The role of phosphorylation and redox regulation of the brain specific hBCAT proteins in vitro and in neuronal cells

El Hindy, Maya

Abstract

Introduction: The hBCAT proteins have a unique redox active CXXC motif which governs its transamination activity. Recent studies have identified that specific neuronal proteins with either redox activity or functions in cell signalling form interactions with the hBCAT proteins that are disrupted when the environment becoming oxidising. However, how hBCAT functions as an oxidoreductase is unknown or how it compares to other known cellular repair enzymes such as thioredoxin, glutaredoxin or protein disulphide isomerase. Moreover, leucine, a substrate for hBCAT regulates protein synthesis through the mTOR pathway, yet the importance of hBCAT itself in this mechanism remains undetermined.

Aims: This thesis firstly investigated the redox substrates for the hBCAT proteins, and their oxidoreductase activity in comparison to the cellular repair enzymes. The second main aim was to establish if hBCAT can be regulated through phosphorylation both in-vitro and in the neuronal cell line (IMR-32). Finally, in addition to understanding their role as oxidoreductases, the importance of the reactive cysteines in redox binding to neuronal proteins was determined.

Results: These studies demonstrated that both hBCAT isoforms have oxidoreductase and isomerase activity, but of lower activity relative to protein disulphide isomerase. The oxidoreductase activity was dependent on a functional CXXC motif, where in particular S-glutathionylation enhanced the ability of hBCAT to catalyse the folding of proteins. It has been demonstrated that hBCATc is regulated through phosphorylation and this is dependent on the redox environment unlike hBCATm which was only affected in the presence of thiol blocker, NEM. Both isoforms required the CXXC motif to be phosphorylated. Although phosphorylation of hBCATc in neuronal cells was observed, the exact mechanism needs to be elucidated. Finally, these studies have identified putative new partners for hBCAT such as inositol polyphosphate multikinase, GRINL1a upstream protein and parvalbumin. These indicate potential new roles for these proteins in cell division, neurotransmitter signal transduction and mTOR pathway, which have previously not been reported.

Conclusion: The hBCAT proteins have oxidoreductase activity and are involved in a number of metabolic pathways such as cell division, neurotransmitter signal transduction and the mTOR pathway. This implies that these proteins may have a protective role in the cell, similarly to previous studies which have shown that upregulation of the hBCAT proteins is protective. Future work will elucidate further the role these proteins have under normal and pathological conditions.

• Thesis Type: Thesis
• Deposit Date: Feb 14, 2014
• Keywords: branched chain aminotransferase, phosphorylation, isomerase, oxidoreductase
• Public URL: https://uwe-repository.worktribe.com/output/821153
• Additional Information: Additional Information : "The Branched-Chain Aminotransferase Proteins: Novel Redox Chaperones for Protein Disulfide Isomerase–Implications in Alzheimer's Disease" is a copy of an article published in the Antioxidants & Redox Signaling © 2013 [copyright Mary Ann Liebert, Inc.]; Antioxidants & Redox Signaling is available online at: http://online.liebertpub.com.
• Contract Date: Jun 5, 2016