The discovery is unusual because the enzymes do not bear a resemblance–in their structures or amino-acid sequences–to any known class of enzymes. The team of scientists nevertheless identified them as \u00aboutlier\u00bb members of the serine\/threonine hydrolase class, using newer techniques that detect biochemical activity.<\/p>\n
\u00abA huge fraction of the human ‘proteome’ remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure,\u00bb said co-senior author Benjamin F. Cravatt, chair of TSRI’s Department of Chemical Physiology.<\/p>\n
\u00abIn this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases,\u00bb said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute.<\/p>\n
The study, which appears as a Nature Chemical Biology Advance Online Publication<\/em> on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine\/threonine hydrolases using fluorophosphonate (FP) probes–molecules that selectively bind and, in effect, label the active sites of these enzymes.<\/p>\n Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP.<\/p>\n \u00abNeither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all,\u00bb said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study.<\/p>\n Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. \u00abIt looks like they have an active site that is novel–it had never been described in the literature,\u00bb said Parsons.<\/p>\n Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases–enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work?<\/p>\n At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation.<\/p>\n \u00abBen’s lab was screening panels of lipids to find the ones that their new enzymes work on,\u00bb said Saghatelian, who is a former research associate in the Cravatt laboratory. \u00abWe suggested they throw FAHFAs in there–and these turned out to be very good substrates.\u00bb<\/p>\n The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes’ functions in vitro and in cultured cells. Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab.<\/p>\n The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid.<\/p>\n In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies. \u00abOur prediction,\u00bb said Saghatelian, \u00abis that if FAHFAs do what we think they’re doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity.\u00bb<\/p>\n The two labs are now collaborating on further studies of the new enzymes–and the potential benefits of inhibiting them–in mouse models of diabetes, inflammation and autoimmune disease.<\/p>\n \u00abOne of the neat things this study shows,\u00bb said Cravatt, \u00abis that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology.\u00bb<\/p>\n Other co-authors of the study, \u00abAIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs<\/em>,\u00bb were Siddhesh S. Kamat, Armand B. Cognetta III, Jonathan J. Hulce and Enrique Saez, of TSRI; and co-senior author Barbara B. Kahn of Beth Israel Deaconess Medical Center and Harvard Medical School.<\/p>\n Funding was provided in part by the U.S. National Institutes of Health (DA033760, DK909810), The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), National Cancer Institute Cancer Center Support (grant P30 [CA014195 MASS core]) and the Dr. Frederick Paulsen Chair\/Ferring Pharmaceuticals.<\/p>\nRegulators of FAHFAs<\/h4>\n