Date of Award

7-13-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Nawab Ali

Abstract

Inositol polyphosphates (InsPs) are signaling molecules that regulate various cellular processes including apoptosis. Among enzymes that metabolize InsPs, Multiple Inositol Polyphosphate Phosphatase1 (Minpp1) is the only enzyme located in the endoplasmic reticulum (ER). It dephosphorylates more phosphorylated InsPs to less phosphorylated InsPs in vitro. Changes in cellular InsPs are known to regulate apoptosis. Minpp1 is a member of the histidine acid phosphatase family of proteins with functional resemblance to phytases found in lower organisms. Its physiological function in higher animals is not clear. Besides its role in dephosphorylating InsPs, it has been shown to have mutase activity. This enzyme is also found in non-mammalian species and therefore has stimulated an interest to explore its phylogenic distribution and functional significance. In this project, an attempt has been made to study an evolutionary diversification in its structure in order to understand its physiological relevance in higher organisms by using a Bioinformatics & Computational Biology approach. The study demonstrates the existence of four isoforms of Minpp1with structural variations. Additionally, there exists a diversification in motifs and thus possible functions through evolution. This study supports the evolutionary adaptability of Minpp1, with simpler function in lower organisms to a more complex function in higher life forms. Apoptosis is a fundamental tissue maintenance process crucial for growth and cell survival. Traditionally, mitochondria are considered the primary site for execution of cellular apoptotic signals followed by the nucleus. Very little is known about the role of ER in this process. Emerging evidences suggest that there is an apoptotic signaling cross talk between the ER and mitochondria, and the ER and nucleus. Calcium release due to ER-stress sensitizes mitochondrial membrane permeability. Alternatively, apoptotic signals originating from mitochondria interfere with ER membrane integrity. This inter-organelle signaling appears to activate death signaling molecules to execute apoptosis. Since Minpp1 is an ER luminal enzyme implicated in InsPs metabolism, which in turn regulate apoptosis, changes in its expression under apoptotic conditions was studied and compared with those of ER-stress. These results suggest that Minpp1 might be involved in ER-stress-mediated apoptosis. In order to implicate a role of other ER proteins in apoptosis, a global proteomic approach of 2D-PAGE analysis was taken. Proteins involved in ER-stress-mediated apoptosis are likely to significantly up regulate or down regulate or redistribute to other cellular locations. Protein profiles of ER on 2D gels were found to alter under apoptotic and ER-stress conditions. Differentially expressed proteins were selected and identified by mass spectrometry. These proteins constitute an important group that can be further explored to study their role in ER-stress-mediated apoptosis. This approach has potential to find novel targets for the development of new therapeutic applications to treat cellular dysfunctions. Additionally, in order to understand the physiological role of Minpp1, its interaction with Hsp40, a potential protein partner identified in a yeast 2 hybrid screening, was followed. A computational approach of protein-protein interaction between Minpp1 and Hsp40 was taken. The specificity of interaction was determined using a related protein Hsp70. Based on molecular docking data, the interaction between Minpp1 and Hsp40 was found stronger and more specific than the interaction between Minpp1 and Hsp70. Minpp1/Hsp40 protein-protein interaction was also validated by co-immunoprecipitation. The overall results suggest that Minpp1 function might be regulated by Hsp40 as the two proteins are co-localized in the ER.

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