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      Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

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          Abstract

          The emerging view of N ε-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 N ε-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 N ε-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.

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          Most cited references53

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          The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.

          The Paragon Algorithm, a novel database search engine for the identification of peptides from tandem mass spectrometry data, is presented. Sequence Temperature Values are computed using a sequence tag algorithm, allowing the degree of implication by an MS/MS spectrum of each region of a database to be determined on a continuum. Counter to conventional approaches, features such as modifications, substitutions, and cleavage events are modeled with probabilities rather than by discrete user-controlled settings to consider or not consider a feature. The use of feature probabilities in conjunction with Sequence Temperature Values allows for a very large increase in the effective search space with only a very small increase in the actual number of hypotheses that must be scored. The algorithm has a new kind of user interface that removes the user expertise requirement, presenting control settings in the language of the laboratory that are translated to optimal algorithmic settings. To validate this new algorithm, a comparison with Mascot is presented for a series of analogous searches to explore the relative impact of increasing search space probed with Mascot by relaxing the tryptic digestion conformance requirements from trypsin to semitrypsin to no enzyme and with the Paragon Algorithm using its Rapid mode and Thorough mode with and without tryptic specificity. Although they performed similarly for small search space, dramatic differences were observed in large search space. With the Paragon Algorithm, hundreds of biological and artifact modifications, all possible substitutions, and all levels of conformance to the expected digestion pattern can be searched in a single search step, yet the typical cost in search time is only 2-5 times that of conventional small search space. Despite this large increase in effective search space, there is no drastic loss of discrimination that typically accompanies the exploration of large search space.
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            Acetylation: a regulatory modification to rival phosphorylation?

            The fact that histones are modified by acetylation has been known for almost 30 years. The recent identification of enzymes that regulate histone acetylation has revealed a broader use of this modification than was suspected previously. Acetylases are now known to modify a variety of proteins, including transcription factors, nuclear import factors and alpha-tubulin. Acetylation regulates many diverse functions, including DNA recognition, protein-protein interaction and protein stability. There is even a conserved structure, the bromodomain, that recognizes acetylated residues and may serve as a signalling domain. If you think all this sounds familiar, it should be. These are features characteristic of kinases. So, is acetylation a modification analogous to phosphorylation? This review sets out what we know about the broader substrate specificity and regulation of acetyl- ases and goes on to compare acetylation with the process of phosphorylation.
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              Acetylation of histones and transcription-related factors.

              The state of chromatin (the packaging of DNA in eukaryotes) has long been recognized to have major effects on levels of gene expression, and numerous chromatin-altering strategies-including ATP-dependent remodeling and histone modification-are employed in the cell to bring about transcriptional regulation. Of these, histone acetylation is one of the best characterized, as recent years have seen the identification and further study of many histone acetyltransferase (HAT) proteins and their associated complexes. Interestingly, most of these proteins were previously shown to have coactivator or other transcription-related functions. Confirmed and putative HAT proteins have been identified from various organisms from yeast to humans, and they include Gcn5-related N-acetyltransferase (GNAT) superfamily members Gcn5, PCAF, Elp3, Hpa2, and Hat1: MYST proteins Sas2, Sas3, Esa1, MOF, Tip60, MOZ, MORF, and HBO1; global coactivators p300 and CREB-binding protein; nuclear receptor coactivators SRC-1, ACTR, and TIF2; TATA-binding protein-associated factor TAF(II)250 and its homologs; and subunits of RNA polymerase III general factor TFIIIC. The acetylation and transcriptional functions of these HATs and the native complexes containing them (such as yeast SAGA, NuA4, and possibly analogous human complexes) are discussed. In addition, some of these HATs are also known to modify certain nonhistone transcription-related proteins, including high-mobility-group chromatin proteins, activators such as p53, coactivators, and general factors. Thus, we also detail these known factor acetyltransferase (FAT) substrates and the demonstrated or potential roles of their acetylation in transcriptional processes.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                22 April 2014
                : 9
                : 4
                : e94816
                Affiliations
                [1 ]Center for Structural Genomics of Infectious Diseases, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America
                [2 ]Department of Microbiology and Immunology, Stritch School of Medicine, Health Sciences Division, Loyola University Chicago, Maywood, Illinois, United States of America
                [3 ]Buck Institute for Research on Aging, Novato, California, United States of America
                [4 ]Departments of Biomedical Engineering, Chemistry, and Cell & Molecular Biology, Northwestern University, Evanston, Illinois, United States of America
                [5 ]Department of Pharmaceutical Chemistry, University of California, San Francisco, California, United States of America
                University of Arizona, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MLK LIH BPL MS MM WFA BWG BS AJW. Performed the experiments: MLK BZ LIH AS DS GM BPL MS BS. Analyzed the data: MLK BZ LIH AS DS GM BPL MS MM WFA BWG BS AJW. Wrote the paper: MLK LIH BPL WFA BWG BS AJW.

                [¤]

                Current address: Department of Oral Biology, University of California Los Angeles, School of Dentistry, Los Angeles, California, United States of America

                Article
                PONE-D-13-52763
                10.1371/journal.pone.0094816
                3995681
                24756028
                789f6b2e-ab29-4fe4-b592-50eb3a13b356
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 December 2013
                : 19 March 2014
                Page count
                Pages: 26
                Funding
                The use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and Michigan Technology Tri-Corridor (Grant 085P100817). This work was supported by grants from the NIH, NIAID & NIDDK; including R24 DK085610 (NIDDK, to BWG), R01 GM066130 (NIGMS, to AJW), Department of Health and Human Services, Contracts HHSN272200700058C and HHSN272201200026C (NIAID, NIH, Department of Health and Human Services to WFA) and U54CA151880 (NCI, to MM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Carbohydrate Metabolism
                Proteins
                Protein Structure
                Proteomics
                Spectrometric Identification of Proteins
                Microbiology
                Bacteriology
                Bacterial Physiology
                Microbial Physiology
                Microbial Metabolism
                Physical Sciences
                Physics
                Condensed Matter Physics
                Solid State Physics
                Crystallography

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                Uncategorized

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