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Figure 2

Figure 2. Serine hydrolases KIAA1363 and MAGL regulate lipid metabolic pathways that support cancer pathogenesis. From: Activity-based protein profiling for biochemical pathway discovery in cancer.

Activity-based protein profiling (ABPP) identified KIAA1363 (part a) and monoacylglycerol (MAG) lipase (MAGL) (part b) as being increased in aggressive human cancer cells from multiple tumour types. Pharmacological and/or RNA interference ablation of KIAA1363 and MAGL coupled with metabolomic analysis revealed specific roles for KIAA1363 and MAGL in cancer metabolism. Disruption of KIAA1363 by the small-molecule inhibitor AS115 lowered monoalkylglycerol ether (MAGE), alkyl lysophosphatidic acid (alkyl LPA) and alkyl lysophosphatidyl choline (alkyl LPC) levels in cancer cells. Disruption of MAGL by the small-molecule inhibitor JZL184 raised MAG levels and reduced free fatty acid, lysophosphatidic acid (LPA) and prostaglandin E2 (PGE2) levels in cancer cells. Disruption of KIAA1363 and MAGL leads to impairments in cancer cell aggressiveness and tumour growth (part c). PAF, platelet-activating factor.

Daniel K. Nomura, et al. Nat Rev Cancer. ;10(9):630-638.
Figure 1

Figure 1. Activity-based protein profiling. From: Activity-based protein profiling for biochemical pathway discovery in cancer.

a) Activity-based protein profiling (ABPP) uses active site-directed chemical probes to assess the functional state of large numbers of enzymes in native biological systems. Activity-based probes consist of a reactive group (red ball) for targeting a specific set of enzymes and a detection handle (a fluorophore, such as a rhodamine (Rh) or biotin (B)). In a typical ABPP experiment, a proteome is reacted with the activity-based probe and probe-labelled proteins detected by either in-gel fluorescence scanning (for fluorophore-conjugated probes; top) or avidin enrichment, on-bead tryptic digest and liquid chromatograpy mass spectrometry (LC–MS) analysis (for biotinylated probes; bottom). b) ABPP can also be used in a competitive format to evaluate the potency and selectivity of enzyme inhibitors in native biological samples. Inhibitors compete with activity-based probes for enzyme targets, and this competition is read out by loss of fluorescence (for fluorophore-conjugated probes) or MS (for biotinylated probes) signals (not shown). m/z, mass to charge ratio.

Daniel K. Nomura, et al. Nat Rev Cancer. ;10(9):630-638.
Figure 3

Figure 3. Proteomic strategies for mapping protease substrates. From: Activity-based protein profiling for biochemical pathway discovery in cancer.

a) Amino terminal labelling techniques use chemical or enzymatic methods to selectively label neo-N termini that are created on protease treatment. The labelled N-terminal peptides can then be enriched through positive selection methods (such as the subtiligase method84) or, alternatively, the internal unmodified peptides can be removed through negative selection methods (such as terminal amine isotopic labelling of substrates (TAILS) methods85). The remaining pool of enriched labelled neo-N termini can then be analysed using tandem mass spectrometry (MS) and the exact sites of proteolytic cleavage can be assigned. b) An alternative proteomic method for protease substrate identification, Protein Topography and Migration Analysis Platform (PROTOMAP86) combines one-dimensional (1D) SDS–PAGE fractionation with liquid chromatograpy (LC)–MS analysis. In a typical PROTOMAP experiment, proteomes from control (Ctrl; red) and experimental (protease-active) (Expt; blue) systems are separated by 1D SDS–PAGE. The lanes are cut into bands at fixed intervals, digested with trypsin and analysed by LC–MS/MS to generate data that are integrated into peptographs, which plot sequence coverage for a given protein in the horizontal dimension (N to C terminus; left to right) versus gel migration in the vertical dimension. Spectral count values for each protein in each gel band provide quantitation. Cleaved proteins are identified by shifts in migration from higher to lower molecular weight (MW) in Ctrl versus Expt systems.

Daniel K. Nomura, et al. Nat Rev Cancer. ;10(9):630-638.

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