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Mahmood, Nada (2018) Molecular and functional characterization of endocannabinoid hydrolases. PhD thesis, University of Nottingham.

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Abstract

Background: It is now widely accepted that cannabinoids (whether synthetic or endogenous ligands) have multiple physiological, psychological and pathological effects. One area of research is investigating how to increase endogenous cannabinoid levels for potential therapeutic benefit, principally by pharmacological inhibition. However, many enzymes within the endocannabinoid system are lacking pharmacological tools such as inhibitors and/or defined substrate/s. Therefore, the development of high throughput screening assays for compound libraries will pave the way for investigation of the biochemical and pharmacological properties of these enzymes and will open the door for further in vivo studies.

Monoacylglycerols, are subjected to hydrolysis by multiple lipases with monoacylglycerol lipase (MAGL) being the principle hydrolysing enzyme in the brain, alongside ‘minority’ enzymes, such as ABHD6 and ABHD12 (Blankman et al., 2007). ABHD6 was recently described as having a potential role in 2-arachidonyl glycerol hydrolysis and its characterization is crucial for understanding and exploitation of endocannabinoid signalling pathways. This project aimed to identify the biochemical characteristics and the cellular and subcellular distribution of these hydrolysing enzymes (particularly ABHD6 and MAGL) and to generate potential high throughput screening assays (HTS).

Methods: A radiometric-based enzyme assay (using [3H]-2-oleoylglycerol as a substrate) was used to measure enzyme activity in rat tissues and cell lines using the reported selective inhibitors for MAGL and ABHD6. Cloning of human recombinant tagged and un-tagged versions of enzymes was done to assess functional activity and sub-cellular distribution of these enzymes. Lentiviral construct expressing ABHD6 was made to allow expression in primary cells. Expression of ABHD6, MAGL and X1MAGL (a splice variant for MAGL in rat) mRNA was determined by quantitative RT-PCR. Immunoblotting and activity based protein profile of over-expressed enzymes in host cells (HEK293) were conducted. A number of colorimetric and fluorescent esterase assays were set up and validated for high through screening of enzymes. Among them, the 4-Methylumbelliferyl heptanoate (MUH) assay was extensively studied as an ABHD6 in vitro assay.

Results: The hydrolysis of 2OG in rat neural tissues appears to be mediated predominantly through MAGL and an as yet unidentified lipase/s, with little contribution from ABHD6. In contrast, high levels of ABHD6 activity were detected in rat intestines. Two isoforms of human MAGL were successfully cloned and characterized. Results for mRNA expression indicated both MAGL and ABHD6 were expressed in all of tissues tested. Notably, ABHD6 showed high level of mRNA expression in rat intestines in comparison to MAGL. Immuno-staining of flag-tagged ABHD6 showed significant cytoplasmic distribution of ABHD6. In addition, nuclear membrane distribution was evident in both transfected and primary cells. MUH hydrolytic assay results proved to be useful for ABHD6 modulators screening in recombinant systems.

Conclusion: The findings from this piece of work provide insight into the functional activities of hydrolase enzymes in a number of rat tissues and cell lines. It also provides a further step in the understanding of cellular and subcellular localization of ABHD6 and MAGLs. MUH assay offers the opportunity for the discovery of a new range of selective and potent ABHD6 inhibitors.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Alexander, Stephen Bennett, Andrew Chapman, Victoria
Keywords:	Endocannabinoids; Hydrolase enzymes; ABHD6
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	55556
Depositing User:	Mahmood, Nada
Date Deposited:	12 Apr 2019 13:14
Last Modified:	13 Dec 2020 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/55556

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