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1. Identification of lysine isobutyrylation as a new histone modification mark

   https://doi.org/10.1093/nar/gkaa1176  

   Zhu, Zhesi; Han, Zhen; Halabelian, Levon; Yang, Xiangkun; Ding, Jun; Zhang, Nawei; Ngo, Liza; Song, Jiabao; Zeng, Hong; He, Maomao; et al (December 2020, Nucleic Acids Research)

   null (Ed.)

   Abstract Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology. 

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2. Dynamic acylome reveals metabolite driven modifications in Syntrophomonas wolfei

   https://doi.org/10.3389/fmicb.2022.1018220  

   Fu, Janine Y.; Muroski, John M.; Arbing, Mark A.; Salguero, Jessica A.; Wofford, Neil Q.; McInerney, Michael J.; Gunsalus, Robert P.; Loo, Joseph A.; Ogorzalek Loo, Rachel R. (November 2022, Frontiers in Microbiology)

   Syntrophomonas wolfei is an anaerobic syntrophic microbe that degrades short-chain fatty acids to acetate, hydrogen, and/or formate. This thermodynamically unfavorable process proceeds through a series of reactive acyl-Coenzyme A species (RACS). In other prokaryotic and eukaryotic systems, the production of intrinsically reactive metabolites correlates with acyl-lysine modifications, which have been shown to play a significant role in metabolic processes. Analogous studies with syntrophic bacteria, however, are relatively unexplored and we hypothesized that highly abundant acylations could exist in S. wolfei proteins, corresponding to the RACS derived from degrading fatty acids. Here, by mass spectrometry-based proteomics (LC–MS/MS), we characterize and compare acylome profiles of two S. wolfei subspecies grown on different carbon substrates. Because modified S. wolfei proteins are sufficiently abundant to analyze post-translational modifications (PTMs) without antibody enrichment, we could identify types of acylations comprehensively, observing six types (acetyl-, butyryl-, 3- hydroxybutyryl-, crotonyl-, valeryl-, and hexanyl-lysine), two of which have not been reported in any system previously. All of the acyl-PTMs identified correspond directly to RACS in fatty acid degradation pathways. A total of 369 sites of modification were identified on 237 proteins. Structural studies and in vitro acylation assays of a heavily modified enzyme, acetyl-CoA transferase, provided insight on the potential impact of these acyl-protein modifications. The extensive changes in acylation-type, abundance, and modification sites with carbon substrate suggest that protein acylation by RACS may be an important regulator of syntrophy. 

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3. DeepSuccinylSite: a deep learning based approach for protein succinylation site prediction.

   Niraj Thapa, Meenal Chaudhari (April 2020, BMC bioinformatics)

   Protein succinylation has recently emerged as an important and common post-translation modification (PTM) that occurs on lysine residues. Succinylation is notable both in its size (e.g., at 100 Da, it is one of the larger chemical PTMs) and in its ability to modify the net charge of the modified lysine residue from + 1 to − 1 at physiological pH. The gross local changes that occur in proteins upon succinylation have been shown to correspond with changes in gene activity and to be perturbed by defects in the citric acid cycle. These observations, together with the fact that succinate is generated as a metabolic intermediate during cellular respiration, have led to suggestions that protein succinylation may play a role in the interaction between cellular metabolism and important cellular functions. For instance, succinylation likely represents an important aspect of genomic regulation and repair and may have important consequences in the etiology of a number of disease states. In this study, we developed DeepSuccinylSite, a novel prediction tool that uses deep learning methodology along with embedding to identify succinylation sites in proteins based on their primary structure. 

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4. The ribosome: A hot spot for the identification of new types of protein methyltransferases

   https://doi.org/10.1074/jbc.AW118.003235  

   Clarke, Steven G. (January 2018, Journal of Biological Chemistry)

   Cellular physiology depends on the alteration of protein structures by covalent modification reactions. Using a combination of bioinformatic, genetic, biochemical, and mass spectrometric approaches, it has been possible to probe ribosomal proteins from the yeast Saccharomyces cerevisiae for posttranslationally methylated amino acid residues and for the enzymes that catalyze these modifications. These efforts have resulted in the identification and characterization of the first protein histidine methyltransferase, the first N-terminal protein methyltransferase, two unusual types of protein arginine methyltransferases, and a new type of cysteine methylation. Two of these enzymes may modify their substrates during ribosomal assembly because the final methylated histidine and arginine residues are buried deep within the ribosome with contacts only with RNA. Two of these modifications occur broadly in eukaryotes, including humans, while the others demonstrate a more limited phylogenetic range. Analysis of strains where the methyltransferase genes are deleted has given insight into the physiological roles of these modifications. These reactions described here add diversity to the modifications that generate the typical methylated lysine and arginine residues previously described in histones and other proteins. 

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5. Amino acid and protein specificity of protein fatty acylation in Caenorhabditis elegans

   https://doi.org/10.1073/pnas.2307515121  

   Zhang, Bingsen; Yu, Yan; Fox, Bennett W; Liu, Yinong; Thirumalaikumar, Venkatesh P; Skirycz, Aleksandra; Lin, Hening; Schroeder, Frank C (January 2024, Proceedings of the National Academy of Sciences)

   Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins ofS-acyl moieties differ fromN- andO-fatty acylation. Here, we show that fatty acylation patterns inCaenorhabditis elegansdiffer markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteineS-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry–capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013S-acylated proteins and 510 hydroxylamine-resistantN- orO-acylated proteins. Subsets ofS-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including theS-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels. 

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