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Alatawi, Abdullah (2025) An investigation of the activity of α/β-Hydrolase domain-containing 6 (ABHD6) in rat brain. MRes thesis, University of Nottingham.

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Abstract

The endocannabinoid system plays a role in various physiological processes, with 2-arachidonoylglycerol (2-AG) and anandamide (AEA) as key signalling molecules. Within this system α/β-Hydrolase Domain-Containing 6 (ABHD6) and α/β-Hydrolase Domain-Containing 12 (ABHD12) regulate 2-AG hydrolysis while also metabolizing other endogenous lipid substrates. ABHD6 hydrolyzes multiple lipid classes, including lysophospholipids, bis(monoacylglycero)phosphate (BMP), and diacylglycerol (DAG), while ABHD12 plays a central role in the metabolism of lysophosphatidylserine (Lyso-PS). Given their involvement in diverse lipid signaling pathways, this thesis aims to investigate the activity of ABHD6 in rat brain tissue using a recently developed spectrophotometric assay based on 4-methylumbelliferylheptanoate (4-MUH) hydrolysis and to validate the selectivity and potency of ABHD6 and ABHD12 inhibitors through the Activity-Based Protein Profiling (ABPP) technique.

This study demonstrates that while the 4-MUH assay was effective in recombinant systems, complete inhibition of 4-MUH hydrolysis in rat brain tissue was not achieved, even with the selective ABHD6 inhibitor KT203. At the highest concentration tested (10 µM), KT203 inhibited 4-MUH hydrolysis but failed to fully suppress enzyme activity, with over 50% of hydrolysis still remaining in both the soluble and particulate fractions. This suggests that other serine hydrolases contribute to 4-MUH hydrolysis in rat brain tissue, limiting the specificity of this assay for measuring ABHD6 activity. In contrast, the ABPP approach allowed for the simultaneous assessment of multiple enzymes, including ABHD6 and ABHD12. Using the probes MB064 and FP-rhodamine, analysis of ABPP gels identified ABHD6 activity and confirmed KT203’s selectivity for ABHD6. Moreover, this method enabled the profiling of ABHD12 and confirmed DO264’s selectivity as an ABHD12 inhibitor. These results suggest that while the 4-MUH assay can effectively measure ABHD6 in recombinant systems, ABPP offers greater reliability for profiling multiple enzymes in complex proteomes.

Item Type:	Thesis (University of Nottingham only) (MRes)
Supervisors:	Alexander, Stephen Roberts, Richard
Keywords:	Endocannabinoid system; ABHD6; 4-methylumbelliferylheptanoate hydrolysis; Activity-Based Protein Profiling Technique; Brain tissue
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	80632
Depositing User:	Alatawi, abdullah
Date Deposited:	24 Jul 2025 04:40
Last Modified:	24 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/80632

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