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).

Mutations in HNF1A cause Maturity Onset Diabetes of the Young type 3, the second most frequent form of diabetes caused by single gene mutation. We generated human stem cell-derived pancreatic endocrine cells with clinically pathogenic mutations in HNF1A and show that HNF1A deficiency impairs endocrine cell fate, insulin granule maturation and the secretion of insulin in response to glucose. Single-cell RNA sequencing reveals that HNF1A orchestrates a network of genes involved in β-cell fate, granule maturation, glucose metabolism, calcium ion binding and hormone exocytosis. In both patients and stem cell-derived β-cells, HNF1A deficiency altered the stoichiometry of secreted insulin to c-peptide. Sulfonylurea, used in the treatment of these patients, restored both insulin secretion and stoichiometry. The uncoupling of insulin and c-peptide secretion as described here questions the common practice of using c-peptide as a proxy to evaluate β-cell function. We also demonstrate that correction of the HNF1A mutations restores function, providing a path to cell-based replacement therapy.