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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
3.
Indian J Med Res ; 143(Supplement): S45-S51, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27748277

RESUMO

BACKGROUND & OBJECTIVES: Number of metastatic lymph nodes has a strong prognostic value in the course of breast cancer treatment, morbidity and mortality. This study was undertaken to determine the association between axillary lymph node metastasis and several variables such as age, tumour size, grade, lymphovascular invasion, oestrogen and progesterone receptor expression and HER2/neu status in patients with breast cancer. METHODS: In this study 426 (with complete information on study variables) patients with breast cancer on treatment during March 2010 to December 2013, were analyzed. TNM (tumour node matastasis) staging was evaluated. The histological grading of tumours was done according to modified Bloom-Richardson Grading System. The immunophenotype of the tumour was determined as the expression of oestrogen (ER) and progesterone (PR) receptors and Her0 2/neu status. Univariate and multivariate analyses were carried out to determine the independent predictors of metastatic lymph node. RESULTS: Among the studied patients, 44.36 per cent (189 of 426) of the patients had nodal metastases. t0 umour histology, tumour grade, size and lympho-vascular invasion were related with node positivity. On univariate analysis, age, menopause, hormone receptor status did not relate with the node metastasis. Age, tumour grade, tumour size, lympho-vascular invasion and HER2/neu expression was likely to be associated with the number of lymph node metastasis. INTERPRETATION & CONCLUSIONS: The lymph node status was associated with clinical stage, tumour grade, tumour histology and HER2/neu status. t0 hese factors may be used for better management of such patients.


Assuntos
Neoplasias da Mama/genética , Linfonodos/patologia , Prognóstico , Receptor ErbB-2/genética , Adulto , Idoso , Axila/patologia , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Índia/epidemiologia , Linfonodos/metabolismo , Metástase Linfática/diagnóstico , Metástase Linfática/patologia , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , Receptores de Progesterona/genética
4.
Nucleic Acids Res ; 44(13): 6409-22, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27131785

RESUMO

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , DNA Mitocondrial/genética , Proteínas de Drosophila/genética , Proteínas Mitocondriais/genética , Ribonuclease P/genética , Animais , Drosophila/genética , Regulação da Expressão Gênica , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , RNA de Transferência/genética , Mutações Sintéticas Letais/genética
5.
Biol Open ; 5(2): 195-203, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26834020

RESUMO

Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. We have previously shown Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1-Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. Here we identify the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, we show Clu interacts with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1-Parkin pathway.

6.
Dis Model Mech ; 8(6): 577-89, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26035866

RESUMO

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD). However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Feminino , Humanos , Proteínas Mitocondriais/metabolismo , Mutação/genética , Fenótipo , Ligação Proteica
7.
J Neurogenet ; 28(3-4): 250-63, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24912380

RESUMO

The horizontal system and vertical system cells of the dipteran optic lobes are well understood regarding their physiology and role in visually guided behavior. Little is known, however, about their development. Drosophila optomotor-blind (omb) is required for the development of the HS/VS cells which are lacking in the adult brain of the In(1)omb[H31] regulatory mutant. We have analyzed the omb regulatory region, required for HS/VS development, for enhancers active in the central nervous system. A 1-kb fragment, ombJb, was identified 114 kb downstream of the omb transcription start site, that could drive expression in much of the presumptive embryonic optic lobe anlage. Expression in these cells is lost in In(1)omb[H31] suggesting that they contain the HS/VS precursor cell(s). We used Laser ablation in the embryonic CNS in order to localize the position of the HS/VS precursor cell(s) in this tissue. An omb-Gal4 enhancer trap line, which showed activity in the optic lobe anlage in a pattern similar to ombJb enhancer, was used to drive GFP expression, thus allowing to focus the Laser beam to the relevant area. We identified a small region in the embryonic brain from which the HS/VS cells are likely to develop. Omb encodes a transcription factor of the T-box family. Since loss of omb disrupts HS/VS cell development, we assume that HS/VS ontogeny is controlled by Omb target genes. As a first step toward their identification, we characterized the Omb DNA-binding specificity.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Lobo Óptico de Animais não Mamíferos/metabolismo , Proteínas com Domínio T/genética , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurópilo/metabolismo , Lobo Óptico de Animais não Mamíferos/citologia , Proteínas com Domínio T/metabolismo
8.
PLoS One ; 8(1): e54283, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23342118

RESUMO

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu's specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu's role in mitochondrial function is not critical during larval development, but is important for pupae and adults.


Assuntos
Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/genética , Larva , Proteínas Nucleares/genética , Fosforilação Oxidativa
9.
Dev Biol ; 343(1-2): 167-77, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20403347

RESUMO

The gene odd paired (opa), a Drosophila homolog of the Zinc finger protein of the cerebellum (Zic) family of mammalian transcription factors, plays roles in embryonic segmentation and development of the adult head. We have determined the preferred DNA binding sequence of Opa by SELEX and shown that it is necessary and sufficient to activate transcription of reporter gene constructs under Opa control in transgenic flies. We have found a related sequence in the enhancer region of an opa-responsive gene, sloppy paired 1. This site also responds to Opa in reporter constructs in vivo. However, nucleotide alterations that abolish the ability of Opa to bind this site in vitro have no effect on the ability of Opa to activate expression from constructs bearing these mutations in vivo. These data suggest that while Opa can function in vivo as a sequence specific transcriptional regulator, it does not require DNA binding for transcriptional activation.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Olho/embriologia , Genes de Insetos , Camundongos , Fatores de Transcrição/genética , Dedos de Zinco
10.
Mol Genet Genomics ; 283(2): 147-56, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20033428

RESUMO

The T-box transcription factors TBX2 and TBX3 are overexpressed in many human cancers raising the need for a thorough understanding of the cellular function of these proteins. In Drosophila, there is one corresponding ortholog, Optomotor-blind (Omb). Currently, only two missense mutations are known for the two human proteins. Making use of the developmental defects caused by inactivation of omb, we have isolated and molecularly characterized four new omb mutations, three of them are missense mutations of amino acids fully conserved in all Tbx proteins. We interpret the functional defects in the framework of the known structure of the human TBX3 protein and provide evidence for loss of Omb DNA-binding activity in all three newly identified missense mutations.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Domínio T/metabolismo , Sequência de Aminoácidos , Animais , Braquiúros/genética , Sequência Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Proteínas com Domínio T/química , Proteínas com Domínio T/genética
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