The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD + -dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis. L ysine acetylation is one of the most common posttranslational modifications among cellular proteins and regulates a variety of physiological processes including enzyme activity, proteinprotein interactions, gene expression, and subcellular localization (1). Large-scale proteomic surveys have demonstrated that lysine acetylation is prevalent within mitochondria (2, 3). As the central regulators of cellular energy production, mitochondria require a coordinated response to changes in nutrient availability to respond to metabolic needs. In addition to ATP production, mitochondria are essential for regulation of fatty acid oxidation, apoptosis, and amino acid catabolism. Disruption of these processes is associated with a variety of neurodegenerative disorders and metabolic diseases (4, 5). Understanding the role of lysine acetylation in mitochondrial function will likely provide insight into altered metabolism in dysregulated or disease states.The sirtuins (SIRT1-7) are an evolutionary conserved family of NAD + -dependent deacetylases (6). SIRT3, SIRT4, and SIRT5 are major, if not exclusively, localized in the mitochondrial matrix (7,8). SIRT3 is the primary regulator of mitochondrial lysine acetylation (9), whereas SIRT5 regulates lysine malonylation and succinylation (10, 11), and SIRT4 has no well established target except weak ADP ribosyltransferase activity (12). SIRT3 is highly expressed in mitochondria-rich tissues and shows differentially regulated expression in liver and skeletal muscle in response to changes in nutrient availability such as fas...
In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl-phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose, and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. Since glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl-phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl-phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.
Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLPproducing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.Cyclic lipopeptides (CLPs) are produced by distinctively different groups of bacteria, both gram-positive (20) and gramnegative (28). The high diversity of CLP-producing microorganisms (28) and differences in chemical structure suggest that the CLP compounds may serve different, and possibly multiple, purposes. This may explain why the specific role of CLP production is often unclear (28,40). For a limited number of CLPs (28), the reported functions include promotion of bacterial swarming (12, 26) and biosurfactant properties (19,24,41). In many cases, CLP compounds are also known to exert a role in antagonistic interactions with other organisms (28), e.g., plant pathogenicity (5) and antifungal (19,30,31,38,44), antibacterial (11), antiviral (49), or cytotoxic (16) activity.Synthesis of CLPs is nonribosomal and catalyzed by large peptide synthetase complexes (27). Various environmental stimuli may affect CLP production, i.e., carbon substrate (36), limitation by C, N, or P (15, 37), Fe limitation (15), growth phase conditions (15), and interaction with interfaces (32). Little information is available on production rates and regulating factors for the compounds in natural environments. Asaka and Shoda (2) detected surfactin and iturine production by Bacillus subtilis RB14 in a sterilized vermiculite-soil system, and Nakayama et al. (31) detected xanthobaccin A production by a Stenotrophomonas sp. strain, SB-K88, in a hydroponic sugar beet rhizosphere system, but documentation for in situ production of CLPs...
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