Predicting protein post-translational modifications using meta-analysis of proteome scale data sets

Protein post-translational modifications are an important biological regulatory mechanism, and the rate of their discovery using high throughput techniques is rapidly increasingly. To make use of this wealth of sequence data, we introduce a new general strategy designed to predict a variety of post-translational modifications in several organisms. We used the motif-x program to determine phosphorylation motifs in yeast, fly, mouse, and man and lysine acetylation motifs in man. These motifs were then scanned against proteomic sequence data using a newly developed tool called scan-x to globally predict other potential modification sites within these organisms. 10-fold cross-validation was used to determine the sensitivity and minimum specificity for each set of predictions, all of which showed improvement over other available tools for phosphoprediction. New motif discovery is a byproduct of this approach, and the phosphorylation motif analyses provide strong evidence of evolutionary conservation of both known and novel kinase motifs.     

Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins

Protein lysine acetylation is a reversible and highly regulated post‐translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology.     

Proteomic Investigations of Lysine Acetylation Identify Diverse Substrates of Mitochondrial Deacetylase Sirt3

Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site-specific acetylation in wild-type murine embryonic fibroblasts to Sirt3 knockout cells. We confirm Sirt3-regulated acetylation of several mitochondrial proteins in human cells by comparing acetylation in U2OS cells overexpressing Sirt3 to U2OS cells in which Sirt3 expression was reduced by shRNA. Our data demonstrate that ablation of Sirt3 significantly increases acetylation at dozens of sites on mitochondrial proteins. Substrates of Sirt3 are implicated in various metabolic pathways, including fatty acid metabolism and the tricarboxylic acid cycle. These results imply broader regulatory roles of Sirt3 in the mitochondria by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases.     

Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum)

Post-translational modification of proteins through lysine succinylation plays important regulatory roles in living cells. Lysine succinylation was recently identified as a novel post-translational modification in Escherichia coli, yeast, Toxoplasma gondii, HeLa cells, and mouse liver. Interestingly, only a few sites of lysine succinylation have been detected in plants to date. In this study, we identified 347 sites of lysine succinylation in 202 proteins in tomato by using high-resolution mass spectrometry. Succinylated proteins are implicated in the regulation of diverse metabolic processes, including chloroplast and mitochondrial metabolism. Bioinformatic analysis showed that succinylated proteins are evolutionarily conserved and involved in various cellular functions such as metabolism and epigenetic regulation. Moreover, succinylated proteins exhibit diverse subcellular localizations. We also defined six types of definitively conserved succinylation motifs. These results provide the first in-depth analysis of the lysine succinylome and novel insights into the role of succinylation in tomato, thereby elucidating lysine succinylation in the context of cellular physiology and metabolite biosynthesis in plants.     

Bioinformatic analysis and post-translational modification crosstalk prediction of lysine acetylation

Recent proteomics studies suggest high abundance and a much wider role for lysine acetylation (K-Ac) in cellular functions. Nevertheless, cross influence between K-Ac and other post-translational modifications (PTMs) has not been carefully examined. Here, we used a variety of bioinformatics tools to analyze several available K-Ac datasets. Using gene ontology databases, we demonstrate that K-Ac sites are found in all cellular compartments. KEGG analysis indicates that the K-Ac sites are found on proteins responsible for a diverse and wide array of vital cellular functions. Domain structure prediction shows that K-Ac sites are found throughout a wide variety of protein domains, including those in heat shock proteins and those involved in cell cycle functions and DNA repair. Secondary structure prediction proves that K-Ac sites are preferentially found in ordered structures such as alpha helices and beta sheets. Finally, by mutating K-Ac sites in silico and predicting the effect on nearby phosphorylation sites, we demonstrate that the majority of lysine acetylation sites have the potential to impact protein phosphorylation, methylation, and ubiquitination status. Our work validates earlier smaller-scale studies on the acetylome and demonstrates the importance of PTM crosstalk for regulation of cellular function.     

Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation,succinylation, ubiquitination,and phosphorylation

Protein lysine acetylation is a highly conserved post-translational modification with various biological functions. However, only a limited number of acetylation sites have been reported in plants, especially in cereals, and the function of non-histone protein acetylation is still largely unknown. In this report, we identified 1003 lysine acetylation sites in 692 proteins of developing rice seeds, which greatly extended the number of known acetylated sites in plants. Seven distinguished motifs were detected flanking acetylated lysines. Functional annotation analyses indicated diverse biological processes and pathways engaged in lysine acetylation. Remarkably, we found that several key enzymes in storage starch synthesis pathway and the main storage proteins were heavily acetylated. A comprehensive comparison of the rice acetylome, succinylome, ubiquitome and phosphorylome with available published data was conducted. A large number of proteins carrying multiple kinds of modifications were identified and many of these proteins are known to be key enzymes of vital metabolic pathways. Our study provides extending knowledge of protein acetylation. It will have critical reference value for understanding the mechanisms underlying PTM mediated multiple signal integration in the regulation of metabolism and development in plants.     

Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite‐specific functions during Plasmodium falciparum intraerythrocytic development

Lysine acetylation has emerged as a major post‐translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine‐acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium‐specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.     

PLMD:An updated data resource of protein lysine modifications

Post-translational modifications(PTMs) occurring at protein lysine residues,or protein lysine modifications(PLMs),play critical roles in regulating biological processes.Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types,here we greatly updated our previous studies,and presented a much more integrative resource of protein lysine modification database(PLMD).In PLMD,we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs,including ubiquitination,acetylation,sumoylation,methylation,succinylation,malonylation,glutarylation,giycation,formylation,hydroxylation,butyrylation,propionylation,crotonylation,pupylation,neddylation,2-hydroxyisobutyrylation,phosphoglycerylation,carboxylation,lipoylation and biotinylation.Using the data set,a motif-based analysis was performed for each PLM type,and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications.Moreover,various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other,and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues.Finally,various options were provided for accessing the data,while original references and other annotations were also present for each PLM substrate.Taken together,we anticipated the PLMD database can serve as a useful resource for further researches of PLMs.PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org.     

赖氨酸乙酰化在代谢相关疾病中的调控机制

赖氨酸乙酰化是把来自于乙酰CoA的乙酰基团转移到靶蛋白赖氨酸的ε-NH3＋上,是蛋白质翻译后的一种可逆修饰过程,受乙酰基转移酶（HAT／KAT）和去乙酰化酶（HDAC／KDAC）的共同调节。赖氨酸乙酰化通过对细胞内多种蛋白质的修饰调节,可以控制体内多种代谢过程,如调节糖类、脂类、氨基酸、核苷酸及次级代谢物的代谢等.因而,细胞内赖氨酸乙酰化失调,可影响与代谢相关的多种疾病,如肥胖症、糖尿病和心血管疾病等。随着对蛋白质乙酰化研究的深入,发现赖氨酸乙酰化与细胞免疫状态及神经退行性疾病,如阿尔茨海默氏症和亨廷顿综合征等也有关。对近年来赖氨酸乙酰化在代谢调控及与代谢相关疾病如心血管疾病和免疫代谢疾病中的分子调控机制进行综述。     

A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles

Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.     

代谢酶乙酰化修饰对新陈代谢的调控

蛋白质的赖氨酸乙酰化修饰可以定义为在蛋白质的赖氨酸残基上添加或移除一个乙酰基团,这个过程是由乙酰化酶和脱乙酰酶调控的.真核生物细胞核内组蛋白和转录因子的可逆乙酰化修饰对基因表达调控的机制早已研究得比较清楚.1996年以来,一些独立的研究也陆续发现,参与到其他生命活动中的蛋白质存在着乙酰化修饰情况,表明乙酰化可能在生命活动中发挥着广泛的调节作用.然而直到2009年,高通量的蛋白质质谱分析技术才使得在蛋白质组水平上研究乙酰化修饰成为可能,并发现蛋白质乙酰化普遍存在.学者们发现,乙酰化修饰是一个在细胞核或细胞质的亚细胞器内广泛存在的翻译后修饰调控机制,可能参与了染色体重塑、细胞周期调控、细胞骨架的大分子运输、新陈代谢等多种生命活动.本文详细总结代谢酶的乙酰化修饰对新陈代谢调控的关键作用,并说明代谢酶的乙酰化修饰是一个从原核生物到真核生物进化上高度保守的调控机制.