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...

Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism. Molecular & Cellular

Although SIRT7 is a member of sirtuin family proteins that are described as NAD+-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD+-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7.

Protein lysine acetylation plays a key role in regulating chromatin dynamics, gene expression and metabolic pathways in eukaryotes, and, thus, contributes to diverse cellular processes like transcription, cell cycle regulation, and apoptosis. Although recent evidence suggests that acetylated proteins impact broadly cellular functions in prokaryotes, the substrates and localization of this modification remain widely unknown due to the limitations of analytical methods. Comprehensive identification of protein acetylation is a major bottleneck due to its dynamic property and pretty low abundance. A complete atlas of acetylome will significantly advance our understanding of this modification functions in prokaryotes. To achieve this goal, we have developed an intergraded approach to identifying lysine acetylation. Combining immunoaffinity enrichment with high sensitive mass spectrometry, we identified 349 acetylated proteins and addressed 1070 acetylation sites in Escherichia coli. To our knowledge, the acetylated proteins and acetylated sites were increased to 3 times and 8 times, respectively, compared to that in previous report. To further characterize this modification, we classified acetylated proteins into several groups according to cell components, molecular functions and biological process. Additionally, interaction networks and high confident domains architectures of acetylated proteins were investigated with the aid of bioinformatics tools. Finally, the acetylated metabolic enzymes were analyzed on the basis of acetylated proteins identified by proteomic survey in E. coli. Our study has demonstrated that the combined approach is powerful for identification and characterization of protein lysine acetylation on a large scale. These results not only greatly expand the number of acetylated proteins, but also provide a series of important information including localization, networks and characterization of acetylome.

SummaryRecently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features.

Background: The threat of drug-resistant Pseudomonas aeruginosa requires great efforts to develop highly effective and safe bactericide. Objective: This study aimed to investigate the antibacterial activity and mechanism of silver nanoparticles (AgNPs) against multidrug-resistant P. aeruginosa. Methods: The antimicrobial effect of AgNPs on clinical isolates of resistant P. aeruginosa was assessed by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). In multidrug-resistant P. aeruginosa, the alterations of morphology and structure were observed by the transmission electron microscopy (TEM); the differentially expressed proteins were analyzed by quantitative proteomics; the production of reactive oxygen species (ROS) was assayed by H 2 DCF-DA staining; the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was chemically measured and the apoptosis-like effect was determined by flow cytometry. Results: Antimicrobial tests revealed that AgNPs had highly bactericidal effect on the drug-resistant or multidrug-resistant P. aeruginosa with the MIC range of 1.406-5.625 µg/mL and the MBC range of 2.813-5.625 µg/mL. TEM showed that AgNPs could enter the multidrug-resistant bacteria and impair their morphology and structure. The proteomics quantified that, in the AgNP-treated bacteria, the levels of SOD, CAT, and POD, such as alkyl hydroperoxide reductase and organic hydroperoxide resistance protein, were obviously high, as well as the significant upregulation of low oxygen regulatory oxidases, including cbb3-type cytochrome c oxidase subunit P2, N2, and O2. Further results confirmed the excessive production of ROS. The antioxidants, reduced glutathione and ascorbic acid, partially antagonized the antibacterial action of AgNPs. The apoptosis-like rate of AgNP-treated bacteria was remarkably higher than that of the untreated bacteria (P,0.01). Conclusion: This study proved that AgNPs could play antimicrobial roles on the multidrug-resistant P. aeruginosa in a concentration-and time-dependent manner. The main mechanism involves the disequilibrium of oxidation and antioxidation processes and the failure to eliminate the excessive ROS.

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to β-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.