A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation
Authors: Matthew D Hirschey
Robert D Stevens
Dhaval P Bhatt
Donald S Backos
Olga R Ilkayeva
Paul A Grimsrud
Thomas M O&#x27;Connell
Grant A Mitchell
Laura G Dubois
Gregory R Wagner
Hao Yang
J Will Thompson
Journal: Cell Metabolism (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: acyl-CoA; chemical biology; non-enzymatic; post-translational modifications; protein acylation
Affiliations: Sarah W. Stedman Nutrition and Metabolism Center, Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC 27710, USA; Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA., Sarah W. Stedman Nutrition and Metabolism Center, Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC 27710, USA., Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA; Duke Proteomics and Metabolomics Shared Resource, Duke University Medical Center, Durham, NC 27710, USA., Duke Proteomics and Metabolomics Shared Resource, Duke University Medical Center, Durham, NC 27710, USA., Computational Chemistry and Biology Core Facility, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA., Division of Medical Genetics, Department of Pediatrics, Université de Montréal and CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, QC H3T 1C5, Canada; College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China., Division of Medical Genetics, Department of Pediatrics, Université de Montréal and CHU Sainte-Justine, 3175 Côte Sainte-Catherine, Montreal, QC H3T 1C5, Canada., Sarah W. Stedman Nutrition and Metabolism Center, Duke Molecular Physiology Institute, Duke University Medical Center, Durham, NC 27710, USA; Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA; Department of Pharmacology & Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA. Electronic address: .

Abstract:  The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergo intramolecular catalysis to form reactive intermediates that non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the "acylomes" of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins and define a new regulatory paradigm in metabolism. 

Retracted: No

PubPeer comments:
 See this tweet storm for a reply, and explanation on how some proteins leave ghosted images when Western blotting. https: twitter.com matthewhirschey status 1225469384782565377 , 
 Figures 1 and 3: WB results have lanes where the lanes are lighter than the background. In figure 1, lanes 1 and 3 for Acetyl-lysine, the shape for the lighter regions are different for each exposure level. The dark spot in the longer exposure is a different shape, perhaps due to different method of smoothing. Figures 3: Lanes 1 and 6, pixels are lighter for Glutaryl-lysine, in the shape of a band, while the surrounding background is darker. ![file] ![file] ![file] ![file] , 
 #2 As an outside observer, however, I do have to wonder how it is you did not anticipate that these features would not trigger questions, especially given that, as mentioned in your twitter thread, * the reviewers of our paper had the same question *. I looked for an explanation in the paper -- in the figure legends, methods, and supplement -- and did not find one. Did I miss where this was explained, or did you expect that for some reason the reviewers would be the only people who would be puzzled by these features? (And if you think the answer is obvious to anyone with experience in western blotting, I can only tell you that it was not immediately obvious to me, despite considerable experience, although I might have figured it out eventually.) , 
 Candidly I didn t think it was that strange of a thing (we see these often, depending on conditions, proteins, etc). Given the reviewers asked about the ghost bands, I guess I could have anticipated more questions. But, as you can imagine, it s impossible to anticipate every possible question. That being said, pubpeer (in theory) is a great platform for this sort of exchange, question asking, sharing information, ideas, etc. Perhaps obvious ghost images might be explained with an asterisk, or something, at the discretion of the editors. But I could also envision soooooo many asterisks. , 
 UPDATE FROM Gregg Wagner (FIRST AUTHOR): Thank you for your comments. Besides the BSA acting as a better blocker than the fish gelatin blocking agent in this scenario, another point that is worth mentioning is that these Westerns were imaged exposed using the Li-Cor imaging system and not using a traditional film exposure. With that in mind, these images are much higher resolution and higher contrast that traditional Western blots, i.e. they will show features that may not be obvious even to those experienced in Western blotting. The Li-Cor approach is advantageous because it gets us closer to quantitative Western blotting, but it can also reveal unusual features that would otherwise go unnoticed when using more traditional methods. In addition, we were using custom-made antibodies for many of the westerns shown in this paper (i.e. we made the antigen and purified the antibody through various selections). As anyone knows who has created their own antibodies, generating the  perfect image can be challenging and usually requires much more optimization relative to antibodies purchased through commercial vendors. ,