Ubiquitin(Ub) and Ubiquitin-like proteins(UBLs) diversify protein functions via reversible posttranslational modification of proteins. Currently, 9 classes of UBLs have been discovered in humans. Remarkably, however, more than half of these UBLs have been discovered serendipitously. This is due to the lack of available methods that can directly interrogate intact proteomes for the presence and the activation states of UBLs.Here we introduce a novel Activity Based Probe(ABP1) that can covalently label UBLs through the conserved mechanism of the UBL activating enzyme(E1). Enzyme based assays prove that ABP1 labels all seven tested UBLs. ABP1 can also form UBL-ABP1 covalent adducts in live A549 cells. The newly formed bond between UBLs and ABP1 is stable in physiological conditions. Interestingly, more than 90% of UBL-ABP1 is hydrolyzed under an acidic condition (pH = 2.0). Therefore, we expect that ABP1 will be useful to interrogate the presence of any UBL protein in a given biological system, and that the Catch-and-Release feature of ABP1 will facilitate subsequent proteomics studies.Because the labeling of UBLs is dependent on the catalytic activity of E1s, ABP1 was expected to measure the relative activity of E1s. Indeed, the intensity of the UBL labeling decreased when cells were treated with small molecule inhibitors for E1s followed by ABP1 treatment. This result suggests that ABP1 can also be used to profile global activity of E1s in cells.

Development of an activity based probe for Ubiquitin and Ubiquitin-like protein signaling pathways

246th National Meeting (2013)

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