanos , Malária/parasitologia , Biogênese de Organelas , Plasmodium falciparum/genética , Proteólise , Proteínas de Protozoários/genética
10.
Chemosphere ; 258: 127305, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32563914

RESUMO

Chronic arsenic toxicity has become a global concern due to its adverse pathophysiological outcome and carcinogenic potential. It is already established that arsenic induced reactive oxygen species alters mitochondrial functionality. Major regulatory genes for mitochondrial biogenesis, i.e., PGC1α, Tfam, NRF1and NRF2 are located in the nucleus. As a result, mitochondria-nucleus crosstalk is crucial for proper mitochondrial function. This previous hypothesis led us to investigateinvolvement of epigenetic alteration behindenhanced mitochondrial biogenesis in chronic arsenic exposure. An extensive case-control study was conducted with 390 study participants (unexposed, exposed without skin lesion, exposed with skin lesion and exposed skin tumour) from highly arsenic exposed areas ofWest Bengal, India. Methylation specific PCRrevealed significant promoter hypomethylation oftwo key biogenesis regulatory genes, PGC1αandTfam in arsenic exposed individuals and also in skin tumour tissues. Linear regression analysis indicated significant negative correlation between urinary arsenic concentration and promoter methylation status. Increased expression of biogenesis regulatory genes wasobtained by quantitative real-time PCR analysis. Moreover, altered mitochondrial fusion-fission regulatory gene expression was also observed in skin tumour tissues. miR663, having tumour suppressor gene like function was known to be epigenetically regulated through mitochondrial retrograde signal. Promoter hypermethylation with significantly decreased expression of miR663 was found in skin cancer tissues compared to non-cancerous control tissue. In conclusion, results indicated crucial role of epigenetic alteration in arsenic induced mitochondrial biogenesis and arsenical skin carcinogenesis for the first time. However, further mechanistic studies are necessary for detailed understanding of mitochondria-nucleus crosstalk in arsenic perturbation.


Assuntos
Arsênico/toxicidade , Epigênese Genética , Mitocôndrias/fisiologia , Arsênico/metabolismo , Intoxicação por Arsênico , Carcinogênese/induzido quimicamente , Estudos de Casos e Controles , Metilação de DNA , Epigenômica , Feminino , Humanos , Índia , Masculino , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Biogênese de Organelas , Regiões Promotoras Genéticas , Dermatopatias/induzido quimicamente , Neoplasias Cutâneas/induzido quimicamente
11.
Proc Natl Acad Sci U S A ; 117(22): 12109-12120, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414919

RESUMO

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteoma/análise , Proteínas de Ligação a Tacrolimo/metabolismo , Cálcio/metabolismo , Humanos , Biogênese de Organelas , Proteoma/metabolismo , Transdução de Sinais
12.
Proc Natl Acad Sci U S A ; 117(22): 12164-12173, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32409604

RESUMO

Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner-membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase extracellular signal-regulated kinase 7 (ERK7). Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7-AC9 complex reveals that AC9 is not only a scaffold but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an "off" state until the specific binding of a true substrate.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/parasitologia , Biogênese de Organelas , Proteínas de Protozoários/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/patologia , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/genética , Humanos , Fosforilação , Conformação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Transdução de Sinais , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia
13.
Int J Nanomedicine ; 15: 2669-2683, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32368048

RESUMO

Background: Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanomaterials in a variety of fields such as industrial, pharmaceutical, and household applications. Increasing evidence suggests that ZnO NPs could elicit unignorable harmful effect to the cardiovascular system, but the potential deleterious effects to human cardiomyocytes remain to be elucidated. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been increasingly used as a promising in vitro model of cardiomyocyte in various fields such as drug cardiac safety evaluation. Herein, the present study was designed to elucidate the cardiac adverse effects of ZnO NPs and explore the possible underlying mechanism using hiPSC-CMs. Methods: ZnO NPs were characterized by transmission electron microscopy and dynamic light scattering. The cytotoxicity induced by ZnO NPs in hiPSC-CMs was evaluated by determination of cell viability and lactate dehydrogenase release. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential were measured by high-content analysis (HCA). Mitochondrial biogenesis was assayed by detection of mtDNA copy number and PGC-1α pathway. Moreover, microelectrode array techniques were used to investigate cardiac electrophysiological alterations. Results: We demonstrated that ZnO NPs concentration- and time-dependently elicited cytotoxicity in hiPSC-CMs. The results from HCA revealed that ZnO NPs exposure at low-cytotoxic concentrations significantly promoted ROS generation and induced mitochondrial dysfunction. We further demonstrated that ZnO NPs could impair mitochondrial biogenesis and inhibit PGC-1α pathway. In addition, ZnO NPs at insignificantly cytotoxic concentrations were found to trigger cardiac electrophysiological alterations as evidenced by decreases of beat rate and spike amplitude. Conclusion: Our findings unveiled the potential harmful effects of ZnO NPs to human cardiomyocytes that involve mitochondrial biogenesis and the PGC-1α pathway that could affect cardiac electrophysiological function.


Assuntos
Coração/fisiopatologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/metabolismo , Nanopartículas/toxicidade , Biogênese de Organelas , Óxido de Zinco/toxicidade , Diferenciação Celular/efeitos dos fármacos , DNA Mitocondrial/genética , Coração/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
14.
Am J Physiol Endocrinol Metab ; 318(6): E943-E955, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369414

RESUMO

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.


Assuntos
Fibronectinas/genética , Músculo Esquelético/metabolismo , Gordura Subcutânea/metabolismo , Animais , Desoxiglucose/metabolismo , Óxido de Deutério , Eletroporação , Fibronectinas/metabolismo , Expressão Gênica , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/genética , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Membro Posterior , Masculino , Proteínas de Desacoplamento Mitocondrial/genética , Biogênese de Organelas , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ratos
15.
Environ Pollut ; 264: 114639, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32388296

RESUMO

Since ambient particulate matter (APM) is closely related to cardiovascular damage with mitochondria being its potential targets, this study was designed to explore the impact of APM on mitochondrial homeostasis, especially on mitochondrial dynamics and biogenesis in human vascular endothelial cells, using a kind of standard material, PM SRM1648a. As a result, internalized particles lead to mitochondrial dysfunction in EA.hy926 human endothelial cells, including mitochondrial reactive oxygen species (mtROS) overproduction, mitochondrial membrane potential (MMP) reduction and adenosine triphosphate (ATP) inhibition, coupled with additional release of mitochondrial DNA (mtDNA) into the cytosol. Moreover, morphological and structural changes in mitochondria are observed in response to PM SRM1648a. In that aspect, according to the evidence of shorter fragmented mitochondria dispersed throughout the cytoplasm, along with aberrant upregulation of fission-related mRNAs/proteins, the mitochondria exhibit a fission phenotype shifting from intact reticular network to fragmentized punctate shapes. Mechanistically, PM SRM1648a facilitates phosphorylation of DRP1 at Ser616 in HUVECs, and triggers its dephosphorylation at Ser637 residue in both EA.hy926 and HUVECs, which are supportive events for mitochondrial fission during particle exposure. Additionally, suppression of a master energy modulator, PGC-1α, reveals that PM SRM1648a has the ability to impair mitochondrial biogenesis. Collectively, it could be well concluded that PM SRM1648a interferes with the equilibrium of mitochondrial dynamics and biogenesis, which is likely to play a pivotal role in mitochondrial dysfunction driven by particles, eventually contributing to endothelial cell damage. Of note, it is more reasonable to conduct risk assessment from both cellular level and subcellular structures, among which mitochondria-targeted toxicity supplements more comprehensive understanding of APM inducible vascular toxicity.


Assuntos
Dinâmica Mitocondrial , Material Particulado , Células Endoteliais , Humanos , Mitocôndrias , Biogênese de Organelas , Espécies Reativas de Oxigênio
16.
J Biomed Sci ; 27(1): 68, 2020 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-32446297

RESUMO

BACKGROUND: Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high-fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP-activated protein kinase (AMPK)-regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)-dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. METHODS: Pregnant female rats were randomly assigned to receive normal diet (ND) or HFD (60% fructose) chow during pregnancy and lactation. Both ND and HFD offspring returned to ND chow after weaning, and blood pressure (BP) was monitored from age 6 to 12 weeks. At age of 8 weeks, ND and HFD offspring received oral administration of simvastatin or metformin; or brain microinfusion of losartan. BP was monitored under conscious condition by the tail-cuff method. Nutrient sensing molecules, AT1R, subunits of NADPH oxidase, mitochondrial biogenesis markers in rostral ventrolateral medulla (RVLM) were measured by Western blot analyses. RVLM oxidative stress was measured by fluorescent probe dihydroethidium and lipid peroxidation by malondialdehyde assay. Mitochondrial DNA copy number was determined by quantitative real-time polymerase chain reaction. RESULTS: Increased systolic BP, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in RVLM where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator-activated receptor γ co-activator α (PGC-1α) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG-CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD-programmed increase in AT1R and decreases in SIRT1, PGC-1α and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. CONCLUSION: Dysfunction of AMPK-regulated AT1R expression and SIRT1-mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and BP in young offspring programmed by excessive maternal fructose consumption.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Frutose/administração & dosagem , Regulação da Expressão Gênica , Mitocôndrias/fisiologia , Receptor Tipo 1 de Angiotensina/genética , Sirtuína 1/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Feminino , Hipertensão/genética , Exposição Materna , Biogênese de Organelas , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Sirtuína 1/metabolismo
17.
Neurology ; 94(23): e2404-e2411, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32457210

RESUMO

OBJECTIVE: To test the hypothesis that ApoE isoforms affect mitochondrial structure and function that are related to cognitive impairment in Alzheimer disease (AD), we systematically investigated the effects of ApoE isoforms on mitochondrial biogenesis and dynamics, oxidative stress, synapses, and cognitive performance in AD. METHODS: We obtained postmortem human brain tissues and measured proteins that are responsible for mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator-1α [PGC-1α] and sirtuin 3 [SIRT3]), for mitochondrial dynamics (mitofusin 1 [MFN1], mitofusin 2 [MFN2], and dynamin-like protein 1 [DLP1]), for oxidative stress (superoxide dismutase 2 [SOD2] and forkhead-box protein O3a [Foxo3a]), and for synapses (postsynaptic density protein 95 [PSD95] and synapsin1 [Syn1]). A total of 46 cases were enrolled, including ApoE-ɛ4 carriers (n = 21) and noncarriers (n = 25). RESULTS: Levels of these proteins were compared between ApoE-ɛ4 carriers and noncarriers. ApoE-ɛ4 was associated with impaired mitochondrial structure and function, oxidative stress, and synaptic integrity in the human brain. Correlation analysis revealed that mitochondrial proteins and the synaptic protein were strongly associated with cognitive performance. CONCLUSION: ApoE isoforms influence mitochondrial structure and function, which likely leads to alteration in oxidative stress, synapses, and cognitive function. These mitochondria-related proteins may be a harbinger of cognitive decline in ApoE-ɛ4 carriers and provide novel therapeutic targets for prevention and treatment of AD.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas E/fisiologia , Mitocôndrias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/psicologia , Apolipoproteína E4/genética , Apolipoproteína E4/fisiologia , Química Encefálica , Feminino , Humanos , Masculino , Testes de Estado Mental e Demência , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Proteínas Mitocondriais/análise , Proteínas do Tecido Nervoso/análise , Plasticidade Neuronal/genética , Biogênese de Organelas , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/análise , Isoformas de Proteínas/fisiologia , Sirtuína 3/análise , Aprendizagem Verbal
18.
Am J Physiol Renal Physiol ; 319(1): F19-F28, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32463728

RESUMO

Scattered tubular-like cells (STCs) are dedifferentiated surviving tubular epithelial cells that repair neighboring injured cells. Experimental renal artery stenosis (RAS) impairs STC reparative potency by inducing mitochondrial injury, but the exact mechanisms of mitochondrial damage remain unknown. We hypothesized that RAS alters expression of mitochondria-related genes, contributing to mitochondrial structural damage and dysfunction in swine STCs. CD24+/CD133+ STCs were isolated from pig kidneys after 10 wk of RAS or sham (n = 3 each). mRNA sequencing was performed, and nuclear DNA (nDNA)-encoded mitochondrial genes and mitochondrial DNA (mtDNA)-encoded genes were identified. Mitochondrial structure, ATP generation, biogenesis, and expression of mitochondria-associated microRNAs were also assessed. There were 96 nDNA-encoded mitochondrial genes upregulated and 12 mtDNA-encoded genes downregulated in RAS-STCs versus normal STCs. Functional analysis revealed that nDNA-encoded and mtDNA-encoded differentially expressed genes were primarily implicated in mitochondrial respiration and ATP synthesis. Mitochondria from RAS STCs were swollen and showed cristae remodeling and loss and decreased ATP production. Immunoreactivity of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and expression of the mitochondria-associated microRNAs miR-15a, miR-181a, miR-196a, and miR-296-3p, which target several mtDNA genes, were higher in RAS-STCs compared with normal STCs, suggesting a potential modulation of mitochondria-related gene expression. These results demonstrate that RAS induces an imbalance in mtDNA- and nDNA-mitochondrial gene expression, impairing mitochondrial structure and function in swine STCs. These observations support development of gene gain- and loss-of-function strategies to ameliorate mitochondrial damage and preserve the reparative potency of STCs in patients with renal ischemia.


Assuntos
Expressão Gênica , Genes Mitocondriais , Isquemia/genética , Rim/irrigação sanguínea , Mitocôndrias/metabolismo , Obstrução da Artéria Renal/metabolismo , Animais , Feminino , Isquemia/metabolismo , Biogênese de Organelas , Obstrução da Artéria Renal/genética , Suínos
19.
Am J Physiol Cell Physiol ; 318(6): C1166-C1177, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32320289

RESUMO

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metabolismo Energético , Células Epiteliais/metabolismo , Lactação/metabolismo , Lisossomos/metabolismo , Glândulas Mamárias Humanas/metabolismo , Leite Humano/metabolismo , Mutação , Trifosfato de Adenosina/metabolismo , Adulto , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Metabolismo Energético/genética , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lactação/genética , Lisossomos/genética , Biogênese de Organelas , Fosforilação , Adulto Jovem
20.
Biochem Biophys Res Commun ; 526(4): 1069-1076, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32305136

RESUMO

Obesity-induced fat ectopic deposition results in mitochondrial dysfunction and oxidative stress in skeletal muscle, which could impair the quality and function of the skeletal muscle. Human fibroblast growth factor 19 (FGF19) acts as a vital metabolic regulator of bile acid synthesis and metabolic homeostasis. Recent studies have shown that FGF19 regulates skeletal muscle mass through the enlargement of muscle fiber size and protects muscles from atrophy. However, the role of FGF19 in regulating mitochondrial function and the antioxidant response in skeletal muscle remains unknown. Therefore, we investigated the effect of FGF19 on palmitic acid (PA)-induced mitochondrial dysfunction and oxidative stress in C2C12 cells. In this study, we found that FGF19 can increase the mRNA and protein expression levels of mitochondrial biogenesis regulators (PGC-1α, Nrf-1, and TFAM) and antioxidant response regulators (Nrf-2 and HO-1), alleviating PA-induced mitochondrial dysfunction and oxidative stress. However, the regulatory effect of FGF19 was blocked by Compound C, an AMP-activated protein kinase (AMPK) inhibitor, and siRNA knockdown of PGC-1a. Taken together, these findings indicate that FGF19 might promote mitochondrial biogenesis and antioxidant response via the AMPK/PGC-1α pathway, attenuating the effect of PA on mitochondrial dysfunction and oxidative stress; therefore, FGF19 might be a potential therapeutic target for the effects of obesity on skeletal muscle.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Palmítico/toxicidade , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , Animais , Antioxidantes/metabolismo , Linhagem Celular , Humanos , Camundongos , Mitocôndrias Musculares/efeitos dos fármacos , Biogênese de Organelas , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
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