Lysine Acetylation Sites Research Articles

Acetylome and Succinylome Profiling of Edwardsiella tarda Reveals Key Roles of Both Lysine Acylations in Bacterial Antibiotic Resistance.

The antibiotic resistance of Edwardsiella tarda is becoming increasingly prevalent, and thus novel antimicrobial strategies are being sought. Lysine acylation has been demonstrated to play an important role in bacterial physiological functions, while its role in bacterial antibiotic resistance remains largely unclear. In this study, we investigated the lysine acetylation and succinylation profiles of E. tarda strain EIB202 using affinity antibody purification combined with LC-MS/MS. A total of 1511 lysine-acetylation sites were identified on 589 proteins, and 2346 lysine-succinylation sites were further identified on 692 proteins of this pathogen. Further bioinformatic analysis showed that both post-translational modifications (PTMs) were enriched in the tricarboxylic acid (TCA) cycle, pyruvate metabolism, biosynthesis, and carbon metabolism. In addition, 948 peptides of 437 proteins had overlapping associations with multiple metabolic pathways. Moreover, both acetylation and succinylation were found in many antimicrobial resistance (AMR) proteins, suggesting their potentially vital roles in antibiotic resistance. In general, our work provides insights into the acetylome and succinylome features responsible for the antibiotic resistance mechanism of E. tarda, and the results may facilitate future investigations into the pathogenesis of this bacterium.

Alterations of Lysine Acetylation Profile in Murine Skeletal Muscles Upon Exercise.

ObjectiveRegular exercise is a powerful tool that enhances skeletal muscle mass and strength. Lysine acetylation is an important post-translational modification (PTM) involved in a broad array of cellular functions. Skeletal muscle protein contains a considerable number of lysine-acetylated (Kac) sites, so we aimed to investigate the effects of exercise-induced lysine acetylation on skeletal muscle proteins.MethodsWe randomly divided 20 male C57BL/6 mice into exercise and control groups. After 6 weeks of treadmill exercise, a lysine acetylation proteomics analysis of the gastrocnemius muscles of mice was performed.ResultsA total of 2,254 lysine acetylation sites in 693 protein groups were identified, among which 1,916 sites in 528 proteins were quantified. The enrichment analysis suggested that protein acetylation could influence both structural and functional muscle protein properties. Moreover, molecular docking revealed that mimicking protein deacetylation primarily influenced the interaction between substrates and enzymes.ConclusionExercise-induced lysine acetylation appears to be a crucial contributor to the alteration of skeletal muscle protein binding free energy, suggesting that its modulation is a potential approach for improving exercise performance.

Novel Regulation of Nitric Oxide Synthase 3 (NOS3) Isoform by Acetylation at Lysine 610

NOS3‐mediated NO regulates endothelial and kidney epithelial function. Lysine acetylation is a dynamic post‐translational modification influencing cell signaling. Our lab and others showed NOS3 is regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites of NOS3 and investigated lysines in the NOS3 reductase domain, K607, K610, K727, and K731. We hypothesized that deacetylation of one or more of these lysines on NOS3 would impact NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation by HPLC in the media over 48 hours. Mutation of all four lysines or lysines K607 and K610 dramatically increased NO production whereas K727 and K731 did not change NO production compared to wildtype (pmol NO2/mg protein, WT: 2271 ± 729.3; K607/610/727/731R: 15176 ± 2759, p=0.046 vs WT; K607/610R: 9431 ± 1772, p=0.0019 vs WT; K727/731R: 3523 ± 805.8, p=0.918). We further investigated single mutation of K607 and K610. We found K610R (10564 ± 1883 pmol NO2/mg protein, p=0.002 vs WT), but not K607R (1589 ± 412.0 pmol NO2/mg protein, p>0.99) to have increased NO production compared to WT (1547 ± 180.8 pmol NO2/mg protein). We then hypothesized that acetylation of K610 on NOS3 would decrease NO production compared to deacetylation of K610. We mutated K610 to glutamine mimicking acetylation of this lysine. Contrary to what we hypothesized, we found K610Q to produce greater NO than both WT and K610R (pmol NO2/mg protein, WT: 2196 ± 606.1, K610Q: 30212 ± 880.2, p<0.0001 WT vs K610Q; K610R:13389 ± 1086, p<0.0001 K610R vs K610Q). Previous reports show that amino acids 605‐612 coordinate regulatory phosphorylation sites of NOS3. We therefore investigated NOS3 K610R and K610Q relative to WT at key phosphorylation regulatory sites by western blot. We found K610R and K610Q to have reduced phosphorylation at T495 (RDU; WT: 1.00 ± 0.252; K610R: 0.820 ± 0.251, p=0.0257; K610Q: 0.711 ± 0.205, p=0.0046), increased phosphorylation at S1177 (WT: 1.00 ± 0.125; K610R: 1.58 ± 0.2921, p=0.038; K610Q: 1.93 ± 0.358, p=0.0082), and no change in phosphorylation at S633 (WT: 1.00 ± 0.054; K610R: 0.885 ± 0.0536, p=0.126; K610Q: 0.955 ± 0.0779, p=0.319). Additionally, K610R but not K610Q was found to have increased S615 phosphorylation (WT: 1.00 ± 0.112; K610R: 2.01 ± 0.259, p=0.0003; K610Q: 1.07 ± 0.112, p=0.682). These data suggest acetylation of K610 would significantly increase NO production and regulate NOS3 phosphorylation sites in endothelial cells and/or kidney epithelial cells. We propose that acetylation/deacetylation of K610 would have dramatic consequences for blood pressure regulation as well as water and electrolyte homeostasis.

A Comprehensive Analysis of the Lysine Acetylome in the Aquatic Animals Pathogenic Bacterium Vibrio mimicus

Protein lysine acetylation is an evolutionarily conserved post-translational modification (PTM), which is dynamic and reversible, playing a crucial regulatory role in almost every aspect of metabolism, of both eukaryotes and prokaryotes. Several global lysine acetylome studies have been carried out in various bacteria, but thus far, there have been no reports of lysine acetylation for the commercially important aquatic animal pathogen Vibrio mimicus. In the present study, we used anti-Ac-K antibody beads to highly sensitive immune-affinity purification and combined high-resolution LC-MS/MS to perform the first global lysine acetylome analysis in V. mimicus, leading to the identification of 1,097 lysine-acetylated sites on 582 proteins, and more than half (58.4%) of the acetylated proteins had only one site. The analysis of acetylated modified peptide motifs revealed six significantly enriched motifs, namely, KacL, KacR, L(-2) KacL, LKacK, L(-7) EKac, and IEKac. In addition, bioinformatic assessments state clearly that acetylated proteins have a hand in many important biological processes in V. mimicus, such as purine metabolism, ribosome, pyruvate metabolism, glycolysis/gluconeogenesis, the TCA cycle, and so on. Moreover, 13 acetylated proteins were related to the virulence of V. mimicus. To sum up, this is a comprehensive analysis whole situation protein lysine acetylome in V. mimicus and provides an important foundation for in-depth study of the biological function of lysine acetylation in V. mimicus.

Investigation of the Importance of Protein 3D Structure for Assessing Conservation of Lysine Acetylation Sites in Protein Homologs.

Acetylation is a protein post-translational modification (PTM) that can affect a variety of cellular processes. In bacteria, two PTM Nε-acetylation mechanisms have been identified: non-enzymatic/chemical acetylation via acetyl phosphate or acetyl coenzyme A and enzymatic acetylation via protein acetyltransferases. Prior studies have shown that extensive acetylation of Nε-lysine residues of numerous proteins from a variety of bacteria occurs via non-enzymatic acetylation. In Escherichia coli, new Nε-lysine acetyltransferases (KATs) that enzymatically acetylate other proteins have been identified, thus expanding the repertoire of protein substrates that are potentially regulated by acetylation. Therefore, we designed a study to leverage the wealth of structural data in the Protein Data Bank (PDB) to determine: (1) the 3D location of lysine residues on substrate proteins that are acetylated by E. coli KATs, and (2) investigate whether these residues are conserved on 3D structures of their homologs. Five E. coli KAT substrate proteins that were previously identified as being acetylated by YiaC and had 3D structures in the PDB were selected for further analysis: adenylate kinase (Adk), isocitrate dehydrogenase (Icd), catalase HPII (KatE), methionyl-tRNA formyltransferase (Fmt), and a peroxide stress resistance protein (YaaA). We methodically compared over 350 protein structures of these E. coli enzymes and their homologs; to accurately determine lysine residue conservation requires a strategy that incorporates both flexible structural alignments and visual inspection. Moreover, our results revealed discrepancies in conclusions about lysine residue conservation in homologs when examining linear amino acid sequences compared to 3D structures.

Recent Trends on the Development of Machine Learning Approaches for the Prediction of Lysine Acetylation Sites.

Acetylation on lysine residues is considered one of the most potent protein post-translational modifications, owing to its crucial role in cellular metabolism and regulatory processes. Recent advances in experimental techniques have unraveled several lysine acetylation substrates and sites. However, owing to its cost-ineffectiveness, cumbersome process, time-consumption, and labor-intensiveness, several efforts have been geared towards the development of computational tools. In particular, machine learning (ML)-based approaches hold great promise in the rapid discovery of lysine acetylation modification sites, which could be witnessed by the growing number of prediction tools. Recently, several ML methods have been developed for the prediction of lysine acetylation sites, owing to their time- and cost-effectiveness. In this review, we present a complete survey of the state-of-the-art ML predictors for lysine acetylation. We discuss a variety of key aspects for developing a successful predictor, including operating ML algorithms, feature selection methods, validation techniques, and software utility. Initially, we review lysine acetylation site databases, current ML approaches, working principles, and their performances. Lastly, we discuss the shortcomings and future directions of ML approaches in the prediction of lysine acetylation sites. This review may act as a useful guide for the experimentalists in choosing the right ML tool for their research. Moreover, it may help bioinformaticians in the development of more accurate and advanced MLbased predictors in protein research.

Global Lysine Acetylome Analysis of Flower bud Development in Catalpa bungei

Lysine acetylation, a reversible and dynamic post-translational modification, plays pivotal roles in regulating many biological processes in plants. However, the information of lysine acetylation during flower development of woody angiosperm remains unclear. Herein, we identified a total of 667 lysine acetylation sites in 467 proteins in flower buds of Catalpa Bungei. The acetylated proteins were mainly involved in the pathways of carbon metabolism, glycometabolism and oxidative phosphorylation. Using functional enrichment analysis, 61 and 13 acetylated proteins were involved in the glycometabolism and oxidative phosphorylation pathways, respectively. This suggests that lysine acetylated proteins play critical roles in the energy metabolism pathways. Accordingly, the protein–protein interactions were also highly enriched in the carbon metabolic pathway. Integrative analysis of lysine acetylome and transcriptome showed that transcript level changes of seven key lysine-acetylated proteins/genes involved in energy metabolism were significant different during floral transition. Importantly, compared with the normal flowering variety, the expression levels of phosphoribulokinase, phosphoenolpyruvate carboxykinase, ATP synthase and pyruvate dehydrogenase complex genes were significantly up-regulated at the stages of vegetative and transition buds during early flowering of C. bungei. These data represent the first lysine acetylome in the C. bungei flowers, and can serve as an essential basis for exploring the function of lysine acetylated proteins in the flower bud development of plants.

Bert-Kace: Prediction of Prokaryotic Lysine Acetylation Sites Based on Pre-Trained Bert Model

Bert-Kace: Prediction of Prokaryotic Lysine Acetylation Sites Based on Pre-Trained Bert Model

CREB1K292 and HINFPK330 as Putative Common Therapeutic Targets in Alzheimer's and Parkinson's Disease.

Integration of omics data and deciphering the mechanism of a biological regulatory network could be a promising approach to reveal the molecular mechanism involved in the progression of complex diseases, including Alzheimer’s and Parkinson’s. Despite having an overlapping mechanism in the etiology of Alzheimer’s disease (AD) and Parkinson’s disease (PD), the exact mechanism and signaling molecules behind them are still unknown. Further, the acetylation mechanism and histone deacetylase (HDAC) enzymes provide a positive direction toward studying the shared phenomenon between AD and PD pathogenesis. For instance, increased expression of HDACs causes a decrease in protein acetylation status, resulting in decreased cognitive and memory function. Herein, we employed an integrative approach to analyze the transcriptomics data that established a potential relationship between AD and PD. Data preprocessing and analysis of four publicly available microarray datasets revealed 10 HUB proteins, namely, CDC42, CD44, FGFR1, MYO5A, NUMA1, TUBB4B, ARHGEF9, USP5, INPP5D, and NUP93, that may be involved in the shared mechanism of AD and PD pathogenesis. Further, we identified the relationship between the HUB proteins and transcription factors that could be involved in the overlapping mechanism of AD and PD. CREB1 and HINFP were the crucial regulatory transcription factors that were involved in the AD and PD crosstalk. Further, lysine acetylation sites and HDAC enzyme prediction revealed the involvement of 15 and 27 potential lysine residues of CREB1 and HINFP, respectively. Our results highlighted the importance of HDAC1(K292) and HDAC6(K330) association with CREB1 and HINFP, respectively, in the AD and PD crosstalk. However, different datasets with a large number of samples and wet lab experimentation are required to validate and pinpoint the exact role of CREB1 and HINFP in the AD and PD crosstalk. It is also possible that the different datasets may or may not affect the results due to analysis parameters. In conclusion, our study potentially highlighted the crucial proteins, transcription factors, biological pathways, lysine residues, and HDAC enzymes shared between AD and PD at the molecular level. The findings can be used to study molecular studies to identify the possible relationship in the AD–PD crosstalk.

Lysine Acetylation Stoichiometry Analysis at the Proteome Level.

Lysine acetylation is a widespread posttranslational modification (PTM) in all kingdoms of live. A large number of proteins involved in most of biological pathways are targets of this PTM. The lysine acetylation is a reversible modification controlled by two main groups of enzymes, lysine acetyltransferases responsible for transferring the acetyl group of acetylCoA to the side chain of lysine residues and lysine deacetylases which effectively remove the acetyl tag. Dysregulation of enzymes that control acetylation and/or target proteins have been associated with a growing number of human pathologies. Lysine acetylation is largely a modification that occurs at low stoichiometry at its target sites. Here we describe a method to identify lysine acetylation sites and estimate their site occupancy at the proteome scale. The method relies on a high-resolution mass spectrometry-based proteomics approach, which includes a specific chemical acetylation reaction on unmodified lysine residues that carry heavy isotopes. The procedures described here have been applied to cell line cultures and to clinically relevant samples stored as both snap-frozen and formalin-fixed paraffin-embedded (FFPE) tissues.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

Acetylation of conserved lysines fine-tunes mitochondrial malate dehydrogenase activity in land plants.

Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

Assays to Study Enzymatic and Non‐Enzymatic Protein Lysine Acetylation In Vitro

Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(ε)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.

Acetylation Profiles in the Metabolic Process of Glioma-Associated Seizures.

Objective: We test the hypothesis that lysine acetylation is involved in the metabolic process of glioma-associated seizures (GAS).Methods: We used label-free mass spectrometry-based quantitative proteomics to quantify dynamic changes of protein acetylation between gliomas with seizure (CA1 group) and gliomas without seizure (CA2 group). Furthermore, differences of acetyltransferase and deacetylase expression between CA1 and CA2 groups were performed by a quantitative proteomic study. We further classified acetylated proteins into groups according to cell component, molecular function, and biological process. In addition, metabolic pathways and protein interaction networks were analyzed. Regulated acetyltransferases and acetylated profiles were validated by PRM and Western blot.Results: We detected 169 downregulated lysine acetylation sites of 134 proteins and 39 upregulated lysine acetylation sites of 35 proteins in glioma with seizures based on acetylome. We detected 407 regulated proteins by proteomics, from which ACAT2 and ACAA2 were the differentially regulated enzymes in the acetylation of GAS. According to the KEGG analysis, the upregulated acetylated proteins within the PPIs were mapped to pathways involved in the TCA cycle, oxidative phosphorylation, biosynthesis of amino acids, and carbon metabolism. The downregulated acetylated proteins within the PPIs were mapped to pathways involved in fatty acid metabolism, oxidative phosphorylation, TCA cycle, and necroptosis. Regulated ACAT2 expression and acetylated profiles were validated by PRM and Western blot.Conclusions: The data support the hypothesis that regulated protein acetylation is involved in the metabolic process of GAS, which may be induced by acetyl-CoA acetyltransferases.

STALLION: a stacking-based ensemble learning framework for prokaryotic lysine acetylation site prediction.

Protein post-translational modification (PTM) is an important regulatory mechanism that plays a key role in both normal and disease states. Acetylation on lysine residues is one of the most potent PTMs owing to its critical role in cellular metabolism and regulatory processes. Identifying protein lysine acetylation (Kace) sites is a challenging task in bioinformatics. To date, several machine learning-based methods for the in silico identification of Kace sites have been developed. Of those, a few are prokaryotic species-specific. Despite their attractive advantages and performances, these methods have certain limitations. Therefore, this study proposes a novel predictor STALLION (STacking-based Predictor for ProkAryotic Lysine AcetyLatION), containing six prokaryotic species-specific models to identify Kace sites accurately. To extract crucial patterns around Kace sites, we employed 11 different encodings representing three different characteristics. Subsequently, a systematic and rigorous feature selection approach was employed to identify the optimal feature set independently for five tree-based ensemble algorithms and built their respective baseline model for each species. Finally, the predicted values from baseline models were utilized and trained with an appropriate classifier using the stacking strategy to develop STALLION. Comparative benchmarking experiments showed that STALLION significantly outperformed existing predictor on independent tests. To expedite direct accessibility to the STALLION models, a user-friendly online predictor was implemented, which is available at: http://thegleelab.org/STALLION.

Comprehensive Proteome and Lysine Acetylome Analysis Reveals the Widespread Involvement of Acetylation in Cold Resistance of Pepper (Capsicum annuum L.).

Pepper is a typical warmth-loving vegetable that lacks a cold acclimation mechanism and is sensitive to cold stress. Lysine acetylation plays an important role in diverse cellular processes, but limited knowledge is available regarding acetylation modifications in the resistance of pepper plants to cold stress. In this study, the proteome and acetylome of two pepper varieties with different levels of cold resistance were investigated by subjecting them to cold treatments of varying durations followed by recovery periods. In total, 6,213 proteins and 4,574 lysine acetylation sites were identified, and this resulted in the discovery of 3,008 differentially expressed proteins and 768 differentially expressed acetylated proteins. A total of 1,988 proteins were identified in both the proteome and acetylome, and the functional differences in these co-identified proteins were elucidated through GO enrichment. KEGG analysis showed that 397 identified acetylated proteins were involved in 93 different metabolic pathways. The dynamic changes in the acetylated proteins in photosynthesis and the “carbon fixation in the photosynthetic organisms” pathway in pepper under low-temperature stress were further analyzed. It was found that acetylation of the PsbO and PsbR proteins in photosystem II and the PsaN protein in photosystem I could regulate the response of pepper leaves to cold stress. The acetylation levels of key carbon assimilation enzymes, such as ribulose bisphosphate carboxylase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, glyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, and triosephosphate isomerase decreased, leading to decreases in carbon assimilation capacity and photosynthetic efficiency, reducing the cold tolerance of pepper leaves. This study is the first to identify the acetylome in pepper, and it greatly expands the catalog of lysine acetylation substrates and sites in Solanaceae crops, providing new insights for posttranslational modification studies.

1258-P: Il-1ß-Induced Transcription of Nf-kB Target Genes Is Repressed by Bet Bromodomain Inhibitors in Pancreatic ß-Cells

Autoimmune diabetes is characterized by chronic inflammation of pancreatic islets which ultimately destroys insulin-producing β-cells. In the islet, elevated proinflammatory cytokines lead to increased transcription of inducible nitric oxide synthase (iNOS) and its downstream product, nitric oxide (NO). Elevated levels of NO for prolonged periods lead to β-cell apoptosis. The exact causes of autoimmune diabetes are not well defined and, although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins indicates a role for epigenetic factors. Recent studies suggest that the bromodomain and extraterminal domain (BET) family of epigenetic regulatory proteins contribute to the onset and progression of autoimmune diabetes. BET proteins are epigenetic readers of sites of lysine acetylation found commonly on histones and have emerged as promising drug targets for a wide variety of diseases. We hypothesize that BET proteins increase proinflammatory cytokine-induced transcription in β-cells leading to β-cell damage and that selective inhibition of BET bromodomains with small molecules will prevent or reverse the progression of autoimmune diabetes. Here, we show that BET bromodomain inhibitors decrease the β-cell response to proinflammatory cytokines in an insulinoma β-cell line (INS832/13) and primary rat islets. BET inhibitors attenuated cytokine-induced transcription of inflammatory mediators, including iNOS. This decreased transcription is mediated through inhibition of the NF-κB signaling pathway by BET inhibitors. Using knockdown of individual BET proteins in a β-cell line, we identified the importance of individual BET family members in the altered transcriptional profile characteristic of autoimmune diabetes. This work uncovers important mechanisms of disease onset and progression of autoimmune diabetes, laying the groundwork for more targeted treatments with drug-like small molecules. Disclosure J. Nord: None. S. Wynia-smith: None. B. C. Smith: None. R. Jones lipinski: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institutes of Health (R01DK119359, T32HL134643)

Quantitative proteomic analysis of aberrant expressed lysine acetylation in gastrointestinal stromal tumors

BackgroundGastrointestinal stromal tumor (GIST) is a common digestive tract tumor with high rate of metastasis and recurrence. Currently, we understand the genome, transcriptome and proteome in GIST. However, posttranscriptional modification features in GIST remain unclear. In the present study, we aimed to construct a complete profile of acetylome in GIST.MethodsFive common protein modifications, including acetylation, succinylation, crotonylation, 2-hydroxyisobutyrylation, and malonylation were tested among GIST subgroups and significantly differentially- expressed lysine acetylation was found. The acetylated peptides labeled with Tandem Mass Tag (TMT)under high sensitive mass spectrometry, and some proteins with acetylation sites were identified. Subsequently, these proteins and peptides were classified into high/moderate (H/M) risk and low (L) risk groups according to the modified NIH classification standard. Furthermore, cell components, molecular function, biological processes, KEGG pathways and protein interaction networks were analyzed.ResultsA total of 2904 acetylation sites from 1319 proteins were identified, of which quantitative information of 2548 sites from 1169 proteins was obtained. Finally, the differentially-expressed lysine acetylation sites were assessed and we found that 42 acetylated sites of 38 proteins were upregulated in the H/M risk group compared with the L risk group, while 48 acetylated sites of 44 proteins were downregulated, of which Ki67 K1063Ac and FCHSD2 K24Ac were the two acetylated proteins that were most changed.ConclusionsOur novel findings provide further understanding of acetylome in GIST and might demonstrate the possibility in the acetylation targeted diagnosis and therapy of GIST.

Regulation of NOS3 by Novel Acetylation Sites

Nitric oxide (NO) is important for cardiovascular and renal health. NO is primarily produced via the enzymatically catalyzed reaction of L-arginine and oxygen to produce NO and L-citrulline by nitric oxide synthase (NOS) isoforms, NOS1, NOS2, and NOS3. NOS3 was first identified in the endothelium but is also present in the kidney epithelium. Endothelial NOS3 maintains cardiovascular health by preventing platelet aggregation, smooth muscle proliferation, and leukocyte adhesion. Epithelial NOS3 controls water and electrolyte homeostasis by regulating ion transporter activity along the nephron. NOS3 activity is tightly controlled by posttranslational modifications. While phosphorylation sites have been well characterized, less is known about regulation by acetylation. Lysine acetylation is a dynamic posttranslational modification observed in both the nucleus and cytosol to regulate intracellular signaling. Our lab and others have shown NOS3 to be regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites on NOS3 which modulate its activity. Based on posttranslational modification prediction software we chose seven lysines to investigate: K486, K497, K504, K607, K610, K727, and K731. We hypothesized that deacetylation of any or all of these lysine sites increases NOS3-mediated NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation in the media over 48 hours. Constructs containing either K486R, K497R, or K504R did not show a significant increase in NO production compared to WT (WT: 1703±499.3 pmol NO2/mg protein; K486R: 1984 ± 1003 pmol NO2/mg protein; K497: 1528 ± 1032 pmol NO2/mg protein; K504: 4337±576.7 pmol NO2/mg protein; p < 0.05 vs WT). However, mutations of four lysines, K607R, K610R, K727R, and K731R (K607/610/727/731R), dramatically increased NO production (21971 ± 1941 pmol NO2/mg protein; WT: 1703±499.3 pmol NO2/mg protein; p < 0.0001 vs WT). To further identify which of the four lysines are responsible for the observed phenotype, we made two new constructs containing either K607/610R or K727/731R. K607/610R was similar to WT (WT: 2271 ± 729.3 pmol NO2/mg protein; K607/610R: 3523 ± 805.8 pmol NO2/mg protein: p > 0.05), yet mutations of K727/731R demonstrated a large increase in NO production (9431 ± 1772 pmol NO2/mg protein; WT: 2271 ± 729.3 pmol NO2/mg protein; p<0.05). These studies suggest K727 and/or K731 is basally acetylated and deacetylation increases NO production uncovering novel posttranslational modifications which regulate NOS3 activity. Future studies will identify whether K727, K731, or both sites are responsible for the increase in NO production.

Discovering and Exploiting Selectivity in Bromodomain Recognition of Epigenetic Lysine Acylation

Bromodomains are promising epigenetic targets in cancer, diabetes, and other inflammatory disorders, and bromodomain inhibitors are in clinical trials for multiple cancers and cardiovascular disease. Bromodomains bind sites of lysine acetylation on histones and transcription factors. Interestingly, nearly half of human bromodomains exist in tandem with at least one other bromodomain on a single polypeptide. These tandem bromodomain-containing proteins are localized disproportionately at super-enhancers, genomic regions with large clusters of elements that enhance gene transcription. The basis of this localization is unknown but important given that super-enhancers are enriched at loci with oncogenic potential. Here, we show that tandem bromodomains can scaffold nucleosomes in an acetylation-dependent manner using biochemical, structural, biophysical, cellular, bioinformatic, and other computational approaches. This scaffolding is consistent with our hypothesis that tandem bromodomains enact acetylation-dependent reorganization of chromatin; for instance, joining promotors with their corresponding distal enhancers to facilitate enhancer-driven oncogenic gene transcription. This mechanism of chromatin reorganization is paradigm-shifting with broad implications on the cellular role of tandem bromodomain proteins. We also hypothesize that changes in acyl-CoA metabolism induce distinct lysine acylations on histones that are selectively recognized by bromodomains, thereby linking metabolism to transcription. Yet, the broader lysine acylation binding specificity of bromodomains is only beginning to be characterized. Here, we address this knowledge gap by determining how metabolically-derived acylations tune bromodomain binding to histones using biophysical, structural biology, and proteomic techniques. To aid mechanistic inquiries, we are removing a critical barrier in the study of bromodomain biology: the lack of inhibitors and chemical probes that selectively target individual bromodomains. This lack of selectivity may be responsible for the dose-limiting toxicity reported in clinical trials of existing bromodomain inhibitors. Here, we report the identification of a novel ligandable site on a bromodomain outside the acetyl-lysine binding site and novel inhibitors that target individual bromodomains. These chemical tools will be necessary to distinguish the differential activities of bromodomain-containing proteins in cell and rodent models of disease and may lead to therapeutics targeting the bromodomain axis in cancer, diabetes, and cardiovascular disease. Overall, the insight gained through these studies will pave the way for safer and more effective therapies targeting bromodomains.