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1.
Nucleic Acids Res ; 49(8): 4689-4704, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33836087

RESUMO

Deficient maturations of mitochondrial transcripts are linked to clinical abnormalities but their pathophysiology remains elusive. Previous investigations showed that pathogenic variants in MTO1 for the biosynthesis of τm5U of tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR) were associated with hypertrophic cardiomyopathy (HCM). Using mto1 knock-out(KO) zebrafish generated by CRISPR/Cas9 system, we demonstrated the pleiotropic effects of Mto1 deficiency on mitochondrial RNA maturations. The perturbed structure and stability of tRNAs caused by mto1 deletion were evidenced by conformation changes and sensitivity to S1-mediated digestion of tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). Notably, mto1KO zebrafish exhibited the global decreases in the aminoacylation of mitochondrial tRNAs with the taurine modification. Strikingly, ablated mto1 mediated the expression of MTPAP and caused the altered polyadenylation of cox1, cox3, and nd1 mRNAs. Immunoprecipitation assay indicated the interaction of MTO1 with MTPAP related to mRNA polyadenylation. These alterations impaired mitochondrial translation and reduced activities of oxidative phosphorylation complexes. These mitochondria dysfunctions caused heart development defects and hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the mto1KO zebrafish recapitulated the clinical phenotypes in HCM patients carrying the MTO1 mutation(s). Our findings highlighted the critical role of MTO1 in mitochondrial transcript maturation and their pathological consequences in hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatia Hipertrófica/genética , Coração/embriologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Cardiomiopatia Hipertrófica/fisiopatologia , Perfilação da Expressão Gênica , Coração/fisiopatologia , Hibridização In Situ , Microscopia Eletrônica de Transmissão , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação Oxidativa , Poliadenilação/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/genética , Aminoacilação de RNA de Transferência/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
2.
Nucleic Acids Res ; 47(10): 5341-5355, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30916346

RESUMO

GTPBP3 is a highly conserved tRNA modifying enzyme for the biosynthesis of τm5U at the wobble position of mitochondrial tRNAGlu, tRNAGln, tRNALys, tRNATrp and tRNALeu(UUR). The previous investigations showed that GTPBP3 mutations were associated with hypertrophic cardiomyopathy (HCM). However, the pathophysiology of GTPBP3 deficiency remains elusively. Using the gtpbp3 knockout zebrafish generated by CRISPR/Cas9 system, we demonstrated the aberrant mitochondrial tRNA metabolism in gtpbp3 knock-out zebrafish. The deletion of gtpbp3 may alter functional folding of tRNA, indicated by conformation changes and sensitivity to S1-mediated digestion of tRNAGlu, tRNALys, tRNATrp and tRNALeu(UUR). Strikingly, gtpbp3 knock-out zebrafish displayed the global increases in the aminoacylated efficiencies of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms impaired mitochondrial translation, produced proteostasis stress and altered activities of respiratory chain complexes. These mitochondria dysfunctions caused the alterations in the embryonic heart development and reduced fractional shortening of ventricles in mutant zebrafish. Notably, the gtpbp3 knock-out zebrafish exhibited hypertrophy of cardiomyocytes and myocardial fiber disarray in ventricles. These cardiac defects in the gtpbp3 knock-out zebrafish recapitulated the clinical phenotypes in HCM patients carrying the GTPBP3 mutation(s). Our findings highlight the fundamental role of defective nucleotide modifications of tRNAs in mitochondrial biogenesis and their pathological consequences in hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Ligação ao GTP/genética , Deleção de Genes , RNA de Transferência/metabolismo , Proteínas de Peixe-Zebra/genética , Aminoacilação , Animais , Sistemas CRISPR-Cas , Cardiomiopatia Hipertrófica/genética , Proteínas de Ligação ao GTP/metabolismo , Hibridização In Situ , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Fenótipo , Conformação Proteica , Transgenes , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Neurodegener Dis ; 18(5-6): 270-280, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30673663

RESUMO

PURPOSE: (1) To investigate atrophy patterns of hippocampal subfield volume and Alzheimer's disease (AD)-signature cortical thickness in mild cognitive impairment (MCI) patients; (2) to explore the association between the neuropsychological (NP) and the brain structure in the MCI and older normal cognition group; (3) to determine whether these associations were modified by the apolipoprotein E (APOE) ε4 gene and cognitive status. METHODS: The FreeSurfer software was used for automated segmentation of hippocampal subfields and AD-signature cortical thickness for 22 MCI patients and 23 cognitive normal controls (NC). The volume, cortical thickness, and the neuropsychological scale were compared with two-sample t tests. Linear regression models were used to determine the association between the NP and the brain structure. RESULTS: Compared with the NC group, MCI patients showed a decreased volume of the left presubiculum, subiculum and right CA2_3 and CA4_DG (p < 0.05, FDR corrected). The volume of these regions was positively correlated with NP scores. Of note, these associations depended on the cognitive status but not on the APOE ε4 status. The left subiculum and presubiculum volume were positively correlated with the Montreal Cognitive Assessment (MoCA) scores only in the MCI patients. CONCLUSION: Atrophy of the hippocampal subfields may be a powerful biomarker for MCI in the Chinese population.


Assuntos
Doença de Alzheimer/diagnóstico , Apolipoproteína E4/genética , Cognição/fisiologia , Imageamento por Ressonância Magnética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Biomarcadores/análise , Disfunção Cognitiva/patologia , Feminino , Hipocampo/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Testes Neuropsicológicos
4.
Dev Cell ; 57(13): 1630-1642.e4, 2022 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-35777354

RESUMO

Maintaining the integrity of the plasma membrane after cellular damage is essential for cell survival. However, it is unclear how cells repair large membrane injuries in vivo. Here, we report that the tetraspanin protein, TSP-15, is recruited to large membrane wounds and forms a ring-like structure in C. elegans epidermis and promotes membrane repair after an injury. TSP-15 recruits from the adjacent region underneath the plasma membrane to the wound site in a RAB-5-dependent manner upon membrane damage. Genetic and live-imaging analysis suggested that the endosomal sorting complex required for transport III (ESCRT III) is necessary for recruiting TSP-15 from the early endosome to the damaged membrane. Moreover, TSP-15 interacts with and is required for the accumulation of t-SNARE protein Syntaxin-2, which facilitates membrane repair. These findings provide valuable insights into the role of the conserved tetraspanin TSP-15 in the cellular repair of large wounds resulting from environmental insults.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Epiderme/metabolismo , Proteínas SNARE/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismo
5.
Cell Rep ; 37(8): 110040, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34818546

RESUMO

Tissue damage induces immediate-early signals, activating Rho small GTPases to trigger actin polymerization essential for later wound repair. However, how tissue damage is sensed to activate Rho small GTPases locally remains elusive. Here, we found that wounding the C. elegans epidermis induces rapid relocalization of CDC-42 into plasma membrane-associated clusters, which subsequently recruits WASP/WSP-1 to trigger actin polymerization to close the wound. In addition, wounding induces a local transient increase and subsequent reduction of H2O2, which negatively regulates the clustering of CDC-42 and wound closure. CDC-42 CAAX motif-mediated prenylation and polybasic region-mediated cation-phospholipid interaction are both required for its clustering. Cysteine residues participate in intermolecular disulfide bonds to reduce membrane association and are required for negative regulation of CDC-42 clustering by H2O2. Collectively, our findings suggest that H2O2-regulated fine-tuning of CDC-42 localization can create a distinct biomolecular cluster that facilitates rapid epithelial wound repair after injury.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Cicatrização/fisiologia , Actinas , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/imunologia , Proteínas de Ciclo Celular/imunologia , Membrana Celular/metabolismo , Células Epidérmicas/metabolismo , Epiderme/metabolismo , Proteínas de Ligação ao GTP/imunologia , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/metabolismo , Oxirredução , Polimerização , Transdução de Sinais , Família de Proteínas da Síndrome de Wiskott-Aldrich/imunologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Cicatrização/imunologia , Proteínas rho de Ligação ao GTP/metabolismo
6.
STAR Protoc ; 1(3): 100175, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33377069

RESUMO

Efficient membrane repair after injury is essential for cell and animal survival. Caenorhabditis elegans epidermal cell hpy7 has emerged as a powerful genetic system to investigate the molecular mechanism of membrane repair in vivo. This protocol describes detailed approaches for how to perform wounding on the epidermis and how to examine membrane repair by trypan blue staining, confocal imaging, and data analysis. For details on the use and execution of this protocol, please refer to Meng et al. (2020).


Assuntos
Caenorhabditis elegans/fisiologia , Técnicas Citológicas/métodos , Epiderme/patologia , Cicatrização , Animais , Proteínas de Fluorescência Verde/metabolismo , Membranas , Microscopia Confocal , Coloração e Rotulagem , Imagem com Lapso de Tempo
7.
Dev Cell ; 54(5): 624-638.e5, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32668210

RESUMO

Membrane repair is essential for cell and organism survival. Exocytosis and endocytosis facilitate membrane repair in small wounds within a single cell; however, it remains unclear how large wounds in the plasma membrane are repaired in metazoans. Here, we show that wounding triggers rapid transcriptional upregulation and dynamic recruitment of the fusogen EFF-1 to the wound site in C. elegans epidermal cells. EFF-1 recruitment at the wounded membrane depends on the actin cytoskeleton and is important for membrane repair. We identified syntaxin-2 (SYX-2) as an essential regulator of EFF-1 recruitment. SYX-2 interacts with the C terminus of EFF-1 to promote its recruitment, facilitating both endoplasmic and exoplasmic membrane repair. Furthermore, we show that SYX-2-EFF-1 repair machinery acts downstream of the ESCRT III signal. Together, our findings identify a key pathway underlying membrane repair and provide insights into tissue repair and regenerative medicine after injury.


Assuntos
Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glicoproteínas de Membrana/metabolismo , Sintaxina 1/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Endocitose/fisiologia , Células Epidérmicas/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-30035262

RESUMO

OBJECTIVE: To investigate whether magnetic resonance imaging (MRI) can be used to directly assess olfactory bulb (OB) lesions and quantify the associated morphological changes of olfactory filaments (OF), also known as fila, in an in vivo OB-lesion rat model of the brain. METHODS: A surgical group (n = 5) of male Sprague-Dawley rats was subjected to the unilateral damage of the OB by a steel needle. The control group (n = 5) did not receive surgery. To assess olfactory system injury in vivo, T2-weighted MRI images were acquired in an oblique plane at a 30° angle from transverse plane one day after surgery. These brain regions were also assessed in the controls. The olfactory function was evaluated using the buried food pellet test (BFPT) 5 days before and after surgery. RESULTS: The OF could be clearly observed on the MRI images from all animals. The left and right OF mean lengths (mm) were similar in the control group (0.81 ± 0.18 vs 0.89 ± 0.17, P > 0.05). In the surgical group, the OB was partially injured in all rats. These rats did not show differences in OF length between left- and right-side (0.83 ± 0.18 vs 0.93 ± 0.24, P > 0.05) at the time of measurement. The time (sec) required to find the food pellets in the BFPT was longer after than before the surgery (83.80 ± 34.37 vs 231.44 ± 53.23, P < 0.05). CONCLUSIONS: MicroMRI may be a feasible tool to evaluate the OF and OBs in rat models. The unilateral partial OB lesion model appears to be an effective post-traumatic olfactory dysfunction model.

9.
Int J Biochem Cell Biol ; 77(Pt A): 1-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27184967

RESUMO

Human mitochondrial DNA (mtDNA) mutations have been associated with a wide spectrum of clinical abnormalities. However, nuclear modifier gene(s) modulate the phenotypic expression of pathogenic mtDNA mutations. In our previous investigation, we identified the human GTPBP3 related to mitochondrial tRNA modification, acting as a modifier to influence of deafness-associated mtDNA mutation. Mutations in GTPBP3 have been found to be associated with other human diseases. However, the pathophysiology of GTPBP3-associated disorders is still not fully understood. Here, we reported the generation and characterization of Gtpbp3 depletion zebrafish model using antisense morpholinos. Zebrafish gtpbp3 has three isoforms localized at mitochondria. Zebrafish gtpbp3 is expressed at various embryonic stages and in multiple tissues. In particular, the gtpbp3 was expressed more abundantly in adult zebrafish ovary and testis. The expression of zebrafish gtpbp3 can functionally restore the growth defects caused by the mss1/gtpbp3 mutation in yeast. A marked decrease of mitochondrial ATP generation accompanied by increased levels of apoptosis and reactive oxygen species were observed in gtpbp3 knockdown zebrafish embryos. The Gtpbp3 morphants exhibited defective in embryonic development including bleeding, melenin, oedema and curved tails within 5days post fertilization, as compared with uninjected controls. The co-injection of wild type gtpbp3 mRNA partially rescued these defects in Gtpbp3 morphants. These data suggest that zebrafish Gtpbp3 is a structural and functional homolog of human and yeast GTPBP3. The mitochondrial dysfunction caused by defective Gtpbp3 may alter the embryonic development in the zebrafish. In addition, this zebrafish model of mitochondrial disease may provide unique opportunities for studying defective tRNA modification, mitochondrial biogenesis, and pathophysiology of mitochondrial disorders.


Assuntos
Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Mitocôndrias/metabolismo , RNA de Transferência/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Especificidade de Órgãos , Domínios Proteicos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
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