Gene_locus Report for: mouse-Q8BLF1

Organophosphorus (OP) insecticides and chemical warfare agents act primarily by inhibiting acetylcholinesterase. There are many secondary targets for OP toxicants as observed for example with the major insecticide chlorpyrifos and its bioactivated metabolite chlorpyrifos oxon (CPO). Therefore, it was surprising that the predominant mouse brain protein labeled in vitro by [(3)H-ethyl]CPO (1 nM) (designated CPO-binding protein or CPO-BP) is not one of these known OP toxicant targets. CPO-BP is a 50-kDa membrane-bound serine hydrolase measured by derivatization with [(3)H]CPO and SDS/PAGE or filtration binding assay. It appears to undergo rapid diethylphosphorylation by [(3)H]CPO followed by either dephosphorylation and reactivation or aging on loss of an ethyl group. CPO and several other OP toxicants potently inhibit CPO-BP in vivo (i.p., 2 h) (50% inhibition at 2-25 mg/kg) and in vitro (50% inhibition at 8-68 nM). Using three chemical labeling reagents, i.e., [(3)H]CPO and the activity-based proteomic probes fluorophosphonate-biotin and fluorophosphonate-rhodamine, mouse brain CPO-BP is identified as serine hydrolase KIAA1363 of unknown function. Brains from KIAA1363(-/-) mice show greatly reduced levels of CPO labeling and hydrolytic metabolism compared to brains from wild-type mice. KIAA1363 therefore is the principal enzyme for metabolizing low levels of CPO in brain and may play a more general role in detoxification of OP nerve poisons.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363(-/-) and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL(-/-) mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363(-/-) but unchanged in HSL(-/-) mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL(-/-) macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1(Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.

Lipases play key roles in nearly all cells and organisms. Potent and selective inhibitors help to elucidate their physiological functions and associated metabolic pathways. Organophosphorus (OP) compounds are best known for their anticholinesterase properties but selectivity for lipases and other targets can also be achieved through structural optimization. This review considers several lipid systems in brain modulated by highly OP-sensitive lipases. Neuropathy target esterase (NTE) hydrolyzes lysophosphatidylcholine (lysoPC) as a preferred substrate. Gene deletion of NTE in mice is embryo lethal and the heterozygotes are hyperactive. NTE is very sensitive in vitro and in vivo to direct-acting OP delayed neurotoxicants and the related NTE-related esterase (NTE-R) is also inhibited in vivo. KIAA1363 hydrolyzes acetyl monoalkylglycerol ether (AcMAGE) of the platelet-activating factor (PAF) de novo biosynthetic pathway and is a marker of cancer cell invasiveness. It is also a detoxifying enzyme that hydrolyzes chlorpyrifos oxon (CPO) and some other potent insecticide metabolites. Monoacylglycerol lipase and fatty acid amide hydrolase regulate endocannabinoid levels with roles in motility, pain and memory. Inhibition of these enzymes in mice by OPs, such as isopropyl dodecylfluorophosphonate (IDFP), leads to dramatic elevation of brain endocannabinoids and distinct cannabinoid-dependent behavior. Hormone-sensitive lipase that hydrolyzes cholesteryl esters and diacylglycerols is a newly recognized in vivo CPO- and IDFP-target in brain. The OP chemotype can therefore be used in proteomic and metabolomic studies to further elucidate the biological function and toxicological significance of lipases in lipid metabolism. Only the first steps have been taken to achieve appropriate selective action for OP therapeutic agents.

Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.

Serine hydrolase KIAA1363 is highly expressed in invasive cancer cells and is the major protein in mouse brain diethylphosphorylated by and hydrolyzing low levels of chlorpyrifos oxon (CPO) (the activated metabolite of a major insecticide). It is also the primary CPO-hydrolyzing enzyme in spinal cord, kidney, heart, lung, testis, and muscle but not liver, a pattern of tissue expression confirmed by fluorophosphonate-rhodamine labeling. KIAA1363 gene deletion using homologous recombination reduces CPO binding, hydrolysis, and metabolism 3-29-fold on incubation with brain membranes and homogenates determined with 1 nM [(3)H-ethyl]CPO and the inhibitory potency for residual CPO with butyrylcholinesterase as a biomarker. Studies with knockout mice further show that KIAA1363 partially protects brain AChE and monoacylglycerol lipase from CPO-induced in vivo inhibition. Surprisingly, mouse brain KIAA1363 and AChE are similar in in vitro sensitivity to seven methyl, ethyl, and propyl but not higher alkyl OP insecticides and analogues, prompting structural comparisons of the active sites of KIAA1363 and AChE relative to OP potency and selectivity. Homology modeling based largely on the Archaeoglobus fulgidus esterase crystal structure indicates that KIAA1363 has a catalytic triad of S191, D348, and H378, a GDSAG motif, and an oxyanion hole of H113, G114, G115, and G116. Excellent selectivity for KIAA1363 is achieved on OP structure optimization with long alkyl chain substituents suggesting that KIAA1363 has larger acyl and leaving group pockets than those of AChE. KIAA1363 reactivates faster than AChE presumably due to differences in the uncoupling of the catalytic triad His upon phosphorylation. The structural modeling of KIAA1363 helps us understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.

Organophosphorus (OP) insecticides and chemical warfare agents act primarily by inhibiting acetylcholinesterase. There are many secondary targets for OP toxicants as observed for example with the major insecticide chlorpyrifos and its bioactivated metabolite chlorpyrifos oxon (CPO). Therefore, it was surprising that the predominant mouse brain protein labeled in vitro by [(3)H-ethyl]CPO (1 nM) (designated CPO-binding protein or CPO-BP) is not one of these known OP toxicant targets. CPO-BP is a 50-kDa membrane-bound serine hydrolase measured by derivatization with [(3)H]CPO and SDS/PAGE or filtration binding assay. It appears to undergo rapid diethylphosphorylation by [(3)H]CPO followed by either dephosphorylation and reactivation or aging on loss of an ethyl group. CPO and several other OP toxicants potently inhibit CPO-BP in vivo (i.p., 2 h) (50% inhibition at 2-25 mg/kg) and in vitro (50% inhibition at 8-68 nM). Using three chemical labeling reagents, i.e., [(3)H]CPO and the activity-based proteomic probes fluorophosphonate-biotin and fluorophosphonate-rhodamine, mouse brain CPO-BP is identified as serine hydrolase KIAA1363 of unknown function. Brains from KIAA1363(-/-) mice show greatly reduced levels of CPO labeling and hydrolytic metabolism compared to brains from wild-type mice. KIAA1363 therefore is the principal enzyme for metabolizing low levels of CPO in brain and may play a more general role in detoxification of OP nerve poisons.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.

By primarily measuring changes in transcript and protein abundance, conventional genomics and proteomics methods may fail to detect significant posttranslational events that regulate protein activity and, ultimately, cell behavior. To address these limitations, activity-based proteomic technologies that measure dynamics in protein function on a global scale would be of particular value. Here, we describe the application of a chemical proteomics strategy to quantitatively compare enzyme activities across a panel of human breast and melanoma cancer cell lines. A global analysis of the activity, subcellular distribution, and glycosylation state for the serine hydrolase superfamily resulted in the identification of a cluster of proteases, lipases, and esterases that distinguished cancer lines based on tissue of origin. Strikingly, nearly all of these enzyme activities were down-regulated in the most invasive cancer lines examined, which instead up-regulated a distinct set of secreted and membrane-associated enzyme activities. These invasiveness-associated enzymes included urokinase, a secreted serine protease with a recognized role in tumor progression, and a membrane-associated hydrolase KIAA1363, for which no previous link to cancer had been made. Collectively, these results suggest that invasive cancer cells share discrete proteomic signatures that are more reflective of their biological phenotype than cellular heritage, highlighting that a common set of enzymes may support the progression of tumors from a variety of origins and thus represent attractive targets for the diagnosis and treatment of cancer.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.