Identification of 'tissue' transglutaminase binding proteins in neural cells committed to apoptosis

Lucia Piredda, Maria Grazia Farrace, Mario L. Lo Bello, Walter Malorni, Gennaro Melino, Raffaele Petruzzelli, Mauro Piacentini

Research output: Contribution to journalArticlepeer-review

Abstract

Overexpression of 'tissue' transglutaminase (tTG) in the human neuroblastoma cells increases spontaneous apoptosis and renders these cells highly susceptible to death induced by various stimuli. We used immunoprecipitation to identify cellular proteins that interact specifically with tTG in SK-N-BE(2)-derived stable transfectants. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis showed that tTG binding proteins have molecular masses of 110, 50, 22, 14, and 12 kDa. Microsequencing and computer search analyses allowed us to identify these polypeptides as the β- tubulin (50 kDa), the histone H2B (14 kDa), and two GST P1-1-truncated forms (22 and 12 kDa). The specificity of the interaction between tTG and these proteins was confirmed by competing tTG binding with purified enzyme and by detecting tTG in immunoprecipitates obtained using β-tubulin or GST P1-1 mAb's. Here we demonstrate that the GST P1-1 acts as an efficient acyl donor as well as acceptor tTG substrate both in cells and in vitro. The tTG- catalyzed polymerization of GST P1-1 leads to its functional inactivation and is competitively inhibited by GSH. By contrast, the tTG-β-tubulin interaction does not result in the cross-linking of this cytoskeletal protein, which suggests that microtubules act as the anchorage site for tTG and GST P1-1 interaction.

Original languageEnglish
Pages (from-to)355-364
Number of pages10
JournalFASEB Journal
Volume13
Issue number2
Publication statusPublished - 1999

Keywords

  • Cell death
  • Chromatin
  • Glutathione
  • Histone H2B
  • Human neuroblastoma cells
  • Microtubules
  • Staurosporine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

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