Activity‐Based Protein Profiling (ABPP) and Click Chemistry (CC)–ABPP by MudPIT Mass Spectrometry

Speers, Anna E; Cravatt, Benjamin F.

doi:10.1002/9780470559277.ch090138

Cited by 110 publications

(104 citation statements)

References 28 publications

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“…To evaluate the usefulness of 1 as a click chemistry probe, we incubated the compound with purified hNAAA (which was preventively activated at acidic pH), coupled an azide-PEG3-biotin conjugate to its terminal alkyne using click chemistry, 49 and visualized protein bands using streptavidin-horseradish peroxidase (HRP). The labeling step was performed either in phosphate buffered saline (PBS, pH 7.4) or in phosphate/ citrate buffer, pH 4.5, while in both cases PBS was used as buffer for the click chemistry reaction.…”

Section: Resultsmentioning

confidence: 99%

Activity-Based Probe for N-Acylethanolamine Acid Amidase

et al. 2015

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N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α–amino–β–lactone (3–aminooxetan–2–one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo, and to investigate the physiological and pathological roles of this enzyme.

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Section: Resultsmentioning

confidence: 99%

Activity-Based Probe for N-Acylethanolamine Acid Amidase

et al. 2015

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“…Following the click reaction that couples a reporter tag to the ABPP probe, labeled proteins can then be analyzed by gel-based ABPP or ABPP-MudPIT. Interested readers are referred to Speers and Cravatt, 2009 for protocols for in situ ABPP.…”

Section: Discussionmentioning

confidence: 99%

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

Galmozzi

Domínguez

Cravatt

et al. 2014

Methods in Enzymology

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Activity-Based Protein Profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes, and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways.

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“…The beads were subsequently washed with 5 ml of 0.2% (w/v) SDS in PBS once, 5 ml of PBS three times and 5 ml of distilled water three times. After transferring the beads to a microfuge tube and removing the supernatant, the captured proteins were on-bead digested with trypsin according to standard protocols 23 and the tryptic peptides were desalted and analyzed by LTQ LC-MS/MS. Peptides were separated on a 75-μm i.d.…”

Section: Methodsmentioning

confidence: 99%

Identification of Palmitoyl Protein Thioesterase 1 in Human THP1 Monocytes and Macrophages and Characterization of Unique Biochemical Activities for This Enzyme

et al. 2013

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The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, both species' macrophages exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed carboxylesterase 1 (CES1) was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes/macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6 and ABHD12, did not have a role because of either limited or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining 2-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)–MudPIT and western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited similar electrophoretic properties to a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was highly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies and chemical inhibitor profiles, we estimated that PPT1 contributed 32-40% of 2-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of 2-AG, PGE2-G and PGF2α-G, although the catalytic efficiency of 2-AG hydrolysis by PPT1 was ∼10-fold lower than CES1's. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte/macrophage cell line and to show PPT1 can hydrolyze the natural substrates 2-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts, and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism.

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Activity‐Based Protein Profiling (ABPP) and Click Chemistry (CC)–ABPP by MudPIT Mass Spectrometry

Cited by 110 publications

References 28 publications

Activity-Based Probe for N-Acylethanolamine Acid Amidase

Activity-Based Probe for N-Acylethanolamine Acid Amidase

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

Identification of Palmitoyl Protein Thioesterase 1 in Human THP1 Monocytes and Macrophages and Characterization of Unique Biochemical Activities for This Enzyme

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