Acyl Modification Research Articles

Acylation modification of konjac glucomannan and its adsorption of Fe (Ⅲ) ion

A biodegradable adsorbent, modified konjac glucomannan (MKGM), was prepared by konjac glucomannan (KGM) acylated with phthalic anhydride catalyzed using concentrated sulfuric acid. The modified conditions such as reaction temperature, mass ratio of phthalic anhydride to KGM, catalyst dosage and reaction time were investigated, respectively. MKGM exhibited preferable adsorption performance for the removal of Fe (Ⅲ) ion. The adsorption behavior was discussed using the Langmuir and Freundlich isotherm models. The results showed that the Freundlich linear model was suitable for describing the adsorption process of Fe (Ⅲ). The maximum adsorption capacity of MKGM for Fe (Ⅲ) ion was 31.87 mg g−1 at 298 K. The kinetics studies suggested that adsorption process followed the pseudo-second-order model and the adsorption process was mainly controlled by both surface reactivity and intra-particle diffusion. Together with the evaluation of the thermodynamic parameters such as Gibbs free energy, enthalpy and entropy changes, the results indicated that the adsorption process of Fe (Ⅲ) was endothermic, feasible, and spontaneous in nature. Hence, as a bioadsorbent, the MKGM has a promising potential for the removal of Fe (Ⅲ) ion from aqueous solutions.

HybridSucc: A Hybrid-learning Architecture for General and Species-specific Succinylation Site Prediction

As an important protein acylation modification, lysine succinylation (Ksucc) is involved in diverse biological processes, and participates in human tumorigenesis. Here, we collected 26,243 non-redundant known Ksucc sites from 13 species as the benchmark data set, combined 10 types of informative features, and implemented a hybrid-learning architecture by integrating deep-learning and conventional machine-learning algorithms into a single framework. We constructed a new tool named HybridSucc, which achieved area under curve (AUC) values of 0.885 and 0.952 for general and human-specific prediction of Ksucc sites, respectively. In comparison, the accuracy of HybridSucc was 17.84%–50.62% better than that of other existing tools. Using HybridSucc, we conducted a proteome-wide prediction and prioritized 370 cancer mutations that change Ksucc states of 218 important proteins, including PKM2, SHMT2, and IDH2. We not only developed a high-profile tool for predicting Ksucc sites, but also generated useful candidates for further experimental consideration. The online service of HybridSucc can be freely accessed for academic research at http://hybridsucc.biocuckoo.org/.

Pan-Specific Antibodies as Novel Tools to Detect Valyllysine

Objective: Amino acyl modification of lysine residues is an essential mechanism of nutrient sensing that regulates various biological functions including reproduction. At present, the lack of pan-specific antibodies for a recently identified lysine valylation hinders the characterization and detection of this modification. The objective of this study is to raise pan-specific antibodies that may facilitate the identification of novel expression patterns of lysine valylation. Methods: Chicken ovalbumin was valylated as an immunogen to raise polyclonal antibodies (PcAbs) in rabbits. The population of the pan-specific antibodies recognizing valylated lysine was purified using the chemically synthesized valylated peptides consisting of random amino acids. The specificity of the antibodies was evaluated using ELISA, dot blots, Western blots, and immunohistochemistry (IHC) staining in human epididymis as well. Results: A preliminary and simple strategy to make an anti-valylated lysine PcAb was developed. The recognition of the antibodies to valyllysine was evaluated as pan specific. This was useful for the detection of the newly identified valyl modification using ELISA, dot blots, and Western blots. The antibodies were also successfully utilized in IHC assays, which revealed novel valyllysine modification patterns in epididymis tissues of human. Conclusions: A new antibody tool was provided for the study of lysine valylation. The novel expression patterns of valyllysine in the epididymis suggest that this modification may be involved in sperm maturation.

Computational Identification of Lysine Glutarylation Sites Using Positive- Unlabeled Learning

Background As a new type of protein acylation modification, lysine glutarylation has been found to play a crucial role in metabolic processes and mitochondrial functions. To further explore the biological mechanisms and functions of glutarylation, it is significant to predict the potential glutarylation sites. In the existing glutarylation site predictors, experimentally verified glutarylation sites are treated as positive samples and non-verified lysine sites as the negative samples to train predictors. However, the non-verified lysine sites may contain some glutarylation sites which have not been experimentally identified yet.Methods In this study, experimentally verified glutarylation sites are treated as the positive samples, whereas the remaining non-verified lysine sites are treated as unlabeled samples. A bioinformatics tool named PUL-GLU was developed to identify glutarylation sites using a positive-unlabeled learning algorithm.Results Experimental results show that PUL-GLU significantly outperforms the current glutarylation site predictors. Therefore, PUL-GLU can be a powerful tool for accurate identification of protein glutarylation sites.Conclusion A user-friendly web-server for PUL-GLU is available at http://bioinform.cn/pul_glu/.

Sensitive fluorogenic substrates for sirtuin deacylase inhibitor discovery

Sirtuins (SIRTs) are NAD+-dependent lysine deacylases, regulating many important biological processes such as metabolism and stress responses. SIRT inhibitors may provide potential benefits against SIRT-driven human diseases. Development of efficient assay platforms based on fluorogenic substrates will facilitate the discovery of high-quality SIRT inhibitors. We here report 16 new fluorogenic peptide substrates (P1–P16) designed with structurally diverse tetrapeptides and acyl modifications. Tests of P1–P16 against SIRT isoforms identified several sensitive substrates for SIRT1, SIRT2, SIRT3 and SIRT5, which manifested lower KM values and higher catalytic efficiency, and particularly had less signal interference in inhibitor screening compared with our previously reported internally quenched fluorescent substrates. Co-crystallization of sensitive substrates P13 and P15 with SIRT5 revealed an unexpected binding mode, involving interactions with residues from active site bordering surfaces, different from that observed for other peptides derived from natural protein substrates. By using SIRT5 sensitive substrates, we found that TW-37, a Bcl-2 inhibitor, displayed low micromolar inhibition to SIRT5, which was further validated by isothermal titration calorimetry analyses, offering a new point to develop dual-action SIRT5/Bcl-2 inhibitors against cancers. This work provides assay platform and structural basis for developing new substrates and inhibitors targeting human SIRTs.

Finding the gas pedal on a slow sirtuin.

The class III histone deacetylase sirtuin 6 (SIRT6) modulates numerous functions in the cell by deacetylating histone lysine residues. Interestingly, SIRT6's efficiency in in vitro experiments is far greater against substrates carrying long-chain fatty acyl modifications such as myristoylated lysine compared with acetylated counterparts, but the deacetylase activity can be stimulated by fatty acids and small-molecule allosteric modulators. A new study helps to explain this puzzling activation using a novel activator, thorough kinetic investigation, and mutagenesis studies. These data help elucidate the molecular requirements for activation of SIRT6 and provide a foundation for development of activators for therapeutic purposes.

Characterization of protein interaction surface on fatty acyl selectivity of starter condensation domain in lipopeptide biosynthesis.

Lipopeptides are important non-ribosomal peptide synthetases (NRPSs) products with broad therapeutic potential in biotechnology and biopharmaceutical applications. Fatty acyl modifications in N-terminal of lipopeptides have attracted wide interest in the engineering processes of altered fatty acyl selectivity. In this study, we focused on the starter condensation domain of antibiotic A54145 (lptC1) and its indiscriminate selectivity of fatty acyl substrates, which results in multi-component products. Using in silico analysis, five site-directed mutations at protein-protein interface were identified with altered activity and selectivity towards wildtype lptC1. The variants Y149W and A330T exhibited changed substrate selectivity to prefer longer branched chain fatty acyl substrate, while T16A and A350D showed improved selectivity for shorter linear chain fatty acyl substrates. Subsequently, molecular dynamics (MD) simulations were performed to analyze the impact of these residues on the changes of catalytic activity and conformation. Through in silico analysis, the altered binding free energy were coincident with the corresponding activity performance of the variants, and surface forces indicated that other factors or processes may influence the activity and selectivity. Moreover, the MD results revealed even altered active center conformations, implying the importance of these interface residues affected on distant active center thus reflected to catalysis activity. Based on the biochemistry and computational results, our work provides detailed insights from molecular and dynamics aspects into the role of C1's interface residues during complex NRPS biosynthesis machinery, prompting further rational engineering for lipopeptide catalysis.

Gcn5 and Esa1 function as histone crotonyltransferases to regulate crotonylation-dependent transcription

Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription.

Genome-wide Profiling of Histone Lysine Butyrylation Reveals its Role in the Positive Regulation of Gene Transcription in Rice

BackgroundHistone modifications play important roles in growth and development of rice (Oryza sativa L.). Lysine butyrylation (Kbu) with a four-carbon chain is a newly-discovered histone acylation modification in rice.Main BodyIn this study, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses, the result showed that major enrichment of histone Kbu located in genebody regions of rice genome, especially in exons. The enrichment level of Kbu histone modification is positively correlated with gene expression. Furthermore, we compared Kbu with DNase-seq and other histone modifications in rice. We found that 60.06% Kub enriched region co-located with DHSs in intergenic regions. The similar profiles were detected among Kbu and several acetylation modifications such as H3K4ac, H3K9ac, and H3K23ac, indicating that Kbu modification is an active signal of transcription. Genes with both histone Kbu and one other acetylation also had significantly increased expression compared with genes with only one acetylation. Gene Ontology (GO) enrichment analysis revealed that these genes with histone Kbu can regulate multiple metabolic process in different rice varieties.ConclusionOur study showed that the lysine butyrylation modificaiton may promote gene expression as histone acetylation and will provide resources for futher studies on histone Kbu and other epigenetic modifications in plants.

Anthocyanin Accumulation in the Leaves of the Purple Sweet Potato (Ipomoea batatas L.) Cultivars.

Sweet potato anthocyanins are water-soluble pigments with many physiological functions. Previous research on anthocyanin accumulation in sweet potato has focused on the roots, but the accumulation progress in the leaves is still unclear. Two purple sweet potato cultivars (Fushu No. 23 and Fushu No. 317) with large quantities of anthocyanin in the leaves were investigated. Anthocyanin composition and content were assessed with ultra-performance liquid chromatography diode-array detection (UPLC-DAD) and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and the expressions of genes were detected by qRT-PCR. The two cultivars contained nine cyanidin anthocyanins and nine peonidin anthocyanins with an acylation modification. The acylation modification of anthocyanins in sweet potato leaves primarily included caffeoyl, p-coumaryl, feruloyl, and p-hydroxy benzoyl. We identified three anthocyanin compounds in sweet potato leaves for the first time: cyanidin 3-p-coumarylsophoroside-5-glucoside, peonidin 3-p-coumarylsophoroside-5-glucoside, and cyanidin 3-caffeoyl-p-coumarylsophoroside-5-glucoside. The anthocyanidin biosynthesis downstream structural genes DFR4, F3H1, anthocyanin synthase (ANS), and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT3), as well as the transcription factor MYB1, were found to be vital regulatory genes during the accumulation of anthocyanins in sweet potato leaves. The composition of anthocyanins (nine cyanidin-based anthocyanins and nine peonidin-based anthocyanins) in all sweet potato leaves were the same, but the quantity of anthocyanins in leaves of sweet potato varied by cultivar and differed from anthocyanin levels in the roots of sweet potatoes. The anthocyanidin biosynthesis structural genes and transcription factor together regulated and controlled the anthocyandin biosynthesis in sweet potato leaves.

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein β-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

Identification, Quantification, and System Analysis of Protein N-ε Lysine Methylation in Anucleate Blood Platelets.

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N-ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl-lysine (meK) proteome of anucleate blood platelets is characterized. With high-resolution, multiplex MS methods, 190 mono-, di-, and tri-meK modifications are identified on 150 different platelet proteins-including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin-like protein (DBNL, Hip-55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.

Characterizing a novel bacterial sirtuin protein in an intact protein system

Sirtuins, proteins that remove acyl-modifications from lysine side chains, have many important medical and biological applications as they are involved in a wide range of cellular processes such as aging, transcriptional regulation through histone tail modification, and stress resistance. They have been suggested to mitigate damage caused by carbon stress and its associated non-enzymatic modification of protein by reactive acyl-CoA species. Protein hyperacylation has been associated with altered metabolic flux or reduced enzymatic activities. Syntrophus aciditrophicus, a bacterium existing in extreme conditions, displays unusually high levels of protein acylation, leading us to wonder whether sirtuins are present in this system to remove these acyl modifications. Sirtuin homologs have been found in these bacteria by genetic homology, but their substrate specificities and activities have not been studied. We developed a novel mass spectrometry based enzymatic assay to test the range of acyl groups S. aciditrophicus sirtuins could recognize and measured their reaction kinetics. Novel sirtuin homologs (Syn42, Syn1020) from S. aciditrophicus were expressed recombinantly. Insulin was chemically modified with acyl-anhydrides corresponding to the desired acyl modification (e.g. acetic anhydride for acetyl-modified insulin, propionic anhydride for propionyl-modified insulin) as a proxy for an acyl-modified substrate. Sirtuin (10 μM) was added to varyied concentrations of acyl-insulin in ammonium acetate buffer (pH ~7.5), with excess NAD+. The reaction was halted at timepoints 1, 3, 5, 10, and 15 minutes by mixing with acetonitrile 1:1 and ubiquitin was added into the mixture as an internal standard for mass spectrometric quantitation. Assay contents were analyzed by mass spectrometry. We have shown that the novel sirtuin Syn42 acts on several acylations including butyryl, glutaryl, succinyl, and propionyllysines. In addition to determining acyl-group substrate specificity, we were also able to measure Michaelis-Menten kinetics, by plotting time courses of sirtuin enzymatic activity. For glutaryl-modified insulin, the Km was determined to be 16.92 μM and the Kcat was determined to be 0.0022 sec−1, which are comparable to previous measurements of mammalian sirtuin SIRT1. In conclusion, we were able to identify and confirm the activity of novel sirtuin from a bacterial source. We were also able to determine its acyl-group substrate specificity as well as its kinetic properties, demonstrating that S. aciditrophicus sirutins have properties on par with those found in mammalian systems. Support or Funding Information This work is funded by the US Department of Energy (UCLA Institute of Genomics and Proteomics, DE-FC03-02ER63421) and the National Institutes of Health (R01GM085402). MALDI-TOF data shows that the bacterial sirtuins will act on a glutaryl modification Comparing of the ratio of the deacylated insulin intensity to that of ubiquitin over time (or the ratio of acylated insulin intensity to ubiquitin) further displays the enzymatic activity on modified insulin. The shift in the ratio of the modified and demodified insulin can be seen at multiple time points using electrospray mass spectrometry. A time course shows the relative intensities of glutaryl-insulin (200 μM) and ubiquitin as seen over a course of 2 hours. Michaelis-Menten kinetics that have been determined for the sirtuin homolog's activity on glutaryllysine. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct Analysis of Protein S-Acylation by Mass Spectrometry.

Dynamic and reversible protein S-acylation, most commonly occurring as S-palmitoylation, plays an important role in protein/membrane association and the regulation of intracellular signaling via cycles of palmitoylation and depalmitoylation. Direct analysis of protein S-acylation by mass spectrometry (MS) offers several benefits over indirect detection methods in that it can definitively determine the location and nature of the acyl modification, and is not prone to false discoveries. However, characterization of acyl proteins is challenging because of the tendency of acyl loss during sample preparation and tandem MS analysis. In this chapter, we present a sample preparation protocol that preserves labile acyl modifications and an LC-MS/MS workflow for detection of S-acylation with high confidence and sensitivity.

DHHC4 and DHHC5 Facilitate Fatty Acid Uptake by Palmitoylating and Targeting CD36 to the Plasma Membrane

Fatty acid uptake is the first step in fatty acid utilization, but it remains unclear how the process is regulated. Protein palmitoylation is a fatty acyl modification that plays a key regulatory role in protein targeting and trafficking; however, its function in regulating fatty acid metabolism is unknown. Here, we show that two of the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases, DHHC4 and DHHC5, regulate fatty acid uptake. DHHC4 and DHHC5 function at different subcellular localizations to control the palmitoylation, plasma membrane localization, and fatty acid uptake activity of the scavenger receptor CD36. Depletion of either DHHC4 or DHHC5 in cells disrupts CD36-dependent fatty acid uptake. Furthermore, both Dhhc4-/- and adipose-specific Dhhc5 knockout mice show decreased fatty acid uptake activity in adipose tissues and develop severe hypothermia upon acute cold exposure. These findings demonstrate a critical role of DHHC4 and DHHC5 in regulating fatty acid uptake.

DNA Methylation and Transcription Factors Competitively Regulate SIRT4 Promoter Activity in Bovine Adipocytes: Roles of NRF1 and CMYB.

Sirtuin 4 (SIRT4) belongs to the mitochondrial sirtuin protein family, a class of NAD+-dependent protein deacylases that remove post-translational acyl modifications from cellular substrates during the regulation of various biological pathways. SIRT4 has been shown to regulate lipid homeostasis. However, the mechanism by which the bovine SIRT4 gene is transcriptionally regulated remains unknown. To explore the molecular mechanism of SIRT4 expression, we obtained a 400-kb fragment of the 5'-regulatory region of bovine SIRT4 by molecular cloning, which contained a CpG island. Electrophoretic mobility shift assays and luciferase reporter gene assays identified the nuclear respiratory factor 1 (NRF1) and myb proto-oncogene protein (CMYB) binding sites as transcriptional repression and activation sites in the SIRT4 promoter region, respectively. We further verified that NRF1 and CMYB bind to the SIRT4 promoter using chromatin immunoprecipitation assays. In addition, from DNA methylation and reporter gene assays, results revealed that SIRT4 promoter activity was enhanced by demethylation. Further, NRF1-mediated transcriptional inhibition and CMYB-mediated transcriptional activation of SIRT4 expression were strengthened by demethylation during bovine adipocyte differentiation. Taken together, our results shed light on the mechanism underlying the promoter methylation and transcriptional regulation of SIRT4 expression in bovine adipocytes.

Whole-Cell Catalytic Synthesis of Puerarin Monoesters and Analysis of Their Antioxidant Activities.

Puerarin, an important isoflavonoid from the edible root of Pueraria lobata, shows multiple bioactivities but suffers from low bioavailability. In this study, a new whole-cell catalytic method for acylation modification of puerarin was developed. Among the 12 strains tested, Aspergillus oryzae showed the highest catalytic activity and selectively catalyzed acylation of puerarin at the 6″-hydroxyl group. The organic solvents used significantly influenced the catalytic efficiency of the cells. In the green solvent 2-methyltetrahydrofuran, the reaction showed high substrate conversion (92.5%) and regioselectivity (95.8%), with results similar to those with tetrahydrofuran (94.2% and 98.5%, respectively) under optimal conditions. The monoester products showed higher liposolubility in comparison to puerarin, and those with C3-C8 fatty acid chain lengths showed evidently improved antioxidant activity toward erythrocyte hemolysis. Considering the operational stability of the cells and efficiency of the scaled-up reactions, this method is efficient and cost effective, with promising applications in the health food industry.

Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding

Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.

The apolipoprotein N-acyl transferase Lnt is dispensable for growth in Acinetobacter species.

Directing the flow of protein traffic is a critical task faced by all cellular organisms. In Gram-negative bacteria, this traffic includes lipoproteins. Lipoproteins are synthesized as precursors in the cytoplasm and receive their acyl modifications upon export across the inner membrane. The third and final acyl chain is added by Lnt, which until recently was thought to be essential in all Gram-negatives. In this report, we show that Acinetobacter species can also tolerate a complete loss-of-function mutation in lnt. Absence of a fully functional Lnt impairs modification of lipoproteins, increases outer membrane permeability and susceptibility to antibiotics, and alters normal cellular morphology. In addition, we show that loss of lnt triggers a global transcriptional response to this added cellular stress. Taken together, our findings provide new insights on and support the growing revisions to the Gram-negative lipoprotein biogenesis paradigm.

Protein Acetylation and Butyrylation Regulate the Phenotype and Metabolic Shifts of the Endospore-forming Clostridium acetobutylicum

Clostridium acetobutylicum is a strict anaerobic, endospore-forming bacterium, which is used for the production of the high energy biofuel butanol in metabolic engineering. The life cycle of C. acetobutylicum can be divided into two phases, with acetic and butyric acids being produced in the exponential phase (acidogenesis) and butanol formed in the stationary phase (solventogenesis). During the transitional phase from acidogenesis to solventogenesis and latter stationary phase, concentration peaks of the metabolic intermediates butyryl phosphate and acetyl phosphate are observed. As an acyl group donor, acyl-phosphate chemically acylates protein substrates. However, the regulatory mechanism of lysine acetylation and butyrylation involved in the phenotype and solventogenesis of C. acetobutylicum remains unknown. In our study, we conducted quantitative analysis of protein acetylome and butyrylome to explore the dynamic change of lysine acetylation and butyrylation in the exponential phase, transitional phase, and stationary phase of C. acetobutylicum Total 458 lysine acetylation sites and 1078 lysine butyrylation sites were identified in 254 and 373 substrates, respectively. Bioinformatics analysis uncovered the similarities and differences between the two acylation modifications in C. acetobutylicum Mutation analysis of butyrate kinase and the central transcriptional factor Spo0A was performed to characterize the unique role of lysine butyrylation in the metabolic pathway and sporulation process of C. acetobutylicum Moreover, quantitative proteomic assays were performed to reveal the relationship between protein features (e.g. gene expression level and lysine acylation level) and metabolites in the three growth stages. This study expanded our knowledge of lysine acetylation and butyrylation in Clostridia and constituted a resource for functional studies on lysine acylation in bacteria.