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Effect of MG-132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes.
Dube, Dipak K; Wang, Jushuo; Fan, Yingli; Dube, Syamalima; Abbott, Lynn; Sanger, Jean M; Channaveerappa, Devika; Darie, Costel C; Poiesz, Bernard J; Sanger, Joseph W.
Afiliação
  • Dube DK; Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Wang J; Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Fan Y; Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Dube S; Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Abbott L; Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Sanger JM; Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Channaveerappa D; Biochemistry and Proteomics Group, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, New York, USA.
  • Darie CC; Biochemistry and Proteomics Group, Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, New York, USA.
  • Poiesz BJ; Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA.
  • Sanger JW; Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, New York, USA.
Cytoskeleton (Hoboken) ; 78(8): 375-390, 2021 08.
Article em En | MEDLINE | ID: mdl-34698442
In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Codorniz / Miofibrilas Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Codorniz / Miofibrilas Tipo de estudo: Prognostic_studies Limite: Animals Idioma: En Ano de publicação: 2021 Tipo de documento: Article