published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

The human MAL2 protein has been demonstrated to regulate secretion in kidney epithelial cells in a lipid raft-dependent manner.  Further, MAL2 interacts with TPD52, a protein that promotes secretion in rat pancreatic acinar cells.  Two MAL2 homologues have recently been identified within rat pancreatic β-cells: MAL2A and MAL2B.  This suggested that MAL2 and TPD52 might interact with each other to coordinate secretion within both human kidney epithelial cells and rat pancreatic β-cells. I report here that both <i>MAL2A </i>and <i>MAL2B </i>are expressed within rat pancreatic β-cells.  Further, the expression of <i>MAL2B </i>appears to be tightly regulated as sequences within the 5’ UTR of the <i>MAL2B </i>transcript inhibit its translation.  Both MAL2A and MAL2B are associated with lipid rafts.  Attempts were made to identify interactions between TPD52 and the MAL2 proteins.  GST pull-down assays indicated that TPD52 interacts with both MAL2A and MAL2B.  This is consistent with previous observations and suggests that TPD52 does not interact with MAL2A or MAL2B via their N-termini, which are distinct between each MAL2 protein.  However, a novel <i>Xenopus </i>egg extract interaction assay failed to detect an interaction between TPD52 and the MAL2 proteins.  Further, immunohistochemistry indicated that TPD52 did not co-localise and either MAL2A or MAL2B within pancreatic β-cells.  These findings indicate that TPD52 and the MAL2 proteins might participate in overlapping secretion pathways.  Further, these data suggest that any interaction between TPD52 and the MAL2 proteins might be weak or transient.  Finally, initial attempts were made to reduce <i>TPD52 </i>expression within β-cells via RNA silencing in order to elucidate the role of TPD52 in directing secretion.  RNA silencing will also be used to examine the role of MAL2A in coordinating secretion.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2008.

Thesis (Ph. D.)--University of Louisville, 2003. / Department of Pharmacology and Toxicology. Vita. "December 2003." Includes bibliographical references (leaves 136-154).

Tumour protein D52 is hypothesised to be involved in regulated secretion in the pancreatic acinar cell, indicated by rapid phosphorylation in response to secretagogue stimulation. The phosphorylation status of TPD52 in response to glucose stimulation in the pancreatic β cell was analysed by an <i>in vitro</i> method involving the incorporation of  ³²P-ATP into the TPD52 protein. Limitations of the system prevented the full confirmation of the rapid phosphorylation of TPD52 in response to glucose stimulation, however preliminary data suggested this was likely to occur. Bio-informatic phosphorylation site prediction was used and we hypothesised that CaMKII was the key enzyme phosphorylating TPD52 in the β cell at the serine phosphorylation site within the motif SPTFKsFEEKV. Proteomic analysis of the phosphorylated TPD52 isoform confirmed the novel identification of the motif phosphorylated by CaMKII. To determine the effect of TPD52 on regulated secretion we reduced TPD52 RNA levels using vector based siRNA. Two stable knockdown cell lines were created  showing a 50 % and 80 % reduction in RNA levels. No difference was observed in glucose stimulated insulin secretion between TPD52 knockdown and control cells. Patch clamp experiments also showed no significant difference in capacitance changes following depolarisation between knockdown and control clones. Quantification of insulin granule number and size from TEM pictures confirmed a high level of inter-clonal variation. Focus shifted to explore the previously published protein interaction between TPD52 and MAL2 as both proteins have been identified within the β cell. The original interaction between MAL2 and TPD52 was confirmed; however, when natural isoforms with shorter N-terminal regions were substituted for TPD52 and MAL2 the interaction was diminished slightly. This suggests that the N-terminal regions are not integral to the interaction between the proteins, but are perhaps required for stability of the complex.

No description available.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xxiv, 185 p.; also includes graphics. Includes bibliographical references (p. 164-185). Available online via OhioLINK's ETD Center

Thesis (Ph. D.)--University of Louisville, 2004. / Department of Pharmacology and Toxicology. Vita. "August 2004." Includes bibliographical references (leaves 124-142).

Type 2 diabetes (T2D) is a growing health-care and economic burden. Obesity is a risk factor for developing T2D, but the underlying molecular mechanisms are not well understood. However, mechanisms such as lipotoxicity, endoplasmic reticulum stress and inflammation are becoming increasingly well-recognised in obesity, and may underlie the development and progression of T2D. A central player in these mechanisms is Protein Kinase R (PKR), proposed to have a role within nutrient- and pathogen-sensing pathways, and is activated by ER stress and lipotoxicity. A small molecule inhibitor Compound-16, adenoviral vectors and RNAi techniques in BRIN-BD11 rodent pancreatic β-cells, were used to demonstrate that PKR knockdown affords significant protection against palmitate-induced cell death. Furthermore, PKR knockdown potentiates palmitoleate cytoprotection during lipotoxicity, suggesting the cytotoxic and cytoprotective actions of long-chain fatty acid species may function via the PKR signalling pathway. The use of a novel 1.1B4 human pancreatic β-cell line has shown that important differences exist between human and rodent cell responses to fatty acids in vitro. In 1.1B4 cells, long-chain saturated and monounsaturated fatty acids do not provide increasing protection as their chain-length increases, in contrast to rodent cell models. Furthermore, methyl-saturated fatty acid species are well tolerated, and methyl-monounsaturated fatty acids are cytoprotective to 1.1B4 β-cells. TXNIP overexpression in an INS-TXNIP β-cell model has a proapoptotic role in conditions of glucotoxicity, but not glucolipotoxicity. Furthermore, in this cell model, succinate is cytoprotective against glucotoxicity, but not glucolipotoxicity. By contrast in 1.1B4 β-cells, succinate significantly protects against apoptosis induced by both glucotoxic and glucolipotoxic conditions. Chronic inflammation has been implicated in the development and progression of T2D. At the centre of this response is the pro-inflammatory cytokine IL-1β. The cellular origin of IL-1β is unclear, but IL-1β secretion has been linked to activation of the NLRP3 inflammasome, recently implicated in pancreatic β-cell death in T2D. Results suggest that IL-1β is secreted by INS-TXNIP and 1.1B4 pancreatic β-cells under lipotoxic conditions, thus offering a potential role for targeted IL-1β therapy in T2D.