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Identification of 'tissue' transglutaminase binding proteins in neural cells committed to apoptosis
L Piredda1, M G Farrace, M Lo Bello
1Department of Biology, University of Rome 'Tor Vergata' Rome, Italy.
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 beta-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 beta-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-beta-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.
Insights
Overexpression of tissue transglutaminase (tTG) increases neuroblastoma cell death. tTG interacts with beta-tubulin and GST P1-1, affecting cell function and potentially serving as an anchorage site.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Tissue transglutaminase (tTG) overexpression in neuroblastoma cells promotes apoptosis.
- Understanding tTG's cellular interactions is crucial for targeted therapies.
Purpose of the Study:
- To identify proteins interacting with tTG in neuroblastoma cells.
- To elucidate the functional consequences of these interactions.
Main Methods:
- Immunoprecipitation to capture tTG-binding proteins.
- SDS-PAGE and microsequencing for protein identification.
- In vitro assays to confirm enzymatic activity and interactions.
Main Results:
- Identified beta-tubulin (50 kDa), histone H2B (14 kDa), and GST P1-1 (22 and 12 kDa) as tTG binding partners.
- Demonstrated GST P1-1 as an acyl donor/acceptor substrate for tTG, leading to polymerization and inactivation.
- Showed tTG-beta-tubulin interaction does not cause cross-linking, suggesting microtubules serve as anchorage sites.
Conclusions:
- tTG interacts with specific cellular proteins, including beta-tubulin and GST P1-1, in neuroblastoma cells.
- tTG-mediated GST P1-1 polymerization inactivates the protein, with potential implications for cellular redox balance.
- The tTG-beta-tubulin interaction may facilitate tTG localization and function via microtubule anchorage.