The ubiquitin and ubiquitin-like proteins regulate a vast array of biological processes. Examples include the ubiquitin that regulates protein degradation, SUMO that regulates stress response pathways, and ATG proteins that regulate autophagy. A total of 20 UBL proteins are known, yet the discovery of these proteins occur mostly serendipitously, and there are few systematic approaches to directly probe the intact eukaryotic proteomes for the presence of UBLs. Bioinformatic approaches to discover UBLs are not always successful, because certain UBL proteins may not share the aminoacid sequence homology. For example ubiquitin does not share aminoacid sequence homology with the ubiquitin-like proteins ATG8, ATG12, UFM1 and URM1, yet all these proteins share a common �-grasp fold. Conversely, not all proteins with the �-grasp fold are known to be activated by the E1 enzymes and serve as UBLs. Therefore, there is a need to develop a new technology that can directly detect a complete set of UBL proteins inside intact eukaryotic proteomes. We developed a mechanism based small molecule probe that covalently labels any UBL protein in intact eukaryotic proteomes. We have shown that the developed probe labels seven known human UBLs in vitro and in cells. Covalently labeled UBLs can be isolated and identified using proteomic techniques. The developed probe can be used to monitor the intracellular activity of ubiquitin-activating E1 enzymes, and profiling the intracellular selectivity of E1 enzyme targeting small molecule inhibitors.

Development of activity based probes for ubiquitin and ubiquitin-like protein signaling pathways

246th National Meeting (2013)

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