Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.
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We are grateful to K. Masuda and L. Bar-Peled for discussions on and assistance with cloning; M. Dix, J. Wu and K. Lum for discussions and technical expertise in designing and analyzing proteomics experiments; J. Teijaro for providing T cells; G. Simon for assistance with the HHpred analysis; and C. Walsh and M. Niphakis for numerous helpful discussions. This work was supported by the US National Institutes of Health (DA033760, DK909810), The Leona M. and Harry B. Helmsley Charitable Trust (grant no. 2012-PG-MED002 to A.S.), National Cancer Institute Cancer Center Support grant P30 (CA014195 MASS core, A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), a Hewitt Foundation for Medical Research Fellowship (to W.H.P.), a Chapman Charitable Trust Fellowship (to M.J.K.), UCSD Medical Scientist Training Program funding (T32 GM007198 to M.J.K.) and an Irving S. Sigal postdoctoral fellowship from the American Chemical Society (to S.S.K.).
B.F.C. is a founder and advisor to Abide Therapeutics, a biotechnology company interested in developing serine hydrolase inhibitors and therapeutics.
Supplementary Results, Supplementary Figures 1–18, Supplementary Table 1 and 2 captions, and Supplementary Tables 3 and 4. (PDF 3492 kb)
Synthetic Procedures. (PDF 270 kb)
Complete ABPP-SILAC data sets. (XLSX 5746 kb)
HHpred search results for AIG1/ADTRP alignment. (XLSX 20 kb)
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Parsons, W., Kolar, M., Kamat, S. et al. AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs. Nat Chem Biol 12, 367–372 (2016). https://doi.org/10.1038/nchembio.2051
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