Gene_locus Report for: human-ABHD3

Heteroatom-rich organoboron compounds have attracted attention as modulators of enzyme function. Driven by the unmet need to develop chemoselective access to boron chemotypes, we report herein the synthesis of alpha- and beta-aminocyano(MIDA)boronates from borylated carbonyl compounds. Activity-based protein profiling of the resulting beta-aminoboronic acids furnishes selective and cell-active inhibitors of the (ox)lipid-metabolizing enzyme alpha/beta-hydrolase domain 3 (ABHD3). The most potent compound displays nanomolar in vitro and in situ IC50 values and fully inhibits ABHD3 activity in human cells with no detectable cross-reactivity against other serine hydrolases. These findings demonstrate that synthetic methods that enhance the heteroatom diversity of boron-containing molecules within a limited set of scaffolds accelerate the discovery of chemical probes of human enzymes.

Genome sequencing efforts have identified many uncharacterized lipase/esterase enzymes that have potential to be drug targets for metabolic diseases such as obesity, diabetes, and atherosclerosis. However, sequence information and associated structural predictions provide only a loose framework for linking enzyme function to disease risk. We are now confronted with the challenge of functionally annotating a large number of uncharacterized lipases, with the goal of generating new therapies for metabolic diseases. This daunting challenge involves gathering not only sequence-driven predictions, but also more importantly structural, biochemical (substrates and products), and physiological data. At the center of such drug discovery efforts are accurately identifying physiologically relevant substrates and products of individual lipases, and determining whether newly identified substrates/products can modulate disease in appropriate preclinical animal model systems. This review describes the importance of coupling in vivo metabolite profiling to in vitro enzymology as a powerful means to assign lipase function in disease specific contexts using animal models. In particular, we highlight recent examples using this multidisciplinary approach to functionally annotate genes within the alpha/beta hydrolase fold domain (ABHD) family of enzymes. These new discoveries within the ABHD enzyme family serve as powerful examples of linking novel lipase function to human disease. This article is part of a Special Issue entitled Tools to study lipid functions.

All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein alpha/beta-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.

Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.

Heteroatom-rich organoboron compounds have attracted attention as modulators of enzyme function. Driven by the unmet need to develop chemoselective access to boron chemotypes, we report herein the synthesis of alpha- and beta-aminocyano(MIDA)boronates from borylated carbonyl compounds. Activity-based protein profiling of the resulting beta-aminoboronic acids furnishes selective and cell-active inhibitors of the (ox)lipid-metabolizing enzyme alpha/beta-hydrolase domain 3 (ABHD3). The most potent compound displays nanomolar in vitro and in situ IC50 values and fully inhibits ABHD3 activity in human cells with no detectable cross-reactivity against other serine hydrolases. These findings demonstrate that synthetic methods that enhance the heteroatom diversity of boron-containing molecules within a limited set of scaffolds accelerate the discovery of chemical probes of human enzymes.

Genome sequencing efforts have identified many uncharacterized lipase/esterase enzymes that have potential to be drug targets for metabolic diseases such as obesity, diabetes, and atherosclerosis. However, sequence information and associated structural predictions provide only a loose framework for linking enzyme function to disease risk. We are now confronted with the challenge of functionally annotating a large number of uncharacterized lipases, with the goal of generating new therapies for metabolic diseases. This daunting challenge involves gathering not only sequence-driven predictions, but also more importantly structural, biochemical (substrates and products), and physiological data. At the center of such drug discovery efforts are accurately identifying physiologically relevant substrates and products of individual lipases, and determining whether newly identified substrates/products can modulate disease in appropriate preclinical animal model systems. This review describes the importance of coupling in vivo metabolite profiling to in vitro enzymology as a powerful means to assign lipase function in disease specific contexts using animal models. In particular, we highlight recent examples using this multidisciplinary approach to functionally annotate genes within the alpha/beta hydrolase fold domain (ABHD) family of enzymes. These new discoveries within the ABHD enzyme family serve as powerful examples of linking novel lipase function to human disease. This article is part of a Special Issue entitled Tools to study lipid functions.

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N=4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value=9.88x10(-204)) and 10 loci for sphingolipids (smallest P-value=3.10x10(-57)). After a correction for multiple comparisons (P-value<2.2x10(-9)), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.

All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein alpha/beta-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.