An activity-based imaging probe for the integral membrane hydrolase KIAA1363.
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ABSTRACT:
SUBMITTER: Chang JW
PROVIDER: S-EPMC3425385 | biostudies-literature | 2012 Jan
REPOSITORIES: biostudies-literature
Publications
An activity-based imaging probe for the integral membrane hydrolase KIAA1363.
Chang Jae Won JW Moellering Raymond E RE Cravatt Benjamin F BF
Angewandte Chemie (International ed. in English) 20111207 4
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Project description: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.
| S-EPMC2936755 | biostudies-literature
Project description:The melting curves of fatty acid amide hydrolase (FAAH) in the presence of 29 reversible inhibitors were measured using a thiol-reactive fluorophore. The thermal stability (T(m)) of the FAAH/inhibitor complex varied significantly depending on the chemical characteristics of the inhibitors, notably variations in the head group. Two separate distributions were observed when T(m) was plotted against K(i). The majority of the inhibitors showed a positive correlation between binding affinity and T(m), however inhibitors with a pyridine carboxylic acid moiety in the head group fell in a distinct and uncorrelated distribution when tail groups were varied.
| S-EPMC2794692 | biostudies-literature
Project description: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.
| S-EPMC2512971 | biostudies-literature
Project description:Oxons are bioactive metabolites of organophosphorus insecticides (OPs) that covalently inactivate serine hydrolases. KIAA1363 is one of the most abundant serine hydrolases in mouse brain. Although the physiological consequences related to the inhibition of KIAA1363 due to environmental exposures to OPs are poorly understood, the enzyme was previously shown to have a role in the detoxification of oxons. Here, we overexpressed human KIAA1363 and CES1 in COS7 cells and compared the potency of inhibition (IC50s, 15 min) of KIAA1363 and CES1 by chlorpyrifos oxon (CPO), paraoxon (PO), and methyl paraoxon (MPO). The order of potency was CPO > PO >> MPO for both enzymes. We also determined the bimolecular rate constants (kinact/Ki) for reactions of CPO and PO with KIAA1363 and CES1. KIAA1363 and CES1 were inactivated by CPO at comparable rates (4.4 × 10(6) s(-1) M(-1) and 6.7 × 10(6) s(-1) M(-1), respectively), whereas PO inactivated both enzymes at slower rates (0.4 × 10(6) s(-1) M(-1) and 1.5 × 10(6) s(-1) M(-1), respectively). Finally, the reactivation rate of KIAA1363 following inhibition by CPO was evaluated. Together, the results define the kinetics of inhibition of KIAA1363 by active metabolites of agrochemicals and indicate that KIAA1363 is highly sensitive to inhibition by these compounds.
| S-EPMC4727981 | biostudies-literature
Project description:Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC50 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.
| S-EPMC2423230 | biostudies-literature
Project description:Lung tumors, as well as normal tumor-adjacent (NTA) tissue of non-small cell lung cancer (NSCLC) patients, were collected and subjected to labeling with a serine hydrolase-directed activity-based probe (ABP) immediately after excision. Labeling with the serine hydrolase-directed ABP allows for the detection of active lipid hydrolases, which are the focus of our investigation. The labeled proteins (corresponding to active enzymes at time of labeling) were enriched using a biotin-containing linker that was covalently attached using strain-promoted alkyne-azide cycloaddition (click chemistry). Shotgun proteomics of the on-bead digested enriched proteins of ABP-treated and control samples enabled identification of enriched proteins as well as comparison of activity profiles between NTA and tumor samples of NSCLC patients.
2023-11-27 | PXD046934 | Pride
Project description:Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.
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Project description:PurposeAbnormal Notch signaling promotes cancer cell growth and tumor progression in various cancers. Targeting γ-secretase, a pivotal regulator in the Notch pathway, has yielded numerous γ-secretase inhibitors (GSIs) for clinical investigation in the last 2 decades. However, GSIs have demonstrated minimal success in clinical trials in part due to the lack of specific and precise tools to assess γ-secretase activity and its inhibition in vivo.Experimental designWe designed an imaging probe based on GSI Semagacestat structure and synthesized the radioiodine-labeled analogues [131I]- or [124I]-PN67 from corresponding trimethyl-tin precursors. Both membrane- and cell-based ligand-binding assays were performed using [131I]-PN67 to determine the binding affinity and specificity for γ-secretase in vitro. Moreover, we evaluated [124I]-PN67 by PET imaging in mammary tumor and glioblastoma mouse models.ResultsThe probe was synthesized through iodo-destannylation using chloramine-T as an oxidant with a high labeling yield and efficiency. In vitro binding results demonstrate the high specificity of this probe and its ability for target replacement study by clinical GSIs. PET imaging studies demonstrated a significant (P < 0.05) increased in the uptake of [124I]-PN67 in tumors versus blocking or sham control groups across multiple mouse models, including 4T1 allograft, MMTV-PyMT breast cancer, and U87 glioblastoma allograft. Ex vivo biodistribution and autoradiography corroborate these results, indicating γ-secretase specific tumor accumulation of [124I]-PN67.Conclusions[124I]-PN67 is a novel PET imaging agent that enables assessment of γ-secretase activity and target engagement of clinical GSIs.
| S-EPMC8610083 | biostudies-literature
Project description:Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.
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Project description:Lipoprotein modification is an essential process in Gram-negative bacteria. The action of three integral membrane proteins that catalyze the transfer of fatty acids derived from membrane phospholipids or cleave the signal peptide of the lipoprotein substrate result in the formation of mature triacylated proteins. Inactivation of the enzymes leads to mis-localization of immature lipoproteins and consequently cell death. Biochemical studies and the development of in vitro assays are challenging due to the fact that the enzymes and substrates are all membrane-embedded proteins difficult to overproduce and purify. Here we describe a sensitive fluorescence-based assay to monitor bacterial apolipoprotein N-acyltransferase activity.
| S-EPMC6828757 | biostudies-literature