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Title: Transglutaminase in the life and death of the pancreatic β-cell
Author: Dookie, S.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2010
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Tissue transglutaminase (TG2) is a ubiquitous enzyme that catalyses both the Ca2+-dependent formation of protein cross-links via intermolecular isopeptide bonds, and the Ca2+-independent hydrolysis of GTP. The multifunctional nature of the TG2 protein has been reported in numerous intracellular mechanisms, cell-surface associations, and extracellular matrix (ECM) interactions. In the pancreas, the expression of TG may be fundamental to the insulin-secretion function of β-cells, and associated diabetic disorders. The functional roles of TG2 in the pancreas were investigated in the present study using in vitro models of rat pancreatic insulinoma β-cells (BRIN-BD11), and ex vivo islet of Langerhans models from human, rat, TG2(-/-) knockout mice and their wild-type counterparts. The importance of ECM-associated TG2 in the maintenance of β-cell survival and function was also investigated using an in vitro human urinary bladder carcinoma support matrix (5637 cells). Biochemical analysis of both clonal BRIN-BD11 and islet β-cells showed a thiol-dependent TG2 activity mechanism that was reciprocally regulated by Ca2+ and GTP. Intracellular TG2 cross-linking activity was up-regulated in the presence of glucose and retinoic acid, while cell-surface associated TG2 activity was reduced in the presence of membrane proteases. The in vitro application of irreversible active-site directed TG inhibitors (R281, R283 and BOC-peptide) strongly supported a role for TG2 in the glucose-stimulated insulin secretion function of BRIN-BD11 β-cells and rat islets, with a possible survival role for the enzyme under conditions of β-cell oxidative stress. Further characterisation of the pancreatic β-cell TG led to the discovery of a novel short-form (~60-kDa) TG2 protein expressed in BRIN-BD11, human and mouse islets that was immunoreactive to CUB7402 antibody, and showed GTP-binding potential. Analysis of the BRIN-BD11 proteome using 2-D SDS-PAGE and western blotting exhibited possible phosphorylation of the TG2 protein at ~60-kDa (pI 4-6), with an additional 120-kDa and ~35-kDa protein. Molecular screening using northern blot analysis of BRIN-BD11 mRNA confirmed the presence of two short-form TG2 transcripts at ~2.5-kb and ~1.0-kb in addition to the full length 3.5-kb TG2 transcript, while RT-PCR analysis using C-terminal directed primers revealed a short-form BRIN-BD11 product similar to an alternatively spliced shorter TG2 isoform found in rat brain. Modifications to ECM-associated TG2 in an in vitro 3-day 5637 support matrix demonstrated the importance of this protein in cell adhesion, cell spreading and aggregation in the maintenance of BRIN-BD11 survival and function. Preliminary studies produced an up-regulation of β-cell focal adhesion kinase, and actin-stained focal adhesion points, in response to the TG2-rich support matrices. The 5637 pre-conditioned matrix also supported greater β-cell viability in response to diabetic conditions such as hyperglycaemia, oxidative stress, and hyperlipidaemia. These findings demonstrate that the primary role for TG2 in pancreatic β-cells may be attributed to insulin secretion and related diabetic stress survival mechanisms. This study also reports the discovery of a shortened TG2 isoform unique to pancreatic β-cells that could lend further insight into TG alternative splicing mechanisms as it relates to structure-function complexity and transcriptional regulation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available