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1.
Clin Sci (Lond) ; 134(2): 239-259, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31943002

RESUMO

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.


Assuntos
/metabolismo , Diabetes Mellitus Experimental/metabolismo , Nefropatias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Albuminúria/genética , Albuminúria/metabolismo , Animais , /genética , Ciclosporina/farmacologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , ATPases Translocadoras de Prótons/metabolismo
2.
Life Sci ; 237: 116941, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31606382

RESUMO

AIMS: Podocytes play an important role in the development of diabetic kidney disease (DKD). Mitochondria are the source of energy for cell survival, and mitochondrial abnormalities have been shown to contribute to podocyte injury in DKD. In high glucose (HG)-treated podocytes, mitochondrial function and dynamics are abnormal, and intracellular metabolism is often disrupted. However, the molecular mechanism is still unclear. Mitochondrial pyruvate carrier 2 (MPC2) mediates pyruvate transport from the cytoplasm to the mitochondrial matrix, which determines the cellular energy supply and cell survival. Here, we hypothesize that MPC2 damages mitochondria and induces apoptosis in HG-treated podocytes. MAIN METHODS: We used Western blotting, immunofluorescence and immunoprecipitation to detect the expression of MPC2 in HG-treated podocytes. Pyruvate levels were measured to evaluate metabolic station. Mitochondrial membrane potential (MMP) was measured by inverted fluorescence microscopy and flow cytometry. Mitochondrial morphology was assayed by MitoTracker Red staining, and cellular apoptosis was examined by flow cytometry. Furthermore, we treated podocytes with UK5099 and MPC2 siRNA to assess the outcomes of UK5099 treatment and MPC2 knockdown. KEY FINDINGS: Intracellular pyruvate accumulated, the mitochondria were damaged and cellular apoptosis increased in podocytes cultured with HG compared to that in control podocytes. MPC2 acetylation was significantly increased in HG-treated podocytes. Furthermore, the mitochondrial morphology changed, the MMP decreased, and cellular apoptosis increased. Inhibition of MPC2 function by UK5099 or MPC2 knockdown by siRNA produced the same abnormal effects observed following treatment with HG. SIGNIFICANCE: MPC2 may mediate mitochondrial dysfunction in HG-treated podocytes, ultimately leading to cell apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Podócitos/patologia , Ácido Pirúvico/metabolismo , Células Cultivadas , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo
3.
Int J Mol Sci ; 20(15)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344855

RESUMO

Breast cancer, the most prevalent cancer type among women worldwide, remains incurable once metastatic. Long noncoding RNA (lncRNA) and microRNA (miRNA) play important roles in breast cancer by regulating specific genes or proteins. In this study, we found miR-133b was silenced in breast cancer cell lines and in breast cancer tissues, which predicted poor prognosis in breast cancer patients. We also confirmed that lncRNA NEAT1 was up-regulated in breast cancer and inhibited the expression of miR-133b, and identified the mitochondrial protein translocase of inner mitochondrial membrane 17 homolog A (TIMM17A) that serves as the target of miR-133b. Both miR-133b knockdown and TIMM17A overexpression in breast cancer cells promoted cell migration and invasion both in vitro and in vivo. In summary, our findings reveal that miR-133b plays a critical role in breast cancer cell metastasis by targeting TIMM17A. These findings may provide new insights into novel molecular therapeutic targets for breast cancer.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , RNA Longo não Codificante/genética , Idoso , Animais , Neoplasias da Mama/patologia , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia
4.
Biochim Biophys Acta Mol Cell Res ; 1866(9): 1433-1449, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31195049

RESUMO

Recent evidences highlight the importance of mitochondria-nucleus communication for the clinical phenotype of oxidative phosphorylation (OXPHOS) diseases. However, the participation of small non-coding RNAs (sncRNAs) in this communication has been poorly explored. We asked whether OXPHOS dysfunction alters the production of a new class of sncRNAs, mitochondrial tRNA fragments (mt tRFs), and, if so, whether mt tRFs play a physiological role and their accumulation is controlled by the action of mt tRNA modification enzymes. To address these questions, we used a cybrid model of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), an OXPHOS disease mostly caused by mutation m.3243A>G in the mitochondrial tRNALeu(UUR) gene. High-throughput analysis of small-RNA-Seq data indicated that m.3243A>G significantly changed the expression pattern of mt tRFs. A functional analysis of potential mt tRFs targets (performed under the assumption that these tRFs act as miRNAs) indicated an association with processes that involve the most common affected tissues in MELAS. We present evidences that mt tRFs may be biologically relevant, as one of them (mt i-tRF GluUUC), likely produced by the action of the nuclease Dicer and whose levels are Ago2 dependent, down-regulates the expression of mitochondrial pyruvate carrier 1 (MPC1), promoting the build-up of extracellular lactate. Therefore, our study underpins the idea that retrograde signaling from mitochondria is also mediated by mt tRFs. Finally, we show that accumulation of mt i-tRF GluUUC depends on the modification status of mt tRNAs, which is regulated by the action of stress-responsive miRNAs on mt tRNA modification enzymes.


Assuntos
Síndrome MELAS/genética , Síndrome MELAS/metabolismo , Mitocôndrias/metabolismo , Mutação , RNA de Transferência de Leucina/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação para Baixo , Proteínas de Ligação ao GTP , Regulação da Expressão Gênica , Células HeLa , Humanos , MicroRNAs/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais , Transportadores de Ácidos Monocarboxílicos/genética , Fosforilação Oxidativa , Pequeno RNA não Traduzido , Proteínas de Ligação a RNA , Transdução de Sinais , Transcriptoma , tRNA Metiltransferases
5.
Oxid Med Cell Longev ; 2019: 4508762, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236191

RESUMO

Much evidence demonstrates that mitochondrial dysfunction plays a crucial role in the pathogenesis of vascular complications of diabetes. However, the signaling pathways through which hyperglycemia leads to mitochondrial dysfunction of endothelial cells are not fully understood. Here, we treated human umbilical vein endothelial cells (HUVECs) with high glucose and examined the role of translocase of mitochondrial outer membrane (Tom) 22 on mitochondrial dynamics and cellular function. Impaired Tom22 expression and protein expression of oxidative phosphorylation (OXPHOS) as well as decreased mitochondrial fusion were observed in HUVECs treated with high glucose. The deletion of Tom22 resulted in reduced mitochondrial fusion and ATP production and increased apoptosis in HUVECs. The overexpression of Tom22 restored the balance of mitochondrial dynamics and OXPHOS disrupted by high glucose. Importantly, we found that Tom22 modulates mitochondrial dynamics and OXPHOS by interacting with mitofusin (Mfn) 1. Taken together, our findings demonstrate for the first time that Tom22 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following high-glucose exposure.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/patologia , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Apoptose , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Transdução de Sinais
6.
mBio ; 10(3)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064825

RESUMO

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/genética , Adaptação Fisiológica , Transporte Biológico , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions , Citosol/química , Citosol/metabolismo , Técnicas de Inativação de Genes , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/patogenicidade
7.
Int J Mol Sci ; 20(8)2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30995827

RESUMO

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).


Assuntos
Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Ativação Transcricional , Fatores Estimuladores Upstream/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Regiões Promotoras Genéticas
8.
EMBO J ; 38(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30979775

RESUMO

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae In contrast to earlier hypotheses, we demonstrate that MPC is a hetero-dimer, not a multimeric complex. When not engaged in hetero-dimers, the yeast Mpc proteins can also form homo-dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero-dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.


Assuntos
Proteínas de Transporte de Ânions , Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Complexos Multiproteicos/fisiologia , Multimerização Proteica/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos/química , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Organismos Geneticamente Modificados , Estrutura Quaternária de Proteína/fisiologia , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Temperatura Ambiente
9.
Cell Physiol Biochem ; 52(3): 435-438, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30873819

RESUMO

BACKGROUND/AIMS: Tachycardiomyopathy (TCM) is a largely reversible form of non-ischemic heart failure. The underlying mechanism are, however, still today poorly understood. Recent data indicate distinct changes in mitochondrial distribution in these patients, compared to other non-ischemic cardiomyopathies.This study investigated underlying mechanisms in mitochondrial dynamics in endomyocardial biopsy samples (EMB) from patients with TCM and compared them to patients with dilated cardiomyopathy (DCM), which show similar clinical features. METHODS: Focused mRNA analyses were performed on routinely obtained paraffinfixed EMB specimen from patients fulfilling TCM diagnosis criteria, as well as patients with DCM to elucidate regulatory changes in mitochondrial fusion, fission and mitophagy. RESULTS: In patients with TCM we were able to identify mRNA of Mitofusin 1 and 2, two effector proteins regulating mitochondrial fusion, to be strongly upregulated compared to patients with DCM. Conclusively, we did not find differences in the mRNA expression of mitochondrial fission regulators including DRP1, Fis1, MFF, MiD49, and MiD51. Furthermore, we did not find significant changes in PINK1 expression, an important mediator for mitochondrial autophagy. CONCLUSION: The mRNA upregulation of Mitofusin 1 and 2 provides first insight into the complex changes of mitochondrial dynamics in cardiomyocytes of patients with reversible heart failure due to TCM.


Assuntos
Cardiomiopatia Dilatada/genética , GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Dinâmica Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/genética , RNA Mensageiro/genética , Biópsia , Cardiomiopatia Dilatada/classificação , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/fisiopatologia , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Frequência Cardíaca/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo
10.
EMBO J ; 38(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842096

RESUMO

Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin-related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1-mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1-/- cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins.


Assuntos
Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Dinaminas/genética , GTP Fosfo-Hidrolases/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/genética
11.
Gene ; 693: 69-75, 2019 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-30708027

RESUMO

The human SLC25A13 gene encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2, commonly named as citrin), which plays a key role in the main NADH-shuttle of human hepatocyte. Biallelic SLC25A13 mutations result in Citrin deficiency (CD). In order to identify the important regulatory region of SLC25A13 gene and elucidate the way how potential promoter mutations affect the citrin expression, we performed promoter deletion analysis and established the reporter constructs of luciferase gene-carrying SLC25A13 promoter containing several mutations located in putative transcription factor-binding sites. The luciferase activities of all promoter constructs were measured using a Dual-Luciferase Reporter Assay System. Bioinformatic analysis showed that the promoter of SLC25A13 gene lacks TATA box and obviously typical initiator element, but contains a CCAAT box and two GC box. Promoter deletion analysis confirmed the region from -221 to -1 upstream ATG was essential for SLC25A13 to maintain the promoter activity. We utilized dual-luciferase reporter system as function analytical model to tentatively assess the effect of artificially constructed promoter mutations on citrin expression, and our analysis revealed that mutated putative CCAAT box and GC box could significantly affect the citrin expression. Our study confirmed the important SLC25A13 promoter regions that influenced citrin expression in HL7702 cells, and constructed a function analytical model. This work may be useful to further identify the pathogenic mutations leading to CD in the promoter region.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Bases , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Biologia Computacional , Humanos , Mutação , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/fisiologia , Regiões Promotoras Genéticas/genética
12.
Mol Cell ; 73(5): 1028-1043.e5, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733118

RESUMO

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.


Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Antiparkinsonianos/farmacologia , Transporte Biológico , Desenho de Drogas , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
13.
Mol Cell ; 73(5): 1044-1055.e8, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30738703

RESUMO

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22. This Por1 association thereby modulates Tom22 integration into the TOM complex, guaranteeing formation of the functional trimeric TOM complex. Por1 sequestration of Tom22 dissociated from the trimeric TOM complex also enhances the dimeric TOM complex, which is preferable for the import of TIM40/MIA-dependent proteins into mitochondria. Furthermore, Por1 appears to contribute to cell-cycle-dependent variation of the functional trimeric TOM complex by chaperoning monomeric Tom22, which arises from the cell-cycle-controlled variation of phosphorylated Tom6.


Assuntos
Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Ciclo Celular , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação , Porinas/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
14.
Mol Cell ; 73(5): 1056-1065.e7, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30738704

RESUMO

The mitochondrial inner membrane harbors a large number of metabolite carriers. The precursors of carrier proteins are synthesized in the cytosol and imported into mitochondria by the translocase of the outer membrane (TOM) and the carrier translocase of the inner membrane (TIM22). Molecular chaperones in the cytosol and intermembrane space bind to the hydrophobic precursors to prevent their aggregation. We report that the major metabolite channel of the outer membrane, termed porin or voltage-dependent anion channel (VDAC), promotes efficient import of carrier precursors. Porin interacts with carrier precursors arriving in the intermembrane space and recruits TIM22 complexes, thus ensuring an efficient transfer of the precursors to the inner membrane translocase. Porin channel mutants impaired in metabolite transport are not disturbed in carrier import into mitochondria. We conclude that porin serves distinct functions as outer membrane channel for metabolites and as coupling factor for protein translocation into the inner membrane.


Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters/genética , Antiporters/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Porinas/genética , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Proc Natl Acad Sci U S A ; 116(9): 3546-3555, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755530

RESUMO

The mitochondrial uniporter is a Ca2+-channel complex resident within the organelle's inner membrane. In mammalian cells the uniporter's activity is regulated by Ca2+ due to concerted action of MICU1 and MICU2, two paralogous, but functionally distinct, EF-hand Ca2+-binding proteins. Here we present the X-ray structure of the apo form of Mus musculus MICU2 at 2.5-Å resolution. The core structure of MICU2 is very similar to that of MICU1. It consists of two lobes, each containing one canonical Ca2+-binding EF-hand (EF1, EF4) and one structural EF-hand (EF2, EF3). Two molecules of MICU2 form a symmetrical dimer stabilized by highly conserved hydrophobic contacts between exposed residues of EF1 of one monomer and EF3 of another. Similar interactions stabilize MICU1 dimers, allowing exchange between homo- and heterodimers. The tight EF1-EF3 interface likely accounts for the structural and functional coupling between the Ca2+-binding sites in MICU1, MICU2, and their complex that leads to the previously reported Ca2+-binding cooperativity and dominant negative effect of mutation of the Ca2+-binding sites in either protein. The N- and C-terminal segments of the two proteins are distinctly different. In MICU2 the C-terminal helix is significantly longer than in MICU1, and it adopts a more rigid structure. MICU2's C-terminal helix is dispensable in vitro for its interaction with MICU1 but required for MICU2's function in cells. We propose that in the MICU1-MICU2 oligomeric complex the C-terminal helices of both proteins form a central semiautonomous assembly which contributes to the gating mechanism of the uniporter.


Assuntos
Canais de Cálcio/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Transporte da Membrana Mitocondrial/química , Conformação Proteica , Animais , Sítios de Ligação , Cálcio/química , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Cristalografia por Raios X , Dimerização , Motivos EF Hand/genética , Células HeLa , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Estrutura Secundária de Proteína
16.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(2): 116-119, 2019 Feb 10.
Artigo em Chinês | MEDLINE | ID: mdl-30703226

RESUMO

OBJECTIVE: To explore the clinical and genetic features of an infant with citrin deficiency (CD). METHODS: Clinical data of the patient was collected and analyzed. Genomic DNA was extracted from peripheral blood samples collected from the patient and her parents. Targeted exome sequencing was performed to explore the genetic cause, and Sanger sequencing was used to confirm the detected variants. SLC25A13 mRNA was extracted from peripheral blood lymphocytes of the infant. The effect of novel mutation of SLC25A13 was analyzed by reverse transcription-PCR, cDNA cloning and Sanger sequencing. RESULTS: The SLC25A13 genotype of the patient was determined as c.845_c.848+1delG/c.1841+3_1841+4delAA, with the latter having not been reported. The mutation has affected the splicing of the SLC25A13 mRNA, giving rise to an aberrant transcript [r.1841_1842ins1841+1_1841+67; 1841+3_c.1841+4del]. CONCLUSION: A novel SLC25A13 mutation c.1841+3_1841+4delAA and the resultant abnormal splicing variant were discovered by combined DNA sequencing and cDNA cloning. The finding has enabled definite diagnosis of CD and enriched the spectrum of SLC25A13 mutations.


Assuntos
Citrulinemia , Proteínas de Transporte da Membrana Mitocondrial/genética , Sequência de Bases , Feminino , Humanos , Mutação , Linhagem
17.
EBioMedicine ; 41: 200-213, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30796006

RESUMO

BACKGROUND: LncRNAs have been found to be involved in various aspects of biological processes. In this study, we aimed to uncover the molecular mechanisms of lncRNA EPB41L4A-AS1 in regulating glycolysis and glutaminolysis in cancer cells. METHODS: The expression of EPB41L4A-AS1 in cancer patients was analyzed in TCGA and GEO datasets. The level of cellular metabolism was determined by extracellular flux analyzer. The relationship between p53 and EPB41L4A-AS1 was explored by qRT-PCR, luciferase assay and ChIP assay. The interactions between EPB41L4A-AS1 and HDAC2 or NPM1 were determined by RNA immunoprecipitation, RNA pull-down assay and RNA-FISH- immunofluorescence. FINDINGS: EPB41L4A-AS1 was a p53-regulated gene. Low expression and deletion of lncRNA EPB41L4A-AS1 were found in a variety of human cancers and associated with poor prognosis of cancer patients. Knock down EPB41L4A-AS1 expression triggered Warburg effect, demonstrated as increased aerobic glycolysis and glutaminolysis. EPB41L4A-AS1 interacted and colocalized with HDAC2 and NPM1 in nucleolus. Silencing EPB41L4A-AS1 reduced the interaction between HDAC2 and NPM1, released HDAC2 from nucleolus and increased its distribution in nucleoplasm, enhanced HDAC2 occupation on VHL and VDAC1 promoter regions, and finally accelerated glycolysis and glutaminolysis. Depletion of EPB41L4A-AS1 increased the sensitivity of tumor to glutaminase inhibitor in tumor therapy. INTERPRETATION: EPB41L4A-AS1 functions as a repressor of the Warburg effect and plays important roles in metabolic reprogramming of cancer.


Assuntos
Núcleo Celular/metabolismo , Glicólise , Histona Desacetilase 2/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Neoplasias/metabolismo , RNA Longo não Codificante/genética , Transporte Ativo do Núcleo Celular , Animais , Glutaminase/metabolismo , Células HeLa , Células Hep G2 , Humanos , Camundongos , Camundongos Nus , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , RNA Longo não Codificante/metabolismo
18.
Proc Natl Acad Sci U S A ; 116(9): 3530-3535, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808746

RESUMO

Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Retina/metabolismo , Visão Ocular/genética , Animais , Glucose/metabolismo , Glicólise/genética , Humanos , Camundongos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos , Ácido Pirúvico/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia
19.
Cancer Sci ; 110(4): 1331-1339, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30801869

RESUMO

Mitochondrial pyruvate carrier (MPC) is known to cause different expressions in normal and cancer cells. We observed a change in phenotype with the suppression of MPC expression. We knocked down MPC1 and/or MPC2 using siRNA or shRNA. We observed its cell morphology and accompanying molecular marker. Furthermore, the radioresistance of the MPC knockdown cell line was examined using a colony formation assay. MPC1-suppressed cells changed their morphology to a spindle shape. Epithelial-mesenchymal transition (EMT) was suspected, and examination of the EMT marker by PCR showed a decrease in E-cadherin and an increase in fibronectin. Focusing on glutamine metabolism as the mechanism of this phenomenon, we knocked down the glutamine-metabolizing enzyme glutaminase (GLS). EMT was also observed in GLS-suppressed cells. Furthermore, when MPC1-suppressed cells were cultured in a glutamine-deficient medium, changes in EMT markers were suppressed. In addition, MPC1-suppressed cells also increased with a significant difference in radioresistance. Decreased MPC1 expression favorably affects EMT and radioresistance of cancer.


Assuntos
Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Transporte da Membrana Mitocondrial/genética , Neoplasias/genética , Neoplasias/patologia , Tolerância a Radiação/genética , Biomarcadores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Biológicos , Transportadores de Ácidos Monocarboxílicos , Neoplasias/metabolismo , Neoplasias/radioterapia
20.
Mol Biol Rep ; 46(1): 271-285, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30712249

RESUMO

Methylmalonic acidemia (MMA), an inherited metabolic disease, results from genetic defects in methylmalonyl-CoA mutase or any of the proteins involved in adenosylcobalamin synthesis. This enzyme is classified into several complementation groups and genotypic classes. In this work we explain the biochemical, structural and genetic analysis of 25 MMA patients, from Iran. The diagnosis was established by the measurement of propionylcarnitine in blood using tandem mass spectrometry and confirmed using a gas chromatography-flame ionization detector. Using clinical, biochemical, structural and molecular analyses we identified 15 mut MMA, three cblA, one cblB, and four cblC-deficient patients. Among mutations identified in the MUT gene (MUT) only one, the c.1874A>C (p.D625A) variant, is likely a mut- mutation. The remaining mutations are probably mut0. Here, we present the first molecular analysis of MMA in Iranian patients and have identified eight novel mutations. Four novel mutations (p.D625A, p.R326G, p.V157F, p.F379L) were seen exclusively in patients from northern Iran. One novel splice site mutation (c.2125-3C>G) in MUT and two novel mutation (p.N225M and p.A99P) in the MMAA gene were associated with patients from eastern Iran. The rs184829210 SNP was recognized only in patients with the novel c.958G>A (p.A320T) mutation. This study confirms pathogenesis of deficient enzyme activity in MUT, MMAA, MMAB, and MMACHC as previous observations. These results could act as a basis for the performance of pharmacological therapies for increasing the activity of proteins derived from these mutations.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Adulto , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Genótipo , Humanos , Lactente , Recém-Nascido , Irã (Geográfico) , Masculino , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação/genética , Oxirredutases
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