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1.
Biochim Biophys Acta ; 1862(9): 1558-69, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27240544

RESUMO

Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.


Assuntos
Ataxina-2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Arsenitos/toxicidade , Ataxina-2/antagonistas & inibidores , Ataxina-2/genética , Linhagem Celular Tumoral , Células Cultivadas , Fatores de Iniciação em Eucariotos/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteína S6 Ribossômica/metabolismo , Inanição/genética , Inanição/metabolismo , Estresse Fisiológico
2.
Cell Signal ; 20(10): 1725-39, 2008 10.
Artigo em Inglês | MEDLINE | ID: mdl-18602463

RESUMO

Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.


Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose , Receptores ErbB/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Aciltransferases/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Ataxinas , Encéfalo/metabolismo , Catálise , Linhagem Celular , Membrana Celular/enzimologia , Retículo Endoplasmático/enzimologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/química , Prolina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Proto-Oncogênicas c-cbl/metabolismo
3.
Neurol Res Pract ; 1: 22, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-33324888

RESUMO

BACKGROUND: Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells and selective loss of neurons in the brainstem. This neurodegenerative disorder is caused by the expansion of a polyglutamine domain in ataxin-2. Ataxin-2 is composed of 1312 amino acids, has a predicted molecular weight of 150-kDa and is widely expressed in neuronal and non-neuronal tissues. To date, the putative functions of ataxin-2 on mRNA translation and endocytosis remain ill-defined. Differential splicing with a lack of exons 10 and 21 was described in humans, and additional splicing of exon 11 in mice. In this study, we observed that the molecular size of transfected full-length wild-type ataxin-2 (22 glutamines) is different from endogenous ataxin-2 and that this variation could not be explained by the previously published splice variants alone. METHODS: Quantitative immunoblots and qualitative reverse-transcriptase polymerase-chain-reaction (RT-PCR) were used to characterize isoform variants, before sequencing was employed for validation. RESULTS: We report the characterization of further splice variants of ataxin-2 in different human cell lines and in mouse and human brain. Using RT-PCR from cell lines HeLa, HEK293 and COS-7 throughout the open reading frame of ataxin-2 together with PCR-sequencing, we found novel splice variants lacking exon 12 and exon 24. These findings were corroborated in murine and human brain. The splice variants were also found in human skin fibroblasts from SCA2 patients and controls, indicating that the polyglutamine expansion does not abolish the splicing. CONCLUSIONS: Given that Ataxin-2 interacts with crucial splice modulators such as TDP-43 and modulates the risk of Amyotrophic Lateral Sclerosis, its own splice isoforms may become relevant in brain tissue to monitor the RNA processing during disease progression and neuroprotective therapy.

4.
Mol Cell Endocrinol ; 372(1-2): 42-8, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23523567

RESUMO

The FOXL2(C134W) mutation has been identified in virtually all adult granulosa cell tumors (GCTs). Here we show that the exogenous FOXL2 expression is necessary for GDF-9 stimulation of follistatin transcription in the human GCT cell line, COV434 that lacks endogenous FOXL2 expression. Interestingly, in the presence of Smad3 co-expression, FOXL2(C134W) negated GDF-9 stimulation of follistatin transcription. However, mutation of the Smad binding element (SBE) located in the intronic enhancer elements in the follistatin gene restored normal FOXL2 activity to FOXL2(C134W), thus the altered activity of FOXL2(C134W) is dependent on the ability of Smad3 to directly bind the SBE. Mutation of the FOXL2 binding element (FBE) or the FBE and SBE completely prevented GDF-9 activity, suggesting that the FBE is essential for GDF-9 stimulation in COV434. Overall, our study supports the view that altered interaction of FOXL2(C134W) with co-factors may underlie the pathogenesis of this mutation in GCTs.


Assuntos
Folistatina/genética , Fatores de Transcrição Forkhead/genética , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Proteína Smad3/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Folistatina/metabolismo , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Tumor de Células da Granulosa , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Ovário/metabolismo , Ovário/patologia , Regiões Promotoras Genéticas , Ratos , Transcrição Gênica , Ativação Transcricional
5.
Mol Cell Endocrinol ; 372(1-2): 57-64, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23567549

RESUMO

A single somatic FOXL2 mutation (FOXL2(C134W)) was identified in almost all granulosa cell tumor (GCT) patients. In the pituitary, FOXL2 and Smad3 coordinately regulate activin stimulation of follistatin transcription. We explored whether a similar regulation occurs in the ovary, and whether FOXL2(C134W) has altered activity. We show that in primary granulosa cells, GDF-9 and activin increase Smad3-mediated follistatin transcription. In contrast to findings in the pituitary, FOXL2 negatively regulates GDF-9 and activin-stimulated follistatin transcription in the ovary. Knockdown of endogenous FOXL2 confirmed this inhibitory role. FOXL2(C134W) displayed enhanced inhibitory activity, completely ablating GDF-9 and activin-induced follistatin transcription. GDF-9 and activin activity was lost when either the smad binding element or the forkhead binding element were mutated, indicating that both sites are required for Smad3 actions. This study highlights that FOXL2 negatively regulates follistatin expression within the ovary, and that the pathogenesis of FOXL2(C134W) may involve an altered interaction with Smad3.


Assuntos
Ativinas/fisiologia , Folistatina/genética , Fatores de Transcrição Forkhead/genética , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Transcrição Gênica , Animais , Células Cultivadas , Feminino , Folistatina/metabolismo , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Tumor de Células da Granulosa , Humanos , Mutação de Sentido Incorreto , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína Smad3/metabolismo , Ativação Transcricional
6.
J Mol Neurosci ; 51(1): 68-81, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23335000

RESUMO

Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.


Assuntos
Proteína Adaptadora GRB2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas ras/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Animais , Ataxinas , Sítios de Ligação , Proliferação de Células , Endocitose , Fibroblastos/metabolismo , Proteína Adaptadora GRB2/genética , Células HEK293 , Humanos , MAP Quinase Quinase 1/metabolismo , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fosforilação , Ligação Proteica
7.
Exp Neurol ; 215(1): 110-8, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18973756

RESUMO

Ataxin-2 is a novel protein, normally with a domain of 22 consecutive glutamine (Q) residues, which may expand beyond a threshold of (Q)(32), causing a neurodegenerative disease named Spinocerebellar ataxia type 2 (SCA2). To obtain clues about the functions of ataxin-2, we used fluorescence microscopy and centrifugation fractionation analyses. Immunocytochemical detection in non-neuronal and neuronal cells showed endogenous and transfected ataxin-2 distributed throughout the cytoplasm, with perinuclear preference and a granular appearance. Triple-labelling and confocal microscopy demonstrated co-localisation with the endoplasmic reticulum (ER) markers calreticulin, calnexin and CFP-ER. The pathogenic form of ataxin-2 with an expanded polyQ domain showed the same distribution pattern. Subcellular fractionation of mouse brain homogenates showed endogenous ataxin-2 associated with rough ER (rER) membranes, in a manner dependent on RNA, salt and phosphorylation. Our data are in agreement with recent findings that ataxin-2 directly interacts with poly(A)-binding protein (PABP), thus associating with polyribosomes under normal conditions and being recruited to stress granules under environmental stress. These data, in conjunction with the presence of Lsm domains within ataxin-2, suggest that ataxin-2 is involved in the processing of mRNA and/or the regulation of translation.


Assuntos
Retículo Endoplasmático Rugoso/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Animais , Ataxinas , Calnexina/metabolismo , Calreticulina/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Embrião de Mamíferos , Proteínas da Matriz do Complexo de Golgi , Hipocampo/citologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Transfecção/métodos , Proteínas de Transporte Vesicular/metabolismo
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