Biochemical and Pharmacological Characterization of the Human Lymphocyte Antigen B-Associated Transcript 5 (BAT5/ABHD16A)

Abstract

BackgroundHuman lymphocyte antigen B-associated transcript 5 (BAT5, also known as ABHD16A) is a poorly characterized 63 kDa protein belonging to the α/β-hydrolase domain (ABHD) containing family of metabolic serine hydrolases. Its natural substrates and biochemical properties are unknown.Methodology/Principal FindingsAmino acid sequence comparison between seven mammalian BAT5 orthologs revealed that the overall primary structure was highly (≥95%) conserved. Activity-based protein profiling (ABPP) confirmed successful generation of catalytically active human (h) and mouse (m) BAT5 in HEK293 cells, enabling further biochemical characterization. A sensitive fluorescent glycerol assay reported hBAT5-mediated hydrolysis of medium-chain saturated (C14∶0), long-chain unsaturated (C18∶1, C18∶2, C20∶4) monoacylglycerols (MAGs) and 15-deoxy-Δ12,14-prostaglandin J2-2-glycerol ester (15d-PGJ2-G). In contrast, hBAT5 possessed only marginal diacylglycerol (DAG), triacylglycerol (TAG), or lysophospholipase activity. The best MAG substrates were 1-linoleylglycerol (1-LG) and 15d-PGJ2-G, both exhibiting low-micromolar Km values. BAT5 had a neutral pH optimum and showed preference for the 1(3)- vs. 2-isomers of MAGs C18∶1, C18∶2 and C20∶4. Inhibitor profiling revealed that β-lactone-based lipase inhibitors were nanomolar inhibitors of hBAT5 activity (palmostatin B > tetrahydrolipstatin > ebelactone A). Moreover, the hormone-sensitive lipase inhibitor C7600 (5-methoxy-3-(4-phenoxyphenyl)-3H-[1], [3], [4]oxadiazol-2-one) was identified as a highly potent inhibitor (IC50 8.3 nM). Phenyl and benzyl substituted analogs of C7600 with increased BAT5 selectivity were synthesized and a preliminary SAR analysis was conducted to obtain initial insights into the active site dimensions.Conclusions/SignificanceThis study provides an initial characterization of BAT5 activity, unveiling the biochemical and pharmacological properties with in vitro substrate preferences and inhibitor profiles. Utilization of glycerolipid substrates and sensitivity to lipase inhibitors suggest that BAT5 is a genuine lipase with preference for long-chain unsaturated MAGs and could in this capacity regulate glycerolipid metabolism in vivo as well. This preliminary SAR data should pave the way towards increasingly potent and BAT5-selective inhibitors.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections – Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

“…We cross-validated the tissue-ABPP findings in gelbased ABPP using homogenates of glioma and control brain. In addition, we used rat cerebellar membranes to facilitate comparison, as previous gel-based ABPP has substantially relied on SH profiling in rodent brain membrane proteomes [14,[19][20][21][22][23][24][25][26][27]. Building on such studies from this and other laboratories [14,[19][20][21][22][23][24][25][26][27], we could identify many of the SH bands based on inhibitor sensitivity and mobility pattern in SDS-PAGE.…”

“…Position of molecular weight markers (in kDa) is indicated on the gel. Based on previous studies from this and other laboratories [14,[19][20][21][22][23][24][25][26][27] many healthy brain-resident SHs were identified, as indicated at left. Note comparable activities of KIAA1363 and LYPLA1/2 doublets (black asterisk) in control brain and glioma.…”

Chemical Methods for Probing Virus–Host Proteomic Interactions

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 and selectivity of the lipase inhibitor palmostatin B. 73 It is plausible that this class of inhibitors exhibiting selectivity towards lipases will find an important use in the ABPP toolkit for studying virus-host cell interactions.…”

The Arabidopsis ABHD11 Mutant Accumulates Polar Lipids in Leaves as a Consequence of Absent Acylhydrolase Activity

“…Vec, Empty vector overexpressed; ABHD11, overexpression of AtABHD11. from various substrates in a 96-well plate (Savinainen et al, 2014). The cellular background activity (both vector and AtABHD11 fraction) was negligible, and the enzyme activity was calculated after normalizing with the background activity.…”

“…700310;Savinainen et al, 2014). Briefly, the enzyme assay was performed by above-mentioned standard conditions, and the released FFA was coupled via three-step enzymatic reaction to produce H 2 O 2 -dependent generation of resorufin and the fluorescence monitored (Ex 530 /Em 590 ) using Thermo Scientific Varioskan Flash Multimode Reader.…”