Functional Characterisation of the Serpin raPIT5a in Neuroendocrine Cells
Abstract (Summary)Restricted Item. Print thesis available in the University of Auckland Library or available through Inter-Library Loan. The serine Proteose inhibitors (serpins) ore a diverse and abundant protein superfamily fundamental to the maintenance of proteolytic homeostasis in virtually all living systems. While the extracellular roles of this highly influential class of protease inhibitors are well defined, few physiological targets or functions have been identified for intracellular serpins-most of which belong to the unique ov-serpin subfamily. Our laboratory has cloned a member of this subfamily, termed raPIT5a, from the rat pituitary gland. The aims of this thesis were to identify intracellular enzyme targets and functions(s) for raPIT5a in neuroendocrine cells. In earlier work I demonstrated that raPIT5a was able to form a stable inhibitory complex with the serine protease granzyme B, characteristic of serpin:protease interaction. To determine if granzyme B-like proteases were expressed in neuroendocrine cells, the rat pituitary gland and a rat phaeochromocytoma (PC12) cell line were screened by reverse transcription PCR using several granzyme-specific primer sets. This identified expression of the rat homologue of granzyme B, rat natural killer protein-1 (RNKP-1), and a novel highly similar granzyme sequence termed rat granzme-like protein-3L (RGP-3L). A homologue of RGP-3L, termed RGP-3L variant (RGP-3LV) was also identified in PC12 cells. These results suggested raPIT5a may regulate the activity of granzyme B-like enzymes expressed in endocrine tissues. To identify other potential enzyme targets for raPlT5a I investigated application of the yeast low-hybrid genetic screen. To establish the efficacy of this approach I conducted proof-of-principle experiments using RNKP-1 as a model target. While compatible raPIt5a and RNKP-l fusion proteins were co-expressed, no interaction was detected. An inactive mutant human granzme B fusion protein in which the active site had been altered to potentially stabilise any substrate interaction was also co-expressed with raPIT5a, but again no interaction was detected. In light of these results no screening of neuroendocrine cDNA libraries was attempted. To investigate the function of raPIT5a, PC12 cell lines overexpressing raPIT5a were established. As serpins closely related to raPIT5a have been shown to inhibit granzyme B and caspase-1, two proteases involved in the cell death pathway, I investigated the involvement of raPIT5a in the process of cell termination. I found overexpression of raPIT5a increased cell viability following insult with neurotoxin 6-hydroxydopamine and the reactive oxygen species (ROS) hydrogen peroxide. I used fluorogenic DNA stains to identify the mode of cell death induced by these insults, with necrotic cell death observed following H2O2 treatment significantly reduced in the raPIT5a overexpressing cell lines. These results suggested that raPIT5a may function to inhibit ROS-induced cell death in neuroendocrine cells. To further investigate a role for raPIT5a in cell death in vivo, expression of the serpin was monitored following severe hypoxic-ischaemic (HI) injury in a rat brain model. Very low levels of raPIT5a were observed previously in the adult rat brain. However, following HI-insult raPIT5a expression was shown to increase, most notably in the hippocampus, with expression restricted primarily to the cytoplasm of cells displaying necrotic cell morphology. As raPIT5a was demonstrated to limit necrotic cell death in vitro, a speculative neuroprotective role for the serpin was proposed. To facilitate further definition of this role and identify enzymes regulated by raPIT5a to reduce neuronal cell death, recombinant raPIT5a a protein was expressed with a polyhistidine tag in the yeast Pichia pastoris and purified by metal chelate affinity chromatography. Based on the research presented in this thesis two potential functions for raPIT5a are proposed; regulation of granzyme activity in endocrine tissue, and regulation of ROS-induced cell death, with raPIT5a upregulated in the brain following insult to limit necrosis and prevent widespread cell loss. Further research into these roles will not only provide insight into raPIT5a function, but will also shed light on the complex pathways leading to cell death and the poorly defined role of intracellular serpins in neuroendocrine tissue.
School Location:New Zealand
Source Type:Master's Thesis
Date of Publication:01/01/2002