组蛋白乙酰化/去乙酰化与基因表达调控

组蛋白是真核生物染色质的主要成分,组蛋白修饰(如甲基化、乙酰化、磷酸化、泛素化等)在真核生物基因表达调控中发挥着重要的作用.在这些修饰中,组蛋白乙酰化/去乙酰化尤为重要.组蛋白乙酰化/去乙酰化可通过改变染色质周围电荷或参与染色质构型重建而影响基因表达;更重要的是组蛋白乙酰化/去乙酰化可形成一种特殊的“密码”,被其它蛋白质识别,影响多种蛋白质因子的活动或与其相互作用,参与到基因表达调控的整个网络中.     

组蛋白乙酰化／去乙酰化作用与真核基因转录调控

核小体组蛋白的翻译后修饰是真核基因转录调控中的关键步骤。对于组蛋白的这类修饰方式 ,近年来研究最为活跃的是组蛋白Ｎ末端区域保守的Ｌｙｓ上ε ＮＨ 3 的乙酰化作用。随着各种组蛋白乙酰化酶 /去乙酰化酶被克隆、鉴定 ,组蛋白乙酰化 /去乙酰化作用与真核基因转录调控之间的关系也开始逐步得以阐明。1 .真核转录相关的组蛋白乙酰化酶和组蛋白去乙酰化酶1 .1　组蛋白乙酰化酶 (ｈｉｓｔｏｎｅａｃｅｔｙｌｔｒａｎｓ ｆｅｒａｓｅ ,ＨＡＴ)　　核小体组蛋白中Ｎ末端区域上保守的Ｌｙｓ的乙酰化是染色质具有转录活性的标志之一。在组蛋白…     

Discriminating between Lysine Sumoylation and Lysine Acetylation Using mRMR Feature Selection and Analysis

Post-translational modifications (PTMs) are crucial steps in protein synthesis and are important factors contributing to protein diversity. PTMs play important roles in the regulation of gene expression, protein stability and metabolism. Lysine residues in protein sequences have been found to be targeted for both types of PTMs: sumoylations and acetylations; however, each PTM has a different cellular role. As experimental approaches are often laborious and time consuming, it is challenging to distinguish the two types of PTMs on lysine residues using computational methods. In this study, we developed a method to discriminate between sumoylated lysine residues and acetylated residues. The method incorporated several features: PSSM conservation scores, amino acid factors, secondary structures, solvent accessibilities and disorder scores. By using the mRMR (Maximum Relevance Minimum Redundancy) method and the IFS (Incremental Feature Selection) method, an optimal feature set was selected from all of the incorporated features, with which the classifier achieved 92.14% accuracy with an MCC value of 0.7322. Analysis of the optimal feature set revealed some differences between acetylation and sumoylation. The results from our study also supported the previous finding that there exist different consensus motifs for the two types of PTMs. The results could suggest possible dominant factors governing the acetylation and sumoylation of lysine residues, shedding some light on the modification dynamics and molecular mechanisms of the two types of PTMs, and provide guidelines for experimental validations.     

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

S-Adenosylhomocysteine hydrolase (SAHH) is an NAD⁺-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys⁴⁰¹ and Lys⁴⁰⁸ but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys⁴⁰¹ and Lys⁴⁰⁸ and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD⁺ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns.     

Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos

Background

As a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts), in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.

Principal Finding

Our study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.

Conclusions/Significance

These results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.     

Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis

The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD⁺-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.Mycobacterium tuberculosis was responsible for 1.3 million deaths and 8.6 million new cases of tuberculosis (TB)¹ worldwide in 2012 (1). This global public health crisis remains a serious problem, with the emergence of drug-resistant M. tuberculosis, especially multidrug-resistant and extensively drug-resistant M. tuberculosis, and also the emergence of coinfections of TB and human immunodeficiency virus (2, 3). To counter the increasing threat of TB, it is critical to understand fundamental aspects of TB-related biology. Such studies will not only provide new drug targets for the design of novel therapeutic agents, but also facilitate the development of novel diagnostic tools and new vaccines.Acetylation is one of the important protein modifications and occurs both co- and post-translationally on the α-amino group at the N terminus of the protein, so-called “N-terminal acetylation,” or on the ε-amino group on the side chain of lysine (4). Lysine acetylation is one of the most common post-translational modifications to proteins in both eukaryotes and prokaryotes. As a dynamic and reversible process, protein acetylation plays important roles in many cellular physiological processes, including cell-cycle regulation and apoptosis, cell morphology (5), metabolic pathways (6–8), protein interactions (9), and enzymatic activity (8, 10). In recent years, great advances have been made in proteomic studies, and a large number of lysine-acetylated proteins have been identified in many eukaryotes, including human (5, 11, 12), rat (13), mouse (11), Drosophila (14), Arabidopsis (15, 16), Saccharomyces cerevisiae (17), and protozoans (18, 19). The global analysis of lysine acetylation has also been reported in bacteria, including Escherichia coli (20–22), Erwinia amylovora (23), Bacillus subtilis (24), and Salmonella enterica (6). These acetylome studies have generated large datasets of bacterial proteins acetylated on lysine residues and have demonstrated the diverse cellular functions of lysine acetylation in bacteria.Increasing evidence shows that protein acetylation occurs and plays an important regulatory role in mycobacteria (8, 25–31). For example, Lange et al. reported the N-terminal acetylation of early secreted antigenic target 6 (ESAT-6) protein (31). Rv1151c is reported to be an NAD⁺-dependent protein deacetylase in M. tuberculosis that deacetylates and thus regulates the activity of acetyl-CoA synthase (25, 32). Two cyclic adenosine monophosphate (cAMP)-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) show similarity to the GNAT family of acetyltransferases and could acetylate a universal stress protein (USP, MSMEG_4207) (30). Subsequent structural studies revealed the fine mechanisms of how cAMP regulates the protein lysine acetyltransferase in mycobacteria (27, 28). Very recently, reversible lysine acetylation was shown to regulate the activity of several fatty acyl-CoA synthetases in M. tuberculosis (8, 26), and also to regulate acetate and propionate metabolism in M. smegmatis (8, 26). However, to the best of our knowledge, only a few acetylated proteins in M. tuberculosis have been identified, presenting a major obstacle to further understanding the regulatory roles of reversible lysine acetylation in this life-threatening pathogen.To fill this gap in our knowledge, we undertook a systematic study of the functional roles of lysine acetylation in M. tuberculosis. We performed an acetylomic analysis of M. tuberculosis H37Ra using high-accuracy MS combined with the identification of 226 unique lysine acetylation sites on 137 proteins. This set of M. tuberculosis proteins acetylated on lysine residues supports the emerging view that lysine acetylation is a general and fundamental regulatory process, and is not restricted to eukaryotes. It also opens the way for its detailed functional and evolutionary analysis of lysine acetylation in M. tuberculosis. The identified acetylated proteins that are involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of acetylated proteins in M. tuberculosis has been constructed, and should allow us to better understand the significance of acetylation in key cellular mechanisms in M. tuberculosis. To further explore the effects of lysine acetylation on the physiology of M. tuberculosis H37Ra, MRA_1161, the gene encoding the only known protein deacetylase in this bacterium, was deleted. The roles of MRA_1161 in the colony morphology, carbon source utilization, heat stress tolerance, and biofilm formation of M. tuberculosis were analyzed. The effect of lysine acetylation on the immunogenicity of a known immunogen, HspX, was also tested.     

组蛋白乙酰基转移酶1在肝癌细胞中介导的蛋白质乙酰化修饰谱分析

赖氨酸乙酰化是重要的蛋白质翻译后修饰之一,广泛存在于细胞的生理和病理过程.组蛋白乙酰基转移酶1(HAT1)作为第一个被鉴定的蛋白ε-氨基赖氨酸乙酰基转移酶,具有介导组蛋白和非组蛋白乙酰化的作用.然而,在肝癌细胞中HAT1介导的乙酰化蛋白质及其修饰位点目前仍不清楚.本研究首先揭示了 HAT1在肝癌组织中呈高表达,并且与预...     

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.     

Biological Modification of Trichothecene Mycotoxins: Acetylation and Deacetylation of Deoxynivalenols by Fusarium spp

Attempts were made to elucidate the acetyl transformation of novel trichothecene mycotoxins, 3a,7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one (deoxynivalenol) and its derivatives, by trichothecene-producing strains of Fusarium nivale, F. roseum, and F. solani. In the peptone-supplemented Czapek-Dox medium, F. roseum converted 3a-acetoxy-7a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (3-acetyldeoxynivalenol) to deoxynivalenol. 3-Acetyldeoxynivalenol was also deacetylated by intact mycelia of the three strains in sugar-free Czapek-Dox medium. The growing F. nivale acetylated deoxynivalenol to afford a small amount of 3-acetyldeoxynivalenol. 3a,7a,15-Triacetoxy-12,13-epoxytrichothec-9-en-8-one (7,15-diacetyl-deoxynivalenol), which was then deacetylated to give 7a-acetoxy-3a,15-dihydroxy-12,13-epoxytrichothec-9-en-8-one (7-acetyldeoxynivalenol). It was noted that the ester at C-7 was not hydrolyzed by the fungal mycelium.     

蛋白质赖氨酸乙酰化修饰对中间代谢的调控研究

生命活动的中间代谢酶存在大量的赖氨酸乙酰化修饰作用,这些在特定位点进行的可逆的赖氨酸乙酰化修饰作用能精确地调控胞内各种代谢路径。因此,对中间代谢酶赖氨酸乙酰化的研究成为了当今热点。对中间代谢酶的乙酰化修饰的研究进展进行综述,并归纳了几种典型的中间代谢酶的可逆乙酰化作用及其乙酰化位点的分布和在中间代谢路径中重要的调控作用,以期为深入研究蛋白质乙酰化修饰提供参考。     

Global Proteome Analysis Links Lysine Acetylation to Diverse Functions in Oryza Sativa

Lysine acetylation (Kac) is an important protein post‐translational modification in both eukaryotes and prokaryotes. Herein, we report the results of a global proteome analysis of Kac and its diverse functions in rice (Oryza sativa). We identified 1353 Kac sites in 866 proteins in rice seedlings. A total of 11 Kac motifs are conserved, and 45% of the identified proteins are localized to the chloroplast. Among all acetylated proteins, 38 Kac sites are combined in core histones. Bioinformatics analysis revealed that Kac occurs on a diverse range of proteins involved in a wide variety of biological processes, especially photosynthesis. Protein–protein interaction networks of the identified proteins provided further evidence that Kac contributes to a wide range of regulatory functions. Furthermore, we demonstrated that the acetylation level of histone H3 (lysine 27 and 36) is increased in response to cold stress. In summary, our approach comprehensively profiles the regulatory roles of Kac in the growth and development of rice.     

Identification of Lysine Acetylation Sites on MERS-CoV Replicase pp1ab

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Highlights

• •Acetylation sites on MERS-CoV protein pp1ab were reported for the first time.
• •Sirt1 was predicted as upstream factor of identified acetylation events.