Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli), yeast (S. cerevisiae), human (HeLa) cells, and mouse liver tissue, demonstrating widespread succinylation in diverse organisms. A majority of succinylation sites in bacteria, yeast, and mouse liver were acetylated at the same position. Quantitative analysis of succinylation in yeast showed that succinylation was globally altered by growth conditions and mutations that affected succinyl-coenzyme A (succinyl-CoA) metabolism in the tricarboxylic acid cycle, indicating that succinylation levels are globally affected by succinyl-CoA concentration. We preferentially detected succinylation on abundant proteins, suggesting that succinylation occurs at a low level and that many succinylation sites remain unidentified. These data provide a systems-wide view of succinylation and its dynamic regulation and show its extensive overlap with acetylation.

Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner.

Antiviral or antitumor immunity requires activation of cytotoxic CD8+ T cells by dendritic cells, which present viral or tumor antigens on major histocompatibility complex (MHC) class I molecules. The intracellular mechanisms facilitating MHC class I-restricted presentation of extracellular antigens ('cross-presentation') are unclear. Here we demonstrate that cross-presentation of soluble antigen occurred in an early endosomal compartment distinct from the endoplasmic reticulum where endogenous antigen is loaded onto MHC class I. Efficient cross-presentation required endotoxin-induced, Toll-like receptor 4- and signaling molecule MyD88-dependent relocation of the transporter associated with antigen processing, essential for loading of MHC class I, to early endosomes. Transport of cross-presented antigen from endosomes to the cell surface was inhibited by primaquine, which blocks endosomal trafficking. Thus, cross-presentation is spatially and mechanistically separated from endogenous MHC class I-restricted antigen presentation and is biased toward antigens containing microbial molecular patterns.

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.

Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acetylation signatures for 19 different KDACIs, covering all 18 human lysine deacetylases. Most KDACIs increased acetylation of a small, specific subset of the acetylome, including sites on histones and other chromatin-associated proteins. Inhibitor treatment combined with genetic deletion showed that the effects of the pan-sirtuin inhibitor nicotinamide are primarily mediated by SIRT1 inhibition. Furthermore, we confirmed that the effects of tubacin and bufexamac on cytoplasmic proteins result from inhibition of HDAC6. Bufexamac also triggered an HDAC6-independent, hypoxia-like response by stabilizing HIF1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases.

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At the same time Fischer and co-workers (Stoller et al., Germany and 1 Max-Planck-Gesellschaft, Arbeitsgruppe 'Enzymologie 1995) searched for a ribosome-bound prolyl isomerase in et al., 1996). This homology is significant is thought to catalyse the folding of newly synthesized only for the residues that are necessary for substrate proteins. In its enzymatic mechanism the trigger factor binding and activity. Proteolytic fragments of trigger follows the Michaelis-Menten equation. The unusually factor, which encompass the putative FKBP domain high folding activity of the trigger factor originates (residues 132-247 and 145-251, respectively) and a from its tight binding to the folding protein substrate, recombinant form of the 148-249 fragment retained as reflected in the low K m value of 0.7 μM. In contrast, the full prolyl isomerase activity of the intact protein, the catalytic constant k cat is small and shows a value when assayed with proline-containing oligopeptides of 1.3 s -1 at 15°C. An unfolded protein inhibits the (Hesterkamp and Bukau, 1996; Stoller et al., 1996). trigger factor in a competitive fashion. The isolatedThe prolyl isomerase function is thought to be important catalytic domain of the trigger factor retains the full for protein folding, and initial experiments (Stoller et al., prolyl isomerase activity towards short peptides, but 1995) showed that the trigger factor is much more effective in a protein folding reaction its activity is 800-fold as a folding catalyst than cyclophilin, FKBP or parvulin. reduced and no longer inhibited by an unfolded protein.These small prolyl isomerases catalyse prolyl isomerizUnlike the prolyl isomerase site, the polypeptide bindations much better in short unstructured oligopeptides ing site obviously extends beyond the FKBP domain. (Stein, 1993;Fischer, 1994) than in refolding protein Together, this suggests that the good substrate binding, chains (Schmid et al., 1993). i.e. the chaperone property, of the intact trigger factorTo understand the basis of the high folding activity of is responsible for its high efficiency as a catalyst of the trigger factor, we developed a procedure to measure proline-limited protein folding.the Michaelis constant (K m ) and the catalytic rate constant Keywords: chaperone/enzyme kinetics/prolyl isomerase/ (k cat ) for a catalysed folding reaction. Unlike the small protein folding/trigger factor substrates of other enzymes, the substrates of folding enzymes are large protein chains, which are in the process of refolding. Therefore it is difficult to elucidate the