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...

Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was successful even when diluted 1000-fold, allowing easy identification of transgenic founders. Given its speed and low cost, we anticipate that the adoption of this method will streamline DNA isolation for zebrafish research.

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Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2'-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation. Here, we review and discuss recent progress in the study of mitochondrial sirtuins and their targets.