Reversible modification of methionine as a mechanism to regualte protein function in the mitochondrion and secretory pathway
Lead Research Organisation:
University of Glasgow
Department Name: College of Medical, Veterinary, Life Sci
Abstract
The function of cells and tissues is often regulated by proteins that can be switched on or off by their reversible modification. These cellular function include critical processes such as cell-division, metabolism and death so it is important that we understand how these reversible modifications occur. This project aims to study one type of poorly characterised modification, called methionine oxidation, which can occur during conditions of cell stress. Specifically we will gain detailed information on the ability of a specific enzyme to catalyse both the oxidation and reduction of proteins - essentially being able to switch on and off protein function. Hence, the research has the potential to unravel a key mechanism for regulating cellular function during normal physiology and during stress.
To understand this enzyme more fully, we will determine its 3-dimensional structure during its catalytic cycle and in that way obtain a detailed understanding of how it works. We will also develop new chemical probes which will enable us to follow its enzymatic activity within live cells providing unprecedented detail on its location and activity under normal and stress conditions. Finally, we will use our knowledge of the enzyme's mechanism to identify the substrates of the enzyme, in other words, the proteins it is able to turn on and off. Using this information we will understand how, where and when the enzyme works and unravel the cellular functions that are controlled by its activity.
To understand this enzyme more fully, we will determine its 3-dimensional structure during its catalytic cycle and in that way obtain a detailed understanding of how it works. We will also develop new chemical probes which will enable us to follow its enzymatic activity within live cells providing unprecedented detail on its location and activity under normal and stress conditions. Finally, we will use our knowledge of the enzyme's mechanism to identify the substrates of the enzyme, in other words, the proteins it is able to turn on and off. Using this information we will understand how, where and when the enzyme works and unravel the cellular functions that are controlled by its activity.
Technical Summary
Redox reactions involving the reversible modification of proteins can give rise to allosteric regulation of proteins - either to control their enzymatic activity or their ability to act as signalling molecules. The focus of much current research is on the reversible modification of thiol groups, however, there are now increasing examples where the oxidation-reduction of methionine side chains provides an alternate mechanism of regulating protein function.
This project will capitalise on our recent discovery that a methionine sulfoxide reductase (MsrB3), localised to the ER or mitochondria, can act as an oxidase as well as a reductase. This insight opens up the possibility that MsrB3's role may be to reversibly modify substrates, thereby indicating a role in the regulation of protein function. As a consequence, the identification of its physiological substrates is important as is our ability to understand more about the mechanism of action to indicate how and when it can act as an oxidase or reductase. As the overexpression of MsrB3 increases cellular resistance to oxidative stress it is highly likely to act as an antioxidant during normal physiology and during specific diseases and ageing. This project aims to increase our understanding of MsrB3 by determining its mechanism of action to provide critical insight into its cellular regulation, developing novel chemical probes to evaluate its intracellular activity and determining its repertoire of client proteins to define its role in the regulation of specific protein function.
This project will capitalise on our recent discovery that a methionine sulfoxide reductase (MsrB3), localised to the ER or mitochondria, can act as an oxidase as well as a reductase. This insight opens up the possibility that MsrB3's role may be to reversibly modify substrates, thereby indicating a role in the regulation of protein function. As a consequence, the identification of its physiological substrates is important as is our ability to understand more about the mechanism of action to indicate how and when it can act as an oxidase or reductase. As the overexpression of MsrB3 increases cellular resistance to oxidative stress it is highly likely to act as an antioxidant during normal physiology and during specific diseases and ageing. This project aims to increase our understanding of MsrB3 by determining its mechanism of action to provide critical insight into its cellular regulation, developing novel chemical probes to evaluate its intracellular activity and determining its repertoire of client proteins to define its role in the regulation of specific protein function.
Planned Impact
The main users who would benefit from this work include researchers interested in investigating how the reversible modification of methionine regulates protein function and how the cell responds to oxidative stress. It is known that a nucleotide exchange factor for one of the major cellular chaperones, Hsp70, has been shown to be regulated by methionine oxidation. Hence, the ER-localised protein Grp170 which regulates the ER Hsp70, BiP, may well be a client for MsrB3. If this proves to be the case then this discovery would impact on companies interested in biopharmaceutical production enabling them to rationally engineer cells to maximise protein folding and production from mammalian cells. Hence any discoveries made will have a very broad and significant impact of the research community.
The major benefit for users will be from the knowledge gained during the project. However, we will make available any cell-lines, DNA constructs or antibodies created during the course of this project. Knowing how the cell regulates the reversible modification of methionine in proteins, will allow users to consider how the process may be manipulated during disease progression and aging.
The discovery of Ero1 and its requirement for oxygen as an electron acceptor has led to an appreciation of the role of the ER in generating reactive oxygen species. A link between methionine oxidation and protein function in the ER will allow a greater understanding of the consequence of oxidative stress on cellular physiology in the context of protein folding and secretion. In summary, the understanding of a fundamental cellular process may have wide benefits for not only greater understanding but also direct applications for users.
The major benefit for users will be from the knowledge gained during the project. However, we will make available any cell-lines, DNA constructs or antibodies created during the course of this project. Knowing how the cell regulates the reversible modification of methionine in proteins, will allow users to consider how the process may be manipulated during disease progression and aging.
The discovery of Ero1 and its requirement for oxygen as an electron acceptor has led to an appreciation of the role of the ER in generating reactive oxygen species. A link between methionine oxidation and protein function in the ER will allow a greater understanding of the consequence of oxidative stress on cellular physiology in the context of protein folding and secretion. In summary, the understanding of a fundamental cellular process may have wide benefits for not only greater understanding but also direct applications for users.
