Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes

Galmozzi, Andrea; Domínguez, Eduardo; Cravatt, Benjamin F.; Sáez, Enrique

doi:10.1016/b978-0-12-800280-3.00009-8

Cited by 18 publications

(13 citation statements)

References 36 publications

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“…For the ABPP assay, we have removed the lipid fractions from the bran sample to minimize the competition from the endogenous lipids with the serine hydrolase probe. Since lipases belong to the serine hydrolase family, the lipids will eventually weaken the interaction of the probe with the lipase active site and lead to loss of potential targets 43 . The labeling of active serine hydrolases was done using 2 µM FP-Rh at pH 8.0 because the optimal SHs labeling was observed at this pH 8.0 in the complex protein lysates 44 .…”

Section: Discussionmentioning

confidence: 99%

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

Dolui

Vijayakumar

Rajasekharan

et al. 2020

Sci Rep

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Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.

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Section: Discussionmentioning

confidence: 99%

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

Dolui

Vijayakumar

Rajasekharan

et al. 2020

Sci Rep

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“…Methyl arachidonyl fluorophosphonate (MAFP) is an irreversible, covalent inhibitor of serine 22 , 23 and threonine 24 lipases. Fluorophosphonate inhibitors, similar to MAFP, inhibit all four lipases that hydrolyse FAHFAs in vitro and in vivo 19 , 25 . We first tested MAFP as a FAHFA hydrolysis inhibitor in 3T3-L1 adipocytes incubated with C17:1 FA and 9-HSA to measure biosynthesis without competing effects from hydrolysis.…”

Section: Fluorophosphonates Inhibit Fahfa Synthesismentioning

confidence: 99%

“…To identify the enzyme(s) responsible for increased 9-FAHFA biosynthesis in AG4OX adipocytes, we performed activity-based protein profiling using a fluorophosphonate (FP)-alkyne probe 23 , 25 that uses the same functional group as MAFP and therefore should inhibit the same enzymes. Covalent labelling with the probe labels the enzyme with an alkyne that can be used to enrich and detect the target proteins through proteomics (Fig.…”

Section: Fluorophosphonates Inhibit Fahfa Synthesismentioning

confidence: 99%

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids

Patel

Santoro

Hofer

et al. 2022

Nature

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Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4–7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80–90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.

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“…The second is a binding group that often resembles the natural substrates of the enzymes and the third is a reporter tag that is commonly a fluorophore or biotin and allows for the detection and enrichment/identification of probe-labeled enzymes. Detection of labeled enzymes is achieved by gel electrophoresis and in-gel fluorescence scanning or LC–MS ( 85 ). The major advantage of ABPP is that only the catalytically active forms of the targeted enzymes are detected.…”

Section: Experimental and Computational Strategies To Identify New Mementioning

confidence: 99%

Confronting the catalytic dark matter encoded by sequenced genomes

Ellens

Christian

Singh

et al. 2017

Nucleic Acids Research

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The post-genomic era has provided researchers with a deluge of protein sequences. However, a significant fraction of the proteins encoded by sequenced genomes remains without an identified function. Here, we aim at determining how many enzymes of uncertain or unknown function are still present in the Saccharomyces cerevisiae and human proteomes. Using information available in the Swiss-Prot, BRENDA and KEGG databases in combination with a Hidden Markov Model-based method, we estimate that >600 yeast and 2000 human proteins (>30% of their proteins of unknown function) are enzymes whose precise function(s) remain(s) to be determined. This illustrates the impressive scale of the ‘unknown enzyme problem’. We extensively review classical biochemical as well as more recent systematic experimental and computational approaches that can be used to support enzyme function discovery research. Finally, we discuss the possible roles of the elusive catalysts in light of recent developments in the fields of enzymology and metabolism as well as the significance of the unknown enzyme problem in the context of metabolic modeling, metabolic engineering and rare disease research.

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Application of Activity-Based Protein Profiling to Study Enzyme Function in Adipocytes

Cited by 18 publications

References 36 publications

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids

Confronting the catalytic dark matter encoded by sequenced genomes

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