Summary Using an integrated approach, we here report the identification of 67 novel histone marks, a discovery that increases the current number of known histone marks by about 70%. We verified one of the newly-identified marks, lysine crotonylation (Kcr), as a novel, evolutionarily-conserved histone post-translational modification. The unique structure and genomic localization of Kcr suggest that it is mechanistically and functionally different from lysine acetylation (Kac), a previously-described post-translational modification. Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in post-meiotic male germ cells.

Protein post-translational modifications (PTMs) at the lysine residue, such as lysine methylation, acetylation, and ubiquitination, are diverse, abundant, and dynamic. They play a key role in the regulation of diverse cellular physiology. Here we report discovery of a new type of lysine PTM, lysine malonylation (Kmal). Kmal was initially detected by mass spectrometry and protein sequence-database searching. The modification was comprehensively validated by Western blot, tandem MS, and high-performance liquid chromatography of synthetic peptides, isotopic labeling, and identification of multiple Kmal substrate proteins. Kmal is a dynamic and evolutionarily conserved PTM observed in mammalian cells and bacterial cells. In addition, we demonstrate that Sirt5, a member of the class III lysine deacetylases, can catalyze lysine demalonylation and lysine desuccinylation reactions both in vitro and in vivo. This result suggests the possibility of nondeacetylation activity of other class III lysine deacetylases, especially those without obvious acetylation protein substrates. Our results therefore reveal a new type of PTM pathway and identify the first enzyme that can regulate lysine malonylation and lysine succinylation status. Molecular & Cellular Proteomics 10: 10.1074/ mcp.M111.012658, 1-12, 2011.Cellular function and physiology are largely determined by the inventory of all proteins in a cell, its proteome. The collection and characterization of the proteome is critical to understanding cellular mechanisms and diseases. Proteomes in eukaryotic cells consist of over a million molecular species of proteins, easily orders of magnitude more complex than the corresponding genomes (1, 2). There are two major mechanisms for expanding the coding capacity of the human genome: mRNA splicing and protein post-translational modifications (PTMs)1 . PTMs (more than 300 types) are complex and fundamental mechanisms of cellular regulation, and have been associated with almost all known cellular pathways and disease processes (1, 2). As an example, protein phosphorylation, the most well-studied PTM, is present in more than one third of human proteins, the phosphorylation status of which can potentially be regulated by ϳ500 human protein kinases and ϳ150 phosphatases (3, 4). The modification mainly occurs at several amino acid residues: serine, threonine, tyrosine, and histidine. Protein phosphorylation makes its substrate residues more acidic, hydrophilic, and induces a charge change from ϩ1 charge to -1 (at physiological pH), which in turn modulates the structure and functions of substrate proteins.The high complexity of PTMs is also reflected by diverse modifications at -amine group of lysine residue, including methylation, acetylation, and ubiquitination. These lysine PTMs have been shown to play an important role in cellular regulations (5, 6). Recently, we identified a new type of PTM at lysine residues, lysine succinylation (7). Like phosporylation, lysine succinylation also induces a change of two negative charges in lysine re...

The combination of high-density transposon-mediated mutagenesis and high-throughput sequencing has led to significant advancements in research on essential genes, resulting in a dramatic increase in the number of identified prokaryotic essential genes under diverse conditions and a revised essential-gene concept that includes all essential genomic elements, rather than focusing on protein-coding genes only. DEG 10, a new release of the Database of Essential Genes (available at http://www.essentialgene.org), has been developed to accommodate these quantitative and qualitative advancements. In addition to increasing the number of bacterial and archaeal essential genes determined by genome-wide gene essentiality screens, DEG 10 also harbors essential noncoding RNAs, promoters, regulatory sequences and replication origins. These essential genomic elements are determined not only in vitro, but also in vivo, under diverse conditions including those for survival, pathogenesis and antibiotic resistance. We have developed customizable BLAST tools that allow users to perform species- and experiment-specific BLAST searches for a single gene, a list of genes, annotated or unannotated genomes. Therefore, DEG 10 includes essential genomic elements under different conditions in three domains of life, with customizable BLAST tools.

Despite of the progress in identifying many Lysine (Lys)1 acetylation is a dynamic, reversible, and evolutionarily conserved protein post-translational modification (PTM). After the discovery of Lys acetylation in histones more than forty years ago (1), early studies mainly focused on histones and transcription factors, establishing the modification's fundamental role in DNA-templated biological processes (2, 3). The discovery of Lys acetylation in tubulin and the presence of sirtuins in mitochondria argued that Lys acetylation may not be restricted to nuclei (4 -6). The complexity of Lys acetylomes outside the nuclei and the high abundance of the PTM in mitochondria revealed by proteomics studies suggested that the regulatory functions of this PTM may mirror those of protein phosphorylation (7-9).Lys acetylation is regulated by two groups of enzymes with opposing activities, lysine acetyltransferases and deacetylases (10). Sirtuins are a family of highly conserved NAD ϩ -dependent deacetylases (11,12). Numerous studies, mainly focusing on the family's founding member, SIRT1 in mammals or Sir2, the enzyme's homolog in yeast, show that sirtuins regulate diverse cellular functions and appear to affect a variety of aging-related diseases, such as cancer, metabolic diseases, and inflammation and are involved in pathways such as metabolisms, oxidative stress, DNA damage, cell cycle, and signaling (12-15). A number of protein substrates for SIRT1 have been identified including p53, DNA methyltransferase 1 (DNMT1), NF-B, forkhead transcription factors, PGC-1␣, and histones (16 -22). Despite significant progress in the past decade, the molecular mechanisms by which SIRT1 regulates cellular physiology are not well understood. A major hurdle in our understanding of SIRT1 biology is our incomplete knowledge of Lys acetylation proteins that mediate SIRT1 functions. Because SIRT1 is a lysine deacetylase, a major mechanism for SIRT1 to exert its functions should be its deacetylation activity. However, SIRT1-induced lysine acetylation proteins are far from complete. This knowledge limitation also exists with other deacetylases as well as with lysine acetyltransferases, representing a major challenge in Lys acetylation biology and in evaluating clinical compounds that target dysregulated Lysacetylation regulatory enzymes.Here we report the first proteomics quantification of Lys acetylation in response to a regulatory enzyme. Our study identified 4623 Lys acetylation sites from SIRT1 ϩ/ϩ and SIRT1 Ϫ/Ϫ MEF cells, among which 4130 Lys acetylation sites were quantified. The data identified multiple pathways that are affected by SIRT1. From this wealth of new information on the SIRT1-modulated Lys acetylome, we discovered consensus motifs for SIRT1-mediated Lys acetylation sites and identified extensive correlation between Lys acetylation and phosphorylation, SUMOylation, and mutations associated with disease,

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G 1 cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

DNA single-strand break repair (SSBR) is important for maintaining genome stability and homeostasis. The current SSBR model derived from an in vitro-reconstituted reaction suggests that the SSBR complex mediated by X-ray repair cross-complementing protein 1 (XRCC1) is assembled sequentially at the site of damage. In this study, we provide biochemical data to demonstrate that two preformed XRCC1 protein complexes exist in cycling HeLa cells. One complex contains known enzymes that are important for SSBR, including DNA ligase 3 (DNL3), polynucleotide kinase 3-phosphatase, and polymerase ␤; the other is a new complex that contains DNL3 and the ataxia with oculomotor apraxia type 1 (AOA) gene product aprataxin. We report the characterization of the new XRCC1 complex. XRCC1 is phosphorylated in vivo and in vitro by CK2, and CK2 phosphorylation of XRCC1 on S518, T519, and T523 largely determines aprataxin binding to XRCC1 though its FHA domain. An acute loss of aprataxin by small interfering RNA renders HeLa cells sensitive to methyl methanesulfonate treatment by a mechanism of shortened half-life of XRCC1. Thus, aprataxin plays a role to maintain the steady-state protein level of XRCC1. Collectively, these data provide insights into the SSBR molecular machinery in the cell and point to the involvement of aprataxin in SSBR, thus linking SSBR to the neurological disease AOA.

Replication of chromosomes is one of the central events in the cell cycle. Chromosome replication begins at specific sites, called origins of replication (oriCs), for all three domains of life. However, the origins of replication still remain unknown in a considerably large number of bacterial and archaeal genomes completely sequenced so far. The availability of increasing complete bacterial and archaeal genomes has created challenges and opportunities for identification of their oriCs in silico, as well as in vivo. Based on the Z-curve theory, we have developed a web-based system Ori-Finder to predict oriCs in bacterial genomes with high accuracy and reliability by taking advantage of comparative genomics, and the predicted oriC regions have been organized into an online database DoriC, which is publicly available at http://tubic.tju.edu.cn/doric/ since 2007. Five years after we constructed DoriC, the database has significant advances over the number of bacterial genomes, increasing about 4-fold. Additionally, oriC regions in archaeal genomes identified by in vivo experiments, as well as in silico analyses, have also been added to the database. Consequently, the latest release of DoriC contains oriCs for >1500 bacterial genomes and 81 archaeal genomes, respectively.

BackgroundPolicy interventions have been taken to protect households from facing unpredictable economic changes that may cause catastrophe in China. This study aims to estimate the change of overall proportion of households incurring catastrophic health care expenditure (CHE) and its income-related inequality in the rural areas of Shaanxi Province from 2008 to 2013.MethodsThe data were drawn from the National Household Health Service Surveys of Shaanxi Province conducted in the years 2008 and 2013. In total, 3,217 households in 2008 and 13,085 households in 2013 were selected for analysis. A “Capacity to pay” approach was used to measure the incidence of CHE. The concentration index was employed to measure the extent of income-related inequality in CHE. A decomposition method, based on a logit model, was used to decompose the concentration index into its determining components.ResultsFrom 2008 to 2013, the overall proportion of households incurring CHE dropped from 17.19 % to 15.83 %, while conversely, the inequality in facing CHE strongly increased. The majority of observed inequalities in CHE were explained by household economic status and household size in 2013. In addition, the absence of commercial health insurance and having elderly members were also important contributors to inequality in CHE.ConclusionsEven though we used a conservative method to measure CHE, the overall proportion of households incurring CHE in Shaanxi Province is still considerably high in both years. Furthermore, there exists a strong pro-rich inequality of CHE in rural areas of Shaanxi Province. Our study suggests that narrowing the gap of household economic status, improving the anti-risk capability of small scale households, establishing prepayment mechanisms in health insurance, strengthening the depth of reimbursement and subsidising vulnerable households in Shaanxi Province are helpful for both reducing the probability of incurring CHE and the pro-rich inequality in CHE.