Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes

Patricelli, Matthew P.; Giang, Dan K.; Stamp, Lisa M.; Burbaum, Jonathan J.

doi:10.1002/1615-9861(200109)1:9<1067::aid-prot1067>3.0.co;2-4

Cited by 275 publications

(232 citation statements)

References 8 publications

Supporting

Mentioning

217

Contrasting

Unclassified

Order By: Relevance

“…We performed these studies with three different activitybased probes: two DAGL-tailored activity-based probes-DH379 (SI Appendix, Fig. S2) and HT-01 (27)-and a broad-spectrum serine hydrolase-directed probe fluorophosphonate-rhodamine (FP-Rh) (32). HT-01 and DH379 provided target engagement assays for DAGLs, and FP-Rh assessed cross-reactivity across a broad array of brain serine hydrolases.…”

Section: Resultsmentioning

confidence: 99%

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition

Ogasawara

Deng

Viader

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

129

190

View full text Add to dashboard Cite

Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

show abstract

Section: Resultsmentioning

confidence: 99%

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition

Ogasawara

Deng

Viader

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

129

190

View full text Add to dashboard Cite

show abstract

“…We used an activity-based probe of serine hydrolases consisting of a fluorophosphonate moiety linked to tetramethyl rhodamine (FP-PEG-TAMRA). The fluorophosphonate warhead tags the active site serine of serine hydrolases, forming an adduct that is stable to SDS gel electrophoresis (29). Activity profiles of three human breast cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-435, were compared (Fig.…”

Section: Serine Hydrolase Profile Of Normal and Neoplasticmentioning

confidence: 99%

A Fatty Acid Synthase Blockade Induces Tumor Cell-cycle Arrest by Down-regulating Skp2

Knowles

Axelrod

Browne

et al. 2004

Journal of Biological Chemistry

138

View full text Add to dashboard Cite

In eukaryotes, fatty acid synthase (FAS) is the enzyme responsible for synthesis of palmitate, the precursor of long-chain nonessential fatty acids. FAS is up-regulated in a wide range of cancers and has been suggested as a relevant drug target. Here, two independent approaches are taken toward knocking down FAS and then probing its connection to tumor cell proliferation. In one approach, Orlistat, a drug approved for treating obesity, is used as a potent inhibitor of the thioesterase function of FAS. In a separate strategy, the expression of FAS is suppressed by targeted knock-down with small interfering RNA. In both circumstances, the ablation of FAS activity causes a dramatic down-regulation of Skp2, a component of the E3 ubiquitin ligase that controls the turnover of p27Kip1 . These effects ultimately tie into the retinoblastoma protein pathway and lead to a cell-cycle arrest at the G 1 /S boundary. Altogether, the findings of the study reveal unappreciated links between fatty acid synthase and ubiquitin-dependent proteolysis of cellcycle regulatory proteins.

show abstract

“…The OP inhibition and reactivation mechanisms are considered based on docking CPO and other inhibitors and structural comparisons of KIAA1363 and AChE. (8). Insecticides and their analogs were from Sigma (St. Louis, MO) or ChemService (West Chester, PA) and highly purified horse serum butyrylcholinesterase (BChE) was from Sigma.…”

Section: Introductionmentioning

confidence: 99%

Serine Hydrolase KIAA1363: Toxicological and Structural Features with Emphasis on Organophosphate Interactions

Nomura

Durkin

Chiang

et al. 2006

Chem. Res. Toxicol.

View full text Add to dashboard Cite

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-to 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 analogs, 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 understand OP structure-activity relationships and the toxicological relevance of this detoxifying enzyme.

show abstract

Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site-directed probes

Cited by 275 publications

References 8 publications

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition

Rapid and profound rewiring of brain lipid signaling networks by acute diacylglycerol lipase inhibition

A Fatty Acid Synthase Blockade Induces Tumor Cell-cycle Arrest by Down-regulating Skp2

Serine Hydrolase KIAA1363: Toxicological and Structural Features with Emphasis on Organophosphate Interactions

Contact Info

Product

Resources

About