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Investigating the molecular mechanisms of protein deCoAlation

Reference	BB/S009027/1
Principal Investigator / Supervisor	Professor Ivan Gout
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Structural Molecular Biology
Funding type	Research
Value (£)	473,705
Status	Current
Type	Research Grant
Start date	01/06/2019
End date	30/06/2023
Duration	49 months

Abstract

Coenzyme A (CoA) is a key metabolic integrator in mammalian cells. CoA and its thioester derivatives participate in diverse metabolic pathways, regulatory interactions and gene expression. Dysregulation of CoA biosynthesis or CoA derivatives homeostasis has been associated with various human pathologies, including cancer, diabetes and neurodegeneration. We have recently uncovered a novel non-canonical function of CoA, involving covalent modification of cellular proteins in redox regulation and termed it protein CoAlation. The development of new reagents and methodologies allowed us to demonstrate that protein CoAlation is a widespread and reversible post-translational modification which occurs in eukaryotic and prokaryotic cells in response to oxidative and metabolic stress. The vast majority of CoAlated proteins were found to be metabolic enzymes, as well as proteins implicated in stress response and protein synthesis. Protein CoAlation alters the molecular mass, charge and the activity of modified proteins, and prevents them from irreversible sulfhydryl overoxidation. This research proposal will further exploit these recent advances to address fundamental questions about the molecular mechanisms of the CoAlation/deCoAlation cycle in redox regulation. Based on our preliminary data we hypothesise that the removal of CoA from CoAlated proteins is an enzymatically-mediated process, occurring when cells recover from oxidative or metabolic stress. The identification and functional characterisation of these enzymes, which we termed CoAredoxins will be the main focus of this proposal. We will also extend a range of new research tools and methodologies for studying protein CoAlation in redox regulation and signalling. The work will provide critical information for understanding the molecular mechanisms of protein deCoAlation and the role of CoA in balancing oxidative stress, redox state and cellular metabolism in health and disease.

Summary

Coenzyme A (CoA) is essential for all living cells. In 1953, F. Lipmann received a Nobel prize for the discovery of CoA and its importance in intermediary metabolism. Since this landmark discovery, numerous functions of CoA and its derivatives (Acetyl CoA, Malonyl CoA, HMG CoA among others) have been revealed, including their significance in the synthesis and oxidation of fatty acids, ATP production, biosynthesis of cholesterol and acetylcholine, degradation of amino acids and the regulation of gene expression and cellular metabolism via protein acetylation. Dysregulation of CoA biosynthesis or CoA derivatives homoeostasis is associated with various human pathologies, including metabolic disorders, cardiac hypertrophy, cancer and neurodegeneration. Prof Gout's research was central to molecular cloning and characterisation of mammalian CoA synthase, and the identification of mutations in this enzyme associated with an aggressive form of a Parkinson's-like neurodegeneration (NBIA, neurodegeneration with brain iron accumulation). Recently, Prof Gout pioneered a new field of research on protein CoAlation and antioxidant function of CoA. He has initiated and led a high profile international consortium on this emerging and significant area of research. These collaborative efforts were essential for demonstrating that protein CoAlation is a reversible post-translational modification induced in bacteria and mammalian cells by oxidising agents and metabolic stress. In the frame of the established consortium, unique reagents and methodologies have been developed and proved to be critical for identifying CoA-modified proteins in cells and tissues, and revealing a widespread nature of protein CoAlation. Protein CoAlation was shown to regulate the subcellular localisation, enzymatic activity and function of modified proteins. The identification of extensive protein CoAlation in eukaryotic and prokaryotic cells, and the reversible nature of this post-translational modification suggest there should also be enzymes which function to mediate the removal of CoA from modified proteins. We have termed these enzymes CoAredoxins. This research proposal leverages novel findings and newly developed methodologies, and will further advance the field of research on protein CoAlation and define the role of CoA as an important antioxidant in cellular response to oxidative and metabolic stress. Key research questions are aimed at: i) the identification and functional characterisation of CoAredoxins from bacteria and mammalian cells; and (ii) the development of novel research tools and methodologies for studying protein CoAlation in redox regulation and signalling. Answering these questions will reveal the molecular basis of protein deCoAlation and thus inform on the fundamental biology of CoA in cellular metabolism and redox regulation. This work will lay the foundation for delineating the role of protein CoAlation in health and disease.

Impact Summary

The main benefit of the proposed project relates to the advance of knowledge of the role protein CoAlation in redox regulation and antioxidant function of coenzyme A, which will be disseminated through peer-reviewed journals, national and international conferences, and free-to-access websites. The project has the potential to generate a diverse range of research reagents and technical expertise, and we make it freely available in order to stimulate the research in this field. Considering that protein CoAlation is an emerging field of research, we believe that the identification and characterisation of deCoAlation enzymes (CoAredoxins) will advance our knowledge on molecular mechanisms of the CoAlation/deCoAlation cycle in redox regulation. Moreover, the proposed research can also contribute to a better understanding of the antioxidant function of CoA in health and disease, and the development of better treatment for pathologies in which deregulation of CoA biosynthesis and homeostasis is implicated, including diabetes, cancer and neurodegeneration with brain iron accumulation (NBIA). Where possible, we will feed findings from the proposed study into our ongoing drug discovery project on ""Utilising coenzyme A scaffold for the development of Aurora kinase A specific inhibitors", aimed at developing novel anticancer drugs. The proposed work therefore has the potential to boost the development of novel therapeutic drugs in the long-term, as well as fostering positive economic outcomes, considering the rapidly growing global pharmaceutical market for protein kinase inhibitors. A diverse range of techniques, including biochemical and molecular biology techniques, bacterial and baculoviral expression of recombinant proteins, lentiviral overexpression and downregulation, mammalian cell biology, in vitro CoAlation assay, mass spectrometry analysis, generation and characterisation of site-specific CoAlation antibodies will be carried out in the frame of this project. Therefore, it offers an excellent opportunity for the staff involved in this project, including undergraduate and postgraduate students, to gain a valuable experience in modern techniques. In addition, effective time-management, co-ordination and communication skills will therefore be critical for successful completion of this multidisciplinary project.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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