WorldWideScience

Sample records for posttranslational modification sites

  1. Proteomic analysis of post-translational modifications

    DEFF Research Database (Denmark)

    Mann, Matthias; Jensen, Ole N

    2003-01-01

    Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now...... systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel...

  2. A homology-based pipeline for global prediction of post-translational modification sites

    Science.gov (United States)

    Chen, Xiang; Shi, Shao-Ping; Xu, Hao-Dong; Suo, Sheng-Bao; Qiu, Jian-Ding

    2016-05-01

    The pathways of protein post-translational modifications (PTMs) have been shown to play particularly important roles for almost any biological process. Identification of PTM substrates along with information on the exact sites is fundamental for fully understanding or controlling biological processes. Alternative computational strategies would help to annotate PTMs in a high-throughput manner. Traditional algorithms are suited for identifying the common organisms and tissues that have a complete PTM atlas or extensive experimental data. While annotation of rare PTMs in most organisms is a clear challenge. In this work, to this end we have developed a novel homology-based pipeline named PTMProber that allows identification of potential modification sites for most of the proteomes lacking PTMs data. Cross-promotion E-value (CPE) as stringent benchmark has been used in our pipeline to evaluate homology to known modification sites. Independent-validation tests show that PTMProber achieves over 58.8% recall with high precision by CPE benchmark. Comparisons with other machine-learning tools show that PTMProber pipeline performs better on general predictions. In addition, we developed a web-based tool to integrate this pipeline at http://bioinfo.ncu.edu.cn/PTMProber/index.aspx. In addition to pre-constructed prediction models of PTM, the website provides an extensional functionality to allow users to customize models.

  3. ReportSites - A Computational Method to Extract Positional and Physico- Chemical Information from Large-Scale Proteomic Post-Translational Modification Datasets

    DEFF Research Database (Denmark)

    Edwards, Alistair; Edwards, Gregory; Larsen, Martin Røssel

    2012-01-01

    -translational modification data sets, wherein patterns of sequence surrounding processed sites may reveal more about the functional and structural requirements of the modification and the biochemical processes that regulate them. Results: We developed Report Sites using a test set of phosphoproteomic data from rat......-chemical environment (local pI and hydrophobicity). These were then also compared to corresponding values extracted from the full database to allow comparison of phosphorylation trends. Conclusions: Report Sites enabled physico-chemical aspects of protein phosphorylation to be deciphered in a test set of eleven...... thousand phospho sites. Basic properties of modified proteins, such as site location in the context of the complete protein, were also documented. This program can be easily adapted to any post-translational modification (or, indeed, to any defined amino acid sequence), or expanded to include more...

  4. Lysine-Directed Post-translational Modifications of Tau Protein in Alzheimer's Disease and Related Tauopathies

    Directory of Open Access Journals (Sweden)

    Christiana Kontaxi

    2017-08-01

    Full Text Available Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions.

  5. Post-Translational Modifications of TRP Channels

    Directory of Open Access Journals (Sweden)

    Olaf Voolstra

    2014-04-01

    Full Text Available Transient receptor potential (TRP channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role.

  6. Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques

    DEFF Research Database (Denmark)

    Zhao, Yingming; Jensen, Ole N

    2009-01-01

    More than 300 different types of protein post-translational modifications (PTMs) have been described, many of which are known to have pivotal roles in cellular physiology and disease. Nevertheless, only a handful of PTMs have been extensively investigated at the proteome level. Knowledge of protein...... substrates and their PTM sites is key to dissection of PTM-mediated cellular processes. The past several years have seen a tremendous progress in developing MS-based proteomics technologies for global PTM analysis, including numerous studies of yeast and other microbes. Modification-specific enrichment...

  7. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

    Science.gov (United States)

    Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

    2013-01-01

    Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

  9. Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

    Directory of Open Access Journals (Sweden)

    Mirko eZaffagnini

    2013-11-01

    Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively-modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively-modified GAPDH in stress signaling pathways in plants.

  10. Posttranslational modifications of proopiomelanocortin in vertebrates and their biological significance

    Directory of Open Access Journals (Sweden)

    Akiyoshi eTakahashi

    2013-10-01

    Full Text Available Proopiomelanocortin (POMC is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived alpha-melanocyte-stimulating hormone (alpha-MSH having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, alpha-MSH, as well as desacety-alpha-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, alpha-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas desacetyl-alpha-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of alpha-MSH as an important consequence of posttranslational modification.

  11. Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola

    OpenAIRE

    Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Mukhtar, Hamid; Lee, Siu Fai; Saleem, Muhammad

    2017-01-01

    Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment o...

  12. Evolutionary constraint and disease associations of post-translational modification sites in human genomes.

    Directory of Open Access Journals (Sweden)

    Jüri Reimand

    2015-01-01

    Full Text Available Interpreting the impact of human genome variation on phenotype is challenging. The functional effect of protein-coding variants is often predicted using sequence conservation and population frequency data, however other factors are likely relevant. We hypothesized that variants in protein post-translational modification (PTM sites contribute to phenotype variation and disease. We analyzed fraction of rare variants and non-synonymous to synonymous variant ratio (Ka/Ks in 7,500 human genomes and found a significant negative selection signal in PTM regions independent of six factors, including conservation, codon usage, and GC-content, that is widely distributed across tissue-specific genes and function classes. PTM regions are also enriched in known disease mutations, suggesting that PTM variation is more likely deleterious. PTM constraint also affects flanking sequence around modified residues and increases around clustered sites, indicating presence of functionally important short linear motifs. Using target site motifs of 124 kinases, we predict that at least ∼180,000 motif-breaker amino acid residues that disrupt PTM sites when substituted, and highlight kinase motifs that show specific negative selection and enrichment of disease mutations. We provide this dataset with corresponding hypothesized mechanisms as a community resource. As an example of our integrative approach, we propose that PTPN11 variants in Noonan syndrome aberrantly activate the protein by disrupting an uncharacterized cluster of phosphorylation sites. Further, as PTMs are molecular switches that are modulated by drugs, we study mutated binding sites of PTM enzymes in disease genes and define a drug-disease network containing 413 novel predicted disease-gene links.

  13. Recent advances on the posttranslational modifications of EXTs and their roles in plant cell walls

    DEFF Research Database (Denmark)

    Velasquez, Melina; Salter, Juan Salgado; Dorosz, Javier Gloazzo

    2012-01-01

    The genetic set up and the enzymes that define the O-glycosylation sites and transfer the activated sugars to cell wall glycoprotein Extensins (EXTs) have remained unknown for a long time. We are now beginning to see the emerging components of the molecular machinery that assembles these complex O......-glycoproteins on the plant cell wall. Genes conferring the posttranslational modifications, i.e., proline hydroxylation and subsequent O-glycosylation, of the EXTs have been recently identified. In this review we summarize the enzymes that define the O-glycosylation sites on the O-glycoproteins, i.e., the prolyl 4......-hydroxylases (P4Hs), the glycosyltransferases that transfer arabinose units (named arabinosyltransferases, AraTs), and the one responsible for transferring a single galactose (galactosyltransferase, GalT) on the protein EXT backbones. We discuss the effects of posttranslational modifications on the structure...

  14. A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

    DEFF Research Database (Denmark)

    Graham, Mark E; Thaysen-Andersen, Morten; Bache, Nicolai

    2011-01-01

    Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved......NAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking...... phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification....

  15. Post-translational modifications regulate signalling by Ror1

    Czech Academy of Sciences Publication Activity Database

    Kaucká, M.; Krejčí, Pavel; Plevová, K.; Pavlová, Š.; Procházková, Jiřina; Janovská, P.; Valnohová, J.; Kozubík, Alois; Pospíšilová, Š.; Bryja, Vítězslav

    2011-01-01

    Roč. 203, č. 3 (2011), s. 351-362 ISSN 1748-1708 Institutional support: RVO:68081707 Keywords : chronic lymphocytic leukaemia * glycosylation * post-translational modification Subject RIV: BO - Biophysics Impact factor: 3.090, year: 2011

  16. Hunting for unexpected post-translational modifications by spectral library searching with tier-wise scoring.

    Science.gov (United States)

    Ma, Chun Wai Manson; Lam, Henry

    2014-05-02

    Discovering novel post-translational modifications (PTMs) to proteins and detecting specific modification sites on proteins is one of the last frontiers of proteomics. At present, hunting for post-translational modifications remains challenging in widely practiced shotgun proteomics workflows due to the typically low abundance of modified peptides and the greatly inflated search space as more potential mass shifts are considered by the search engines. Moreover, most popular search methods require that the user specifies the modification(s) for which to search; therefore, unexpected and novel PTMs will not be detected. Here a new algorithm is proposed to apply spectral library searching to the problem of open modification searches, namely, hunting for PTMs without prior knowledge of what PTMs are in the sample. The proposed tier-wise scoring method intelligently looks for unexpected PTMs by allowing mass-shifted peak matches but only when the number of matches found is deemed statistically significant. This allows the search engine to search for unexpected modifications while maintaining its ability to identify unmodified peptides effectively at the same time. The utility of the method is demonstrated using three different data sets, in which the numbers of spectrum identifications to both unmodified and modified peptides were substantially increased relative to a regular spectral library search as well as to another open modification spectral search method, pMatch.

  17. Tubulin post-translational modifications in the primitive protist Trichomonas vaginalis.

    Science.gov (United States)

    Delgado-Viscogliosi, P; Brugerolle, G; Viscogliosi, E

    1996-01-01

    Using several specific monoclonal antibodies, we investigated the occurrence and distribution of different post-translationally modified tubulin during interphase and division of the primitive flagellated protist Trichomonas vaginalis. Immunoblotting and immunofluorescence experiments revealed that interphasic microtubular structures of T. vaginalis contained acetylated and glutamylated but non-tyrosinated and non-glycylated [Brugerolle and Adoutte, 1988: Bio Systems 21: 255-268] tubulin. Immunofluorescence studies performed on dividing cells showed that the extranuclear mitotic spindle (or paradesmosis) was acetylated and glutamylated, which contrast with the ephemeral nature of this structure. Newly formed short axostyles also contained acetylated and glutamylated tubulin suggesting that both post-translational modifications might take place very early after assembly of microtubular structures. Our results indicate that acetylation and glutamylation of tubulin appeared early in the history of eukaryotes and could reflect the occurrence of post-translational modifications of tubulin in the primitive eukaryotic cells. These cells probably had a highly ordered cross-linked microtubular cytoskeleton in which microtubules showed a low level of subunit exchange dynamics.

  18. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

    DEFF Research Database (Denmark)

    Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

    2016-01-01

    and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

  19. Prediction of protein post-translational modifications: main trends and methods

    Science.gov (United States)

    Sobolev, B. N.; Veselovsky, A. V.; Poroikov, V. V.

    2014-02-01

    The review summarizes main trends in the development of methods for the prediction of protein post-translational modifications (PTMs) by considering the three most common types of PTMs — phosphorylation, acetylation and glycosylation. Considerable attention is given to general characteristics of regulatory interactions associated with PTMs. Different approaches to the prediction of PTMs are analyzed. Most of the methods are based only on the analysis of the neighbouring environment of modification sites. The related software is characterized by relatively low accuracy of PTM predictions, which may be due both to the incompleteness of training data and the features of PTM regulation. Advantages and limitations of the phylogenetic approach are considered. The prediction of PTMs using data on regulatory interactions, including the modular organization of interacting proteins, is a promising field, provided that a more carefully selected training data will be used. The bibliography includes 145 references.

  20. Trafficking and function of the cystic fibrosis transmembrane conductance regulator: a complex network of posttranslational modifications

    Science.gov (United States)

    McClure, Michelle L.; Barnes, Stephen; Brodsky, Jeffrey L.

    2016-01-01

    Posttranslational modifications add diversity to protein function. Throughout its life cycle, the cystic fibrosis transmembrane conductance regulator (CFTR) undergoes numerous covalent posttranslational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and posttranslational CFTR modifications and their significance with regard to protein biogenesis. PMID:27474090

  1. Cell signaling, post-translational protein modifications and NMR spectroscopy

    International Nuclear Information System (INIS)

    Theillet, Francois-Xavier; Smet-Nocca, Caroline; Liokatis, Stamatios; Thongwichian, Rossukon; Kosten, Jonas; Yoon, Mi-Kyung; Kriwacki, Richard W.; Landrieu, Isabelle; Lippens, Guy; Selenko, Philipp

    2012-01-01

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy.

  2. Tipping the balance by manipulating posttranslational modifications

    Science.gov (United States)

    Broberg, Christopher A.; Orth, Kim

    2010-01-01

    Summary Bacteria use a variety of mechanisms during infection to ensure their survival including the delivery of virulence factors via a type III secretion system into the infected cell. The factors exhibit diverse activities that in many cases mimic eukaryotic mechanisms used by the host to defend against infection. Herein we describe a class of effectors that use posttranslational modifications, some reversible and others irreversible, to manipulate host signaling systems to subvert the host response. PMID:20071215

  3. Multiple post-translational modifications in hepatocyte nuclear factor 4α

    International Nuclear Information System (INIS)

    Yokoyama, Atsushi; Katsura, Shogo; Ito, Ryo; Hashiba, Waka; Sekine, Hiroki; Fujiki, Ryoji; Kato, Shigeaki

    2011-01-01

    Highlights: → We performed comprehensive PTM analysis for HNF4α protein. → We identified 8 PTMs in HNF4α protein including newly identified PTMs. → Among them, we found acetylation at lysine 458 was one of the prime PTMs for HNF4α function. → Acetylation at lysine 458 was inhibitory for HNF4α transcription function. → This modification fluctuated in response to extracellular condition. -- Abstract: To investigate the role of post-translational modifications (PTMs) in the hepatocyte nuclear factor 4α (HNF4α)-mediated transcription, we took a comprehensive survey of PTMs in HNF4α protein by massspectrometry and identified totally 8 PTM sites including newly identified ubiquitilation and acetylation sites. To assess the impact of identified PTMs in HNF4α-function, we introduced point mutations at the identified PTM sites and, tested transcriptional activity of the HNF4α. Among the point-mutations, an acetylation site at lysine 458 was found significant in the HNF4α-mediated transcriptional control. An acetylation negative mutant at lysine 458 showed an increased transcriptional activity by about 2-fold, while an acetylation mimic mutant had a lowered transcriptional activation. Furthermore, this acetylation appeared to be fluctuated in response to extracellular nutrient conditions. Thus, by applying an comprehensive analysis of PTMs, multiple PTMs were newly identified in HNF4α and unexpected role of an HNF4α acetylation could be uncovered.

  4. Spatial and Temporal Effects in Protein Post-translational Modification Distributions in the Developing Mouse Brain

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Edwards, Gregory J; Schwämmle, Veit

    2014-01-01

    Protein post-translational modification (PTM) is a powerful way to modify the behavior of cellular proteins and thereby cellular behavior. Multiple recent studies of evolutionary trends have shown that certain pairs of protein post-translational modifications tend to occur closer to each other than...... for observations of increasingly frequent and diverse protein modification in cell biology. In this study, we use mass spectrometry and proteomic strategies to present biological data showing spatiotemporal PTM co-localization across multiple PTM categories, which display changes over development of the brain...

  5. Golgi structure formation, function, and post-translational modifications in mammalian cells.

    Science.gov (United States)

    Huang, Shijiao; Wang, Yanzhuang

    2017-01-01

    The Golgi apparatus is a central membrane organelle for trafficking and post-translational modifications of proteins and lipids in cells. In mammalian cells, it is organized in the form of stacks of tightly aligned flattened cisternae, and dozens of stacks are often linked laterally into a ribbon-like structure located in the perinuclear region of the cell. Proper Golgi functionality requires an intact architecture, yet Golgi structure is dynamically regulated during the cell cycle and under disease conditions. In this review, we summarize our current understanding of the relationship between Golgi structure formation, function, and regulation, with focus on how post-translational modifications including phosphorylation and ubiquitination regulate Golgi structure and on how Golgi unstacking affects its functions, in particular, protein trafficking, glycosylation, and sorting in mammalian cells.

  6. Large scale analysis of co-existing post-translational modifications in histone tails reveals global fine structure of cross-talk

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Aspalter, Claudia-Maria; Sidoli, Simone

    2014-01-01

    Mass spectrometry (MS) is a powerful analytical method for the identification and quantification of co-existing post-translational modifications in histone proteins. One of the most important challenges in current chromatin biology is to characterize the relationships between co-existing histone...... sample-specific patterns for the co-frequency of histone post-translational modifications. We implemented a new method to identify positive and negative interplay between pairs of methylation and acetylation marks in proteins. Many of the detected features were conserved between different cell types...... sites but negative cross-talk for distant ones, and for discrete methylation states at Lys-9, Lys-27, and Lys-36 of histone H3, suggesting a more differentiated functional role of methylation beyond the general expectation of enhanced activity at higher methylation states....

  7. Targeting post-translational modifications of histones for cancer therapy.

    Science.gov (United States)

    Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H

    2015-10-30

    Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.

  8. Effect of posttranslational modifications on enzyme function and assembly

    Czech Academy of Sciences Publication Activity Database

    Ryšlavá, H.; Doubnerová, V.; Kavan, Daniel; Vaněk, O.

    2013-01-01

    Roč. 92, OCT 2013 (2013), s. 80-109 ISSN 1874-3919 R&D Projects: GA ČR(CZ) GAP207/10/1040; GA ČR GA303/09/0477; GA ČR GD305/09/H008; GA ČR(CZ) GAP504/11/0394 Institutional support: RVO:61388971 Keywords : Enzyme * Posttranslational modification * Structure Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.929, year: 2013

  9. Elucidating Host-Pathogen Interactions Based on Post-Translational Modifications Using Proteomics Approaches

    DEFF Research Database (Denmark)

    Ravikumar, Vaishnavi; Jers, Carsten; Mijakovic, Ivan

    2015-01-01

    can be efficiently applied to gain an insight into the molecular mechanisms involved. The measurement of the proteome and post-translationally modified proteome dynamics using mass spectrometry, results in a wide array of information, such as significant changes in protein expression, protein...... display host specificity through a complex network of molecular interactions that aid their survival and propagation. Co-infection states further lead to complications by increasing the microbial burden and risk factors. Quantitative proteomics based approaches and post-translational modification analysis...... pathogen interactions....

  10. Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins

    Czech Academy of Sciences Publication Activity Database

    Stepper, J.; Shastri, S.; Loo, T. S.; Preston, J. C.; Novák, Petr; Man, Petr; Moore, Ch. H.; Havlíček, Vladimír; Patchett, M. L.; Norris, G. E.

    2011-01-01

    Roč. 585, č. 4 (2011), s. 645-650 ISSN 0014-5793 Institutional research plan: CEZ:AV0Z50200510 Keywords : Post-translational modification * Glycosylation * Bacteriocin Subject RIV: CE - Biochemistry Impact factor: 3.538, year: 2011

  11. Nitric Oxide-Mediated Posttranslational Modifications: Impacts at the Synapse

    Directory of Open Access Journals (Sweden)

    Sophie A. Bradley

    2016-01-01

    Full Text Available Nitric oxide (NO is an important gasotransmitter molecule that is involved in numerous physiological processes throughout the nervous system. In addition to its involvement in physiological plasticity processes (long-term potentiation, LTP; long-term depression, LTD which can include NMDAR-mediated calcium-dependent activation of neuronal nitric oxide synthase (nNOS, new insights into physiological and pathological consequences of nitrergic signalling have recently emerged. In addition to the canonical cGMP-mediated signalling, NO is also implicated in numerous pathways involving posttranslational modifications. In this review we discuss the multiple effects of S-nitrosylation and 3-nitrotyrosination on proteins with potential modulation of function but limit the analyses to signalling involved in synaptic transmission and vesicular release. Here, crucial proteins which mediate synaptic transmission can undergo posttranslational modifications with either pre- or postsynaptic origin. During normal brain function, both pathways serve as important cellular signalling cascades that modulate a diverse array of physiological processes, including synaptic plasticity, transcriptional activity, and neuronal survival. In contrast, evidence suggests that aging and disease can induce nitrosative stress via excessive NO production. Consequently, uncontrolled S-nitrosylation/3-nitrotyrosination can occur and represent pathological features that contribute to the onset and progression of various neurodegenerative diseases, including Parkinson’s, Alzheimer’s, and Huntington’s.

  12. Comparison at the peptide level with post-translational modification consideration reveals more differences between two unenriched samples.

    Science.gov (United States)

    Yin, Jianrui; Shao, Chen; Jia, Lulu; Gao, Youhe

    2014-06-30

    In shotgun strategies, peptide sequences are first identified from tandem mass (MS/MS) spectra, and the existence and abundance of the proteins are then inferred from the peptide information. However, the protein inference step can produce errors and a loss of information. To identify the information that is lost using the traditional approaches, this study compared the proteomic data of two leukemia cell lines (Jurkat and K562) at the peptide level with consideration of post-translational modifications (PTMs). The raw files from the two cell lines were searched against the decoy IPI-human database version 3.68, which contains forward and reverse sequences. Then the observed modification name in the results was matched with the modification classification on the Unimod website by a manual search. Only the peptides with 'post-translational' modifications were compared between the two cell lines. After searching the database with consideration of PTMs, a total of 44046 non-redundant peptides were identified in both the Jurkat and K562 cell lines. Of these peptides, even without specific PTM enrichment, 11.43% of them (with at least two spectra in one cell line) existed in different PTM forms between the two cell lines, and 1.73% of the peptides were modified in both cell lines, but with different modifications or possibly on different sites. Comparing proteomic data at the peptide level with consideration of PTMs can reveal more differences between two unenriched samples. Copyright © 2014 John Wiley & Sons, Ltd.

  13. Global turnover of histone post-translational modifications and variants in human cells

    Directory of Open Access Journals (Sweden)

    Zee Barry M

    2010-12-01

    Full Text Available Abstract Background Post-translational modifications (PTMs on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement. Results In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same

  14. Variable elimination in post-translational modification reaction networks with mass-action kinetics

    DEFF Research Database (Denmark)

    Feliu, Elisenda; Wiuf, Carsten

    2013-01-01

    We define a subclass of chemical reaction networks called post-translational modification systems. Important biological examples of such systems include MAPK cascades and two-component systems which are well-studied experimentally as well as theoretically. The steady states of such a system...

  15. Posttranslational Modification Biology of Glutamate Receptors and Drug Addiction

    Directory of Open Access Journals (Sweden)

    Li-Min eMao

    2011-03-01

    Full Text Available Posttranslational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues on their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling and protein-protein interactions, subcellular redistribution (trafficking, endocytosis, synaptic delivery and clustering, and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamines. Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction.

  16. Post-Translational Modifications of RelB NF-κB Subunit and Associated Functions

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    Véronique Baud

    2016-05-01

    Full Text Available The family of NF-κB transcription factors plays a key role in diverse biological processes, such as inflammatory and immune responses, cell survival and tumor development. Beyond the classical NF-κB activation pathway, a second NF-κB pathway has more recently been uncovered, the so-called alternative NF-κB activation pathway. It has been shown that this pathway mainly controls the activity of RelB, a member of the NF-κB family. Post-translational modifications, such as phosphorylation, acetylation, methylation, ubiquitination and SUMOylation, have recently emerged as a strategy for the fine-tuned regulation of NF-κB. Our review discusses recent progress in the understanding of RelB regulation by post-translational modifications and the associated functions in normal and pathological conditions.

  17. Regulation of the tumor suppressor PML by sequential posttranslational modifications

    Directory of Open Access Journals (Sweden)

    Lienhard eSchmitz

    2012-12-01

    Full Text Available Posttranslational modifications (PTMs regulate multiple biological functions of the PML (promyelocytic leukemia protein and also the fission, disassembly and rebuilding of PML nuclear bodies (PML-NBs during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitute typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα.

  18. Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth

    DEFF Research Database (Denmark)

    Boskey, Adele L.; Christensen, Brian Søndergaard; Taleb, Hayat

    2012-01-01

    The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA...

  19. Post-Translational Modifications of Desulfovibrio vulgaris Hildenborough Sulfate Reduction Pathway Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gaucher, S.P.; Redding, A.M.; Mukhopadhyay, A.; Keasling, J.D.; Singh, A.K.

    2008-03-01

    Recent developments in shotgun proteomics have enabled high-throughput studies of a variety of microorganisms at a proteome level and provide experimental validation for predicted open reading frames in the corresponding genome. More importantly, advances in mass spectrometric data analysis now allow mining of large proteomics data sets for the presence of post-translational modifications(PTMs). Although PTMs are a critical aspectof cellular activity, such information eludes cell-wide studies conducted at the transcript level. Here, we analyze several mass spectrometric data sets acquired using two-dimensional liquid chromatography tandem mass spectrometry, 2D-LC/MS/MS, for the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough. Our searches of the raw spectra led us to discover several post-translationally modified peptides in D. vulgaris. Of these, several peptides containing a lysine with a +42 Da modification were found reproducibly across all data sets. Both acetylation and trimethylation have the same nominal +42 Da mass, and are therefore candidates for this modification. Several spectra were identified having markers for trimethylation, while one is consistent with an acetylation. Surprisingly, these modified peptides predominantly mapped to proteins involved in sulfate respiration. Other highly expressed proteins in D. vulgaris, such as enzymes involved in electron transport and other central metabolic processes, did not contain this modification. Decoy database searches were used to control for random spectrum/sequence matches. Additional validation for these modifications was provided by alternate workflows, for example, two-dimensional gel electrophoresis followed by mass spectrometry analysis of the dissimilatory sulfite reductase gamma-subunit(DsrC) protein. MS data for DsrC in this alternate workflow also contained the +42 Da modification at the same loci. Furthermore, the DsrC homologue in another sulfate reducing bacterium

  20. Regulation of the tumor suppressor PML by sequential post-translational modifications

    International Nuclear Information System (INIS)

    Schmitz, M. Lienhard; Grishina, Inna

    2012-01-01

    Post-translational modifications (PTMs) regulate multiple biological functions of the promyelocytic leukemia (PML) protein and also the fission, disassembly, and rebuilding of PML nuclear bodies (PML-NBs) during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitutes typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα.

  1. Posttranslational Modifications and the Immunogenicity of Biotherapeutics

    Directory of Open Access Journals (Sweden)

    Roy Jefferis

    2016-01-01

    Full Text Available Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs, including quality control (QC in the endoplasmic reticulum (ER and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA; aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs, a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

  2. Post-Translational Modifications of Histones in Human Sperm.

    Science.gov (United States)

    Krejčí, Jana; Stixová, Lenka; Pagáčová, Eva; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, Gabriela; Zdráhal, Zbyněk; Sehnalová, Petra; Dabravolski, Siarhei; Hejátko, Jan; Bártová, Eva

    2015-10-01

    We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. © 2015 Wiley Periodicals, Inc.

  3. Modification site localization scoring integrated into a search engine.

    Science.gov (United States)

    Baker, Peter R; Trinidad, Jonathan C; Chalkley, Robert J

    2011-07-01

    Large proteomic data sets identifying hundreds or thousands of modified peptides are becoming increasingly common in the literature. Several methods for assessing the reliability of peptide identifications both at the individual peptide or data set level have become established. However, tools for measuring the confidence of modification site assignments are sparse and are not often employed. A few tools for estimating phosphorylation site assignment reliabilities have been developed, but these are not integral to a search engine, so require a particular search engine output for a second step of processing. They may also require use of a particular fragmentation method and are mostly only applicable for phosphorylation analysis, rather than post-translational modifications analysis in general. In this study, we present the performance of site assignment scoring that is directly integrated into the search engine Protein Prospector, which allows site assignment reliability to be automatically reported for all modifications present in an identified peptide. It clearly indicates when a site assignment is ambiguous (and if so, between which residues), and reports an assignment score that can be translated into a reliability measure for individual site assignments.

  4. The interplay of post-translational modification and gene therapy

    Directory of Open Access Journals (Sweden)

    Osamor VC

    2016-02-01

    Full Text Available Victor Chukwudi Osamor,1–3 Shalom N Chinedu,3,4 Dominic E Azuh,3,5 Emeka Joshua Iweala,3,4 Olubanke Olujoke Ogunlana3,4 1Covenant University Bioinformatics Research (CUBRe Unit, Department of Computer and Information Sciences, College of Science and Technology (CST, Covenant University, Ota, Ogun State, Nigeria; 2Institute of Informatics (Computational biology and Bioinformatics, Faculty of Mathematics, Informatics and Mechanics, University of Warsaw (Uniwersytet Warszawski, Warszawa, Poland; 3Covenant University Public Health and Well-being Research Group (CUPHWERG, Covenant University, 4Biochemistry and Molecular Biology Unit, Department of Biological Sciences, College of Science and Technology, Covenant University, Canaan Land, 5Department of Economics and Development Studies, Covenant University, Ota, Ogun State, Nigeria Abstract: Several proteins interact either to activate or repress the expression of other genes during transcription. Based on the impact of these activities, the proteins can be classified into readers, modifier writers, and modifier erasers depending on whether histone marks are read, added, or removed, respectively, from a specific amino acid. Transcription is controlled by dynamic epigenetic marks with serious health implications in certain complex diseases, whose understanding may be useful in gene therapy. This work highlights traditional and current advances in post-translational modifications with relevance to gene therapy delivery. We report that enhanced understanding of epigenetic machinery provides clues to functional implication of certain genes/gene products and may facilitate transition toward revision of our clinical treatment procedure with effective fortification of gene therapy delivery. Keywords: post-translational modification, gene therapy, epigenetics, histone, methylation

  5. Analysis of the post-translational modifications of the individual amino acids in lens proteins which were induced by aging and irradiation

    International Nuclear Information System (INIS)

    Fujii, Noriko; Kim, Ingu; Saito, Takeshi; Takata, Takumi

    2017-01-01

    The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, β- and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500 Gy) and extracted the water-soluble (WS) and insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation of asparagine and glutamine, and isomerization of aspartyl in rat α- and β-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 Gy and 5 Gy; however, isomerization did not occur in any crystallin even after exposure to 500 Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts. (author)

  6. Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions.

    Science.gov (United States)

    Su, Min-Gang; Weng, Julia Tzu-Ya; Hsu, Justin Bo-Kai; Huang, Kai-Yao; Chi, Yu-Hsiang; Lee, Tzong-Yi

    2017-12-21

    Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM's function is critical to our ability to manipulate the biological mechanisms of protein. In this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking

  7. Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola

    Directory of Open Access Journals (Sweden)

    Rabia Arif

    2017-01-01

    Full Text Available Posttranslational modifications (PTMs occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope of Evolution Canyon (EC, Israel. Based on the whole genome sequence of S. macrospora, we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs. Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution.

  8. Genetic Variation and Its Reflection on Posttranslational Modifications in Frequency Clock and Mating Type a-1 Proteins in Sordaria fimicola.

    Science.gov (United States)

    Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Mukhtar, Hamid; Lee, Siu Fai; Saleem, Muhammad

    2017-01-01

    Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora , we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution.

  9. Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

    Science.gov (United States)

    Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-15

    Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to

  10. Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Mowei; Wu, Si; Stenoien, David L.; Zhang, Zhaorui; Connolly, Lanelle; Freitag, Michael; Pasa-Tolic, Ljiljana

    2016-11-11

    Top-down mass spectrometry is a valuable tool for charactering post-translational modifications on histones for understanding of gene control and expression. In this protocol, we describe a top-down workflow using liquid chromatography coupled to mass spectrometry for fast global profiling of changes in histone proteoforms between a wild-type and a mutant of a fungal species. The proteoforms exhibiting different abundances can be subjected to further targeted studies by other mass spectrometric or biochemical assays. This method can be generally adapted for preliminary screening for changes in histone modifications between samples such as wild-type vs. mutant, and control vs. disease.

  11. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns

    DEFF Research Database (Denmark)

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone

    2015-01-01

    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties...... hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly...

  12. Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors.

    Science.gov (United States)

    Olson, Michael F

    2018-05-04

    The 20 members of the Rho GTPase family are key regulators of a wide-variety of biological activities. In response to activation, they signal via downstream effector proteins to induce dynamic alterations in the organization of the actomyosin cytoskeleton. In this review, post-translational modifications, mechanisms of dysregulation identified in human pathological conditions, and the ways that Rho GTPases might be targeted for chemotherapy will be discussed.

  13. Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

    Directory of Open Access Journals (Sweden)

    Shisheng Li

    2012-09-01

    Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

  14. Autoantibodies to Posttranslational Modifications in Rheumatoid Arthritis

    Science.gov (United States)

    Burska, Agata N.; Hunt, Laura; Strollo, Rocky; Ryan, Brent J.; Vital, Ed; Nissim, Ahuva; Winyard, Paul G.; Emery, Paul; Ponchel, Frederique

    2014-01-01

    Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell development in vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential. PMID:24782594

  15. Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives

    Science.gov (United States)

    Edelmann, Mariola J.

    2011-01-01

    Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide. PMID:22174558

  16. Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.

    Science.gov (United States)

    Moraes, Izabel; Yuan, Zuo-Fei; Liu, Shichong; Souza, Glaucia Mendes; Garcia, Benjamin A; Casas-Mollano, J Armando

    2015-01-01

    Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in

  17. Molecular dynamics simulation of phosphorylated KID post-translational modification.

    Directory of Open Access Journals (Sweden)

    Hai-Feng Chen

    2009-08-01

    Full Text Available Kinase-inducible domain (KID as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX. NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood.To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD for both bound and apo phosphorylated KID (pKID. Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding.Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B, folding of helix alpha(A, completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A and alpha(B are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.

  18. Site-selective protein-modification chemistry for basic biology and drug development.

    Science.gov (United States)

    Krall, Nikolaus; da Cruz, Filipa P; Boutureira, Omar; Bernardes, Gonçalo J L

    2016-02-01

    Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.

  19. Use of synthetic biology techniques to site-selective introduce posttranslational modifactions in proteins

    NARCIS (Netherlands)

    Bosmans, R.P.G.; Brunsveld, L.; Ryadnov, M.; Brunsveld, L.; Suga, H.

    2014-01-01

    Unravelling the influence of posttranslational modifications (PTMs) on protein functioning is of key interest to get understanding how complex cellular networks are regulated. The current biological toolbox to synthesize these modified proteins in a single form in decent quantities is insufficient,

  20. PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

    Science.gov (United States)

    Chaudhuri, Rima; Sadrieh, Arash; Hoffman, Nolan J; Parker, Benjamin L; Humphrey, Sean J; Stöckli, Jacqueline; Hill, Adam P; James, David E; Yang, Jean Yee Hwa

    2015-08-19

    Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites

  1. A sensitive mass spectrometric method for hypothesis-driven detection of peptide post-translational modifications: multiple reaction monitoring-initiated detection and sequencing (MIDAS).

    Science.gov (United States)

    Unwin, Richard D; Griffiths, John R; Whetton, Anthony D

    2009-01-01

    The application of a targeted mass spectrometric workflow to the sensitive identification of post-translational modifications is described. This protocol employs multiple reaction monitoring (MRM) to search for all putative peptides specifically modified in a target protein. Positive MRMs trigger an MS/MS experiment to confirm the nature and site of the modification. This approach, termed MIDAS (MRM-initiated detection and sequencing), is more sensitive than approaches using neutral loss scanning or precursor ion scanning methodologies, due to a more efficient use of duty cycle along with a decreased background signal associated with MRM. We describe the use of MIDAS for the identification of phosphorylation, with a typical experiment taking just a couple of hours from obtaining a peptide sample. With minor modifications, the MIDAS method can be applied to other protein modifications or unmodified peptides can be used as a MIDAS target.

  2. How Posttranslational Modification of Nitrogenase Is Circumvented in Rhodopseudomonas palustris Strains That Produce Hydrogen Gas Constitutively

    Science.gov (United States)

    Heiniger, Erin K.; Oda, Yasuhiro; Samanta, Sudip K.

    2012-01-01

    Nitrogenase catalyzes the conversion of dinitrogen gas (N2) and protons to ammonia and hydrogen gas (H2). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris have been described that express nitrogenase genes constitutively and produce H2 when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of nitrogenase. Microarray, mutation analysis, and gene expression studies showed that posttranslational regulation of nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated PII protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 nitrogenase modification enzyme. In addition, the NifA* strain had elevated nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H2 production by an nifA* mutant grown with ammonium. Inactivation of the nitrogenase posttranslational modification system by mutation of draT2 resulted in increased H2 production by ammonium-grown NifA* cells. PMID:22179236

  3. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

    Science.gov (United States)

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

    2016-01-21

    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Multiple γ-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

    International Nuclear Information System (INIS)

    Hasegawa, Mai; Ikeda, Yuka; Kanzawa, Hideaki; Sakamoto, Mika; Goto, Mina; Tsunasawa, Susumu; Uchiumi, Toshio; Odani, Shoji

    2010-01-01

    A highly hydrophilic, glutamate-rich protein was identified in the aqueous phenol extract from the cytosolic fraction of brine shrimp (Artemia franciscana) diapausing cysts and termed Artemia phenol soluble protein (PSP). Mass spectrometric analysis revealed the presence of many protein peaks around m/z 11,000, separated by 129 atomic mass units; this value corresponds to that of glutamate, which is strongly suggestive of heterogeneous polyglutamylation. Polyglutamylation has long been known as the functionally important post-translational modification of tubulins, which carry poly(L-glutamic acid) chains of heterogeneous length branching off from the main chain at the γ-carboxy groups of a few specific glutamate residues. In Artemia PSP, however, Edman degradation of enzymatic peptides revealed that at least 13, and presumably 16, glutamate residues were modified by the attachment of a single L-glutamate, representing a hitherto undescribed type of post-translational modification: namely, multiple γ-glutamylation or the addition of a large number of glutamate residues along the polypeptide chain. Although biological significance of PSP and its modification is yet to be established, suppression of in vitro thermal aggregation of lactate dehydrogenase by glutamylated PSP was observed.

  5. Prediction of human protein function from post-translational modifications and localization features

    DEFF Research Database (Denmark)

    Jensen, Lars Juhl; Gupta, Ramneek; Blom, Nikolaj

    2002-01-01

    a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects......We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from...

  6. Electrospray mass spectrometry characterization of post-translational modifications of barley alpha-amylase 1 produced in yeast

    DEFF Research Database (Denmark)

    Søgaard, M; Andersen, Jens S.; Roepstorff, P

    1993-01-01

    We have used electrospray mass spectrometry (ESMS) in combination with protein chemistry and genetics to delineate post-translational modifications in yeast of barley alpha-amylase 1 (AMY1), a 45 kD enzyme crucial for production of malt, an important starting material in the manufacture of beer...

  7. Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes

    DEFF Research Database (Denmark)

    Lyles, J M; Linnemann, D; Bock, E

    1984-01-01

    Posttranslational modifications and intracellular transport of the D2-cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A...

  8. Beyond gene expression: the impact of protein post-translational modifications in bacteria.

    Science.gov (United States)

    Cain, Joel A; Solis, Nestor; Cordwell, Stuart J

    2014-01-31

    The post-translational modification (PTM) of proteins plays a critical role in the regulation of a broad range of cellular processes in eukaryotes. Yet their role in governing similar systems in the conventionally presumed 'simpler' forms of life has been largely neglected and, until recently, was thought to occur only rarely, with some modifications assumed to be limited to higher organisms alone. Recent developments in mass spectrometry-based proteomics have provided an unparalleled power to enrich, identify and quantify peptides with PTMs. Additional modifications to biological molecules such as lipids and carbohydrates that are essential for bacterial pathophysiology have only recently been detected on proteins. Here we review bacterial protein PTMs, focusing on phosphorylation, acetylation, proteolytic degradation, methylation and lipidation and the roles they play in bacterial adaptation - thus highlighting the importance of proteomic techniques in a field that is only just in its infancy. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 Elsevier B.V. All rights reserved.

  9. POSTRANSLATIONAL MODIFICATIONS OF P53: UPSTREAM SIGNALING PATHWAYS.

    Energy Technology Data Exchange (ETDEWEB)

    ANDERSON,C.W.APPELLA,E.

    2003-10-23

    The p53 tumor suppressor is a tetrameric transcription factor that is posttranslational modified at >20 different sites by phosphorylation, acetylation, or sumoylation in response to various cellular stress conditions. Specific posttranslational modifications, or groups of modifications, that result from the activation of different stress-induced signaling pathways are thought to modulate p53 activity to regulate cell fate by inducing cell cycle arrest, apoptosis, or cellular senescence. Here we review recent progress in characterizing the upstream signaling pathways whose activation in response to various genotoxic and non-genotoxic stresses result in p53 posttranslational modifications.

  10. Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

    Science.gov (United States)

    Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

    2016-02-15

    The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Post-Translational Modifications of H2A Histone Variants and Their Role in Cancer

    Directory of Open Access Journals (Sweden)

    David Corujo

    2018-02-01

    Full Text Available Histone variants are chromatin components that replace replication-coupled histones in a fraction of nucleosomes and confer particular characteristics to chromatin. H2A variants represent the most numerous and diverse group among histone protein families. In the nucleosomal structure, H2A-H2B dimers can be removed and exchanged more easily than the stable H3-H4 core. The unstructured N-terminal histone tails of all histones, but also the C-terminal tails of H2A histones protrude out of the compact structure of the nucleosome core. These accessible tails are the preferential target sites for a large number of post-translational modifications (PTMs. While some PTMs are shared between replication-coupled H2A and H2A variants, many modifications are limited to a specific histone variant. The present review focuses on the H2A variants H2A.Z, H2A.X, and macroH2A, and summarizes their functions in chromatin and how these are linked to cancer development and progression. H2A.Z primarily acts as an oncogene and macroH2A and H2A.X as tumour suppressors. We further focus on the regulation by PTMs, which helps to understand a degree of context dependency.

  12. Overview of xeroderma pigmentosum proteins architecture, mutations and post-translational modifications.

    Science.gov (United States)

    Feltes, Bruno César; Bonatto, Diego

    2015-01-01

    The xeroderma pigmentosum complementation group proteins (XPs), which include XPA through XPG, play a critical role in coordinating and promoting global genome and transcription-coupled nucleotide excision repair (GG-NER and TC-NER, respectively) pathways in eukaryotic cells. GG-NER and TC-NER are both required for the repair of bulky DNA lesions, such as those induced by UV radiation. Mutations in genes that encode XPs lead to the clinical condition xeroderma pigmentosum (XP). Although the roles of XPs in the GG-NER/TC-NER subpathways have been extensively studied, complete knowledge of their three-dimensional structure is only beginning to emerge. Hence, this review aims to summarize the current knowledge of mapped mutations and other structural information on XP proteins that influence their function and protein-protein interactions. We also review the possible post-translational modifications for each protein and the impact of these modifications on XP protein functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1 in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Stefan Burén

    Full Text Available BACKGROUND: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. METHODOLOGY/PRINCIPAL FINDINGS: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. CONCLUSIONS/SIGNIFICANCE: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native

  14. Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

    2017-01-01

    Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein, we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.

  15. Posttranslational modification of bioaerosol protein by common gas pollutants: NO2 and O3

    Science.gov (United States)

    Abdullahi Mahmood, Marliyyah; Bloss, William; Pope, Francis

    2016-04-01

    Air pollution can exacerbate several medical conditions, for example, hay fever and asthma. The global incidence of hay fever has been rising for decades; however, the underlying reasons behind this rise remain unclear. It is hypothesized that the exposure of pollen to common gas phase pollutants, such as nitrogen dioxide (NO2) and ozone (O3), increases the allergenicity of the pollen and thus increases hay fever incidence (Reinmuth-Selzle et al., 2014, Franze, et al., 2005). Since atmospheric pollutants often have greater concentrations within urban areas (in particular NO2) the hypothesis suggests that greater allergenicity should occur in urban areas. Certainly, several studies do suggest higher hay fever incidence within urban areas compared to rural areas (Schröder et al., 2015). Previous published work suggests a link between increased allergies and changes in the chemical composition of pollen protein via posttranslational modification of the protein (Reinmuth-Selzle et al., 2014). This study investigates the posttranslational modification of two highly allergenic pollen species (Birch and Ragweed) that are common in Europe. Within the laboratory, we expose pollen grains to atmospherically relevant exposures of gas phase NO2, O3 and other common gas phase oxidants under a range of environmentally relevant conditions. The effects of the exposures on the biochemistry of the pollen grains were probed using a proteomic approach (liquid chromatography coupled ultra-high resolution spectrometer). Our findings indicate the interaction between gas phase pollutants and pollen cause protein specific modifications; in particular nitration that occurs upon tyrosine residues and nitrosylation on cysteine residues. These modifications may affect human immune response to the pollen protein, which may suggest a possible reason for increased allergies in reaction to such chemically altered protein. Quantification of the relative degree of PTMs, from a variety of

  16. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  17. Tyrosine Sulfation as a Protein Post-Translational Modification

    Directory of Open Access Journals (Sweden)

    Yuh-Shyong Yang

    2015-01-01

    Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

  18. Posttranslational modifications in human plasma MBL and human recombinant MBL

    DEFF Research Database (Denmark)

    Jensen, Pia Hønnerup; Laursen, Inga; Matthiesen, Finn

    2007-01-01

    the intact protein in its active conformation. For the first time, positions and occupation frequency of partial hydroxylations and partial glycosylations are reported in MBL. Hydroxylation and glycosylation patterns of both recombinant and plasma derived MBL were determined, using a combination of mass......Mannan-binding lectin (MBL) is a complex serum protein that plays an important role in innate immunity. In addition to assuming several different oligomeric forms, the polypeptide itself is highly heterogeneous. This heterogeneity is due to post-translational modifications, which help to stabilize......(202)) was modified in trace amounts to dehydroalanine, as detected by a 34 Da mass loss. This impairs the formation of a disulphide bond in the carbohydrate recognition domain. The dehydroalanine was identified in similar small amounts in both recombinant and plasma-derived MBL....

  19. Identification and characterization of HTLV-1 HBZ post-translational modifications.

    Directory of Open Access Journals (Sweden)

    Nathan Dissinger

    Full Text Available Human T-cell leukemia virus type-1 (HTLV-1 is estimated to infect 15-25 million people worldwide, with several areas including southern Japan and the Caribbean basin being endemic. The virus is the etiological agent of debilitating and fatal diseases, for which there is currently no long-term cure. In the majority of cases of leukemia caused by HTLV-1, only a single viral gene, hbz, and its cognate protein, HBZ, are expressed and their importance is increasingly being recognized in the development of HTLV-1-associated disease. We hypothesized that HBZ, like other HTLV-1 proteins, has properties and functions regulated by post-translational modifications (PTMs that affect specific signaling pathways important for disease development. To date, PTM of HBZ has not been described. We used an affinity-tagged protein and mass spectrometry method to identify seven modifications of HBZ for the first time. We examined how these PTMs affected the ability of HBZ to modulate several pathways, as measured using luciferase reporter assays. Herein, we report that none of the identified PTMs affected HBZ stability or its regulation of tested pathways.

  20. Computational and statistical methods for high-throughput analysis of post-translational modifications of proteins

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Braga, Thiago Verano; Roepstorff, Peter

    2015-01-01

    The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines...... for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis...

  1. Histone posttranslational modifications predict specific alternative exon subtypes in mammalian brain.

    Directory of Open Access Journals (Sweden)

    Qiwen Hu

    2017-06-01

    Full Text Available A compelling body of literature, based on next generation chromatin immunoprecipitation and RNA sequencing of reward brain regions indicates that the regulation of the epigenetic landscape likely underlies chronic drug abuse and addiction. It is now critical to develop highly innovative computational strategies to reveal the relevant regulatory transcriptional mechanisms that may underlie neuropsychiatric disease. We have analyzed chromatin regulation of alternative splicing, which is implicated in cocaine exposure in mice. Recent literature has described chromatin-regulated alternative splicing, suggesting a novel function for drug-induced neuroepigenetic remodeling. However, the extent of the genome-wide association between particular histone modifications and alternative splicing remains unexplored. To address this, we have developed novel computational approaches to model the association between alternative splicing and histone posttranslational modifications in the nucleus accumbens (NAc, a brain reward region. Using classical statistical methods and machine learning to combine ChIP-Seq and RNA-Seq data, we found that specific histone modifications are strongly associated with various aspects of differential splicing. H3K36me3 and H3K4me1 have the strongest association with splicing indicating they play a significant role in alternative splicing in brain reward tissue.

  2. In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

    Science.gov (United States)

    Kurotani, Atsushi; Sakurai, Tetsuya

    2015-08-20

    Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

  3. In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae

    Directory of Open Access Journals (Sweden)

    Atsushi Kurotani

    2015-08-01

    Full Text Available Recent proteome analyses have reported that intrinsically disordered regions (IDRs of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.

  4. Regulation of Wnt/β-catenin signaling by posttranslational modifications

    Science.gov (United States)

    2014-01-01

    The canonical Wnt signaling pathway (or Wnt/β-catenin pathway) plays a pivotal role in embryonic development and adult homeostasis; deregulation of the Wnt pathway contributes to the initiation and progression of human diseases including cancer. Despite its importance in human biology and disease, how regulation of the Wnt/β-catenin pathway is achieved remains largely undefined. Increasing evidence suggests that post-translational modifications (PTMs) of Wnt pathway components are essential for the activation of the Wnt/β-catenin pathway. PTMs create a highly dynamic relay system that responds to Wnt stimulation without requiring de novo protein synthesis and offer a platform for non-Wnt pathway components to be involved in the regulation of Wnt signaling, hence providing alternative opportunities for targeting the Wnt pathway. This review highlights the current status of PTM-mediated regulation of the Wnt/β-catenin pathway with a focus on factors involved in Wnt-mediated stabilization of β-catenin. PMID:24594309

  5. Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

    Science.gov (United States)

    Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

    2016-06-01

    P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  6. Comparative proteomic analysis of histone post-translational modifications upon ischemia/reperfusion-induced retinal injury

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Sidoli, Simone; Wang, Leilei

    2014-01-01

    We present a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in rat retinas affected by ischemia and reperfusion (I/R) injury. Retinal I/R injury contributes to serious ocular diseases, which can lead to vision loss and blindness. We applied linear...... ion trap-orbitrap hybrid tandem mass spectrometry (MS/MS) to quantify 131 single histone marks and 143 combinations of multiple histone marks in noninjured and injured retinas. We observed 34 histone PTMs that exhibited significantly (p

  7. Quantitative proteomic analysis of post-translational modifications of human histones

    DEFF Research Database (Denmark)

    Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune

    2006-01-01

    , and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational...

  8. The Role of Posttranslational Protein Modifications in Rheumatological Diseases: Focus on Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Andrea Mastrangelo

    2015-01-01

    Full Text Available The definition of posttranslational modification (PTM encompasses a wide group of chemical reactions that allow modification and modulation of protein functions. The regulation of PTMs is crucial for the activity and survival of the cells. Dysregulation of PTMs has been observed in several pathological conditions, including rheumatoid arthritis (RA. RA is a systemic autoimmune disease primarily targeting the joints. The three PTMs mainly involved in this disease are glycosylation, citrullination, and carbamylation. Glycosylation is essential for antigen processing and presentation and can modulate immunoglobulin activity. Citrullination of self-antigens is strongly associated with RA, as demonstrated by the presence of antibodies directed to anti-citrullinated proteins in patients’ sera. Carbamylation and its dysregulation have been recently associated with RA. Aim of this review is to illustrate the most significant alterations of these PTMs in RA and to evaluate their possible involvement in the pathogenesis of the disease.

  9. Prediction of protein modification sites of pyrrolidone carboxylic acid using mRMR feature selection and analysis.

    Directory of Open Access Journals (Sweden)

    Lu-Lu Zheng

    Full Text Available Pyrrolidone carboxylic acid (PCA is formed during a common post-translational modification (PTM of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR and incremental feature selection (IFS. We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations.

  10. Mass-spectrometry analysis of histone post-translational modifications in pathology tissue using the PAT-H-MS approach

    Directory of Open Access Journals (Sweden)

    Roberta Noberini

    2016-06-01

    Full Text Available Aberrant histone post-translational modifications (hPTMs have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification patterns in patient samples (Noberini, 2015 [1], are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002669.

  11. The measurement of reversible redox dependent post-translational modifications and their regulation of mitochondrial and skeletal muscle function

    Directory of Open Access Journals (Sweden)

    Philip A Kramer

    2015-11-01

    Full Text Available Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar and excitation-contraction (EC coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.

  12. The Measurement of Reversible Redox Dependent Post-translational Modifications and Their Regulation of Mitochondrial and Skeletal Muscle Function

    Energy Technology Data Exchange (ETDEWEB)

    Kramer, Philip A.; Duan, Jicheng; Qian, Wei-Jun; Marcinek, David J.

    2015-11-25

    Mitochondrial oxidative stress is a common feature of skeletal myopathies across multiple conditions; however, the mechanism by which it contributes to skeletal muscle dysfunction remains controversial. Oxidative damage to proteins, lipids, and DNA has received the most attention, yet an important role for reversible redox post-translational modifications (PTMs) in pathophysiology is emerging. The possibility that these PTMs can exert dynamic control of muscle function implicates them as a mechanism contributing to skeletal muscle dysfunction in chronic disease. Herein, we discuss the significance of thiol-based redox dependent modifications to mitochondrial, myofibrillar and excitation-contraction (EC) coupling proteins with an emphasis on how these changes could alter skeletal muscle performance under chronically stressed conditions. A major barrier to a better mechanistic understanding of the role of reversible redox PTMs in muscle function is the technical challenges associated with accurately measuring the changes of site-specific redox PTMs. Here we will critically review current approaches with an emphasis on sample preparation artifacts, quantitation, and specificity. Despite these challenges, the ability to accurately quantify reversible redox PTMs is critical to understanding the mechanisms by which mitochondrial oxidative stress contributes to skeletal muscle dysfunction in chronic diseases.

  13. Protein redox chemistry: post-translational cysteine modifications that regulate signal transduction and drug pharmacology

    Directory of Open Access Journals (Sweden)

    Revati eWani

    2014-10-01

    Full Text Available The perception of reactive oxygen species (ROS has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g. cancer. New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically-distinct alterations to the protein (e.g. sulfenic/sulfinic/sulfonic acid, disulfides. These post-translational modifications (PTM are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.

  14. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    Science.gov (United States)

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

  15. A Design Principle for a Posttranslational Biochemical Oscillator

    Directory of Open Access Journals (Sweden)

    Craig C. Jolley

    2012-10-01

    Full Text Available Multisite phosphorylation plays an important role in biological oscillators such as the circadian clock. Its general role, however, has been elusive. In this theoretical study, we show that a simple substrate with two modification sites acted upon by two opposing enzymes (e.g., a kinase and a phosphatase can show oscillations in its modification state. An unbiased computational analysis of this oscillator reveals two common characteristics: a unidirectional modification cycle and sequestering of an enzyme by a specific modification state. These two motifs cause a substrate to act as a coupled system in which a unidirectional cycle generates single-molecule oscillators, whereas sequestration synchronizes the population by limiting the available enzyme under conditions in which substrate is in excess. We also demonstrate the conditions under which the oscillation period is temperature compensated, an important feature of the circadian clock. This theoretical model will provide a framework for analyzing and synthesizing posttranslational oscillators.

  16. Osteopontin binding to the alpha 4 integrin requires highest affinity integrin conformation, but is independent of post-translational modifications of osteopontin

    DEFF Research Database (Denmark)

    Hui, Tommy; Sørensen, Esben Skipper; Rittling, Susan R.

    2015-01-01

    Osteopontin (OPN) is a ligand for the α4 integrin, but the physiological importance of this binding is not well understood. Here, we have assessed the effect of posttranslational modifications on OPN binding to the α4 integrin on cultured human leukocyte cell lines, and compared OPN interaction...

  17. Status of large-scale analysis of post-translational modifications by mass spectrometry

    DEFF Research Database (Denmark)

    Olsen, Jesper V; Mann, Matthias

    2013-01-01

    Cellular function can be controlled through the gene expression program but often protein post translations modifications (PTMs) provide a more precisely and elegant mechanism. Key functional roles of specific modification events for instance during the cell cycle have been known for decades...... of protein modifications. For many PTMs, including phosphorylation, ubiquitination, glycosylation and acetylation, tens of thousands of sites can now be confidently identified and localized in the sequence of the protein. Quantitation of PTM levels between different cellular states is likewise established......, with label-free methods showing particular promise. It is also becoming possible to determine the absolute occupancy or stoichiometry of PTMS sites on a large scale. Powerful software for the bioinformatic analysis of thousands of PTM sites has been developed. However, a complete inventory of sites has...

  18. GPS-Lipid: a robust tool for the prediction of multiple lipid modification sites

    OpenAIRE

    Xie, Yubin; Zheng, Yueyuan; Li, Hongyu; Luo, Xiaotong; He, Zhihao; Cao, Shuo; Shi, Yi; Zhao, Qi; Xue, Yu; Zuo, Zhixiang; Ren, Jian

    2016-01-01

    As one of the most common post-translational modifications in eukaryotic cells, lipid modification is an important mechanism for the regulation of variety aspects of protein function. Over the last decades, three classes of lipid modifications have been increasingly studied. The co-regulation of these different lipid modifications is beginning to be noticed. However, due to the lack of integrated bioinformatics resources, the studies of co-regulatory mechanisms are still very limited. In this...

  19. Posttranslational regulation of phosphatase and tensin homolog (PTEN and its functional impact on cancer behaviors

    Directory of Open Access Journals (Sweden)

    Xu WT

    2014-10-01

    Full Text Available Wenting Xu,1 Zhen Yang,1 Shu-Feng Zhou,2 Nonghua Lu1 1Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA Abstract: The incidence of cancer is increasing worldwide, but the biochemical mechanisms for the occurrence of cancer is not fully understood, and there is no cure for advanced tumors. Defects of posttranslational modifications of proteins are linked to a number of important diseases, such as cancer. This review will update our knowledge on the critical role of posttranscriptional regulation of phosphatase and tensin homolog (PTEN and its activities and the functional impact on cancer behaviors. PTEN is a tumor suppressor gene that occupies a key position in regulating cell growth, proliferation, apoptosis, mobility, signal transduction, and other crucial cellular processes. The activity and function of PTEN are regulated by coordinated epigenetic, transcriptional, posttranscriptional, and posttranslational modifications. In particular, PTEN is subject to phosphorylation, ubiquitylation, somoylation, acetylation, and active site oxidation. Posttranslational modifications of PTEN can dynamically change its activity and function. Deficiency in the posttranslational regulation of PTEN leads to abnormal cell proliferation, apoptosis, migration, and adhesion, which are associated with cancer initiation, progression, and metastasis. With increasing information on how PTEN is regulated by multiple mechanisms and networked proteins, its exact role in cancer initiation, growth, and metastasis will be revealed. PTEN and its functionally related proteins may represent useful targets for the discovery of new anticancer drugs, and gene therapy and the therapeutic potentials should be fully explored. Keywords: phosphorylation, ubiquitination, acetylation, oxidation

  20. Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer

    Directory of Open Access Journals (Sweden)

    Gopinath Prakasam

    2018-02-01

    Full Text Available Cancer cells rewire metabolism to meet biosynthetic and energetic demands. The characteristic increase in glycolysis, i.e., Warburg effect, now considered as a hallmark, supports cancer in various ways. To attain such metabolic reshuffle, cancer cells preferentially re-express the M2 isoform of pyruvate kinase (PKM2, M2-PK and alter its quaternary structure to generate less-active PKM2 dimers. The relatively inactive dimers cause the accumulation of glycolytic intermediates that are redirected into anabolic pathways. In addition, dimeric PKM2 also benefits cancer cells through various non-glycolytic moonlight functions, such as gene transcription, protein kinase activity, and redox balance. A large body of data have shown that several distinct posttranslation modifications (PTMs regulate PKM2 in a way that benefits cancer growth, e.g., formation of PKM2 dimers. This review discusses the recent advancements in our understanding of various PTMs and the benefits they impart to the sustenance of cancer. Understanding the PTMs in PKM2 is crucial to assess their therapeutic potential and to design novel anticancer strategies.

  1. Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

    Science.gov (United States)

    Bussiere, Renaud; Lacampagne, Alain; Reiken, Steven; Liu, Xiaoping; Scheuerman, Valerie; Zalk, Ran; Martin, Cécile; Checler, Frederic; Marks, Andrew R; Chami, Mounia

    2017-06-16

    Alteration of ryanodine receptor (RyR)-mediated calcium (Ca 2+ ) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca 2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca 2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca 2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca 2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca 2+ leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

    Directory of Open Access Journals (Sweden)

    Takashi Onikubo

    2015-03-01

    Full Text Available Nucleoplasmin (Npm is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

  3. Posttranslational modifications of desmin and their implication in biological processes and pathologies.

    Science.gov (United States)

    Winter, Daniel L; Paulin, Denise; Mericskay, Mathias; Li, Zhenlin

    2014-01-01

    Desmin, the muscle-specific intermediate filament, is involved in myofibrillar myopathies, dilated cardiomyopathy and muscle wasting. Desmin is the target of posttranslational modifications (PTMs) such as phosphorylation, ADP-ribosylation and ubiquitylation as well as nonenzymatic modifications such as glycation, oxidation and nitration. Several PTM target residues and their corresponding modifying enzymes have been discovered in human and nonhuman desmin. The major effect of phosphorylation and ADP-ribosylation is the disassembly of desmin filaments, while ubiquitylation of desmin leads to its degradation. The regulation of the desmin filament network by phosphorylation and ADP-ribosylation was found to be implicated in several major biological processes such as myogenesis, myoblast fusion, muscle contraction, muscle atrophy, cell division and possibly desmin interactions with its binding partners. Phosphorylation of desmin is also implicated in many forms of desmin-related myopathies (desminopathies). In this review, we summarize the findings on desmin PTMs and their implication in biological processes and pathologies, and discuss the current knowledge on the regulation of the desmin network by PTMs. We conclude that the desmin filament network can be seen as an intricate scaffold for muscle cell structure and biological processes and that its dynamics can be affected by PTMs. There are now precise tools to investigate PTMs and visualize cellular structures that have been underexploited in the study of desminopathies. Future studies should focus on these aspects.

  4. Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach

    Energy Technology Data Exchange (ETDEWEB)

    Dykstra, Andrew B [ORNL; Rodriguez, Jr., Miguel [ORNL; Raman, Babu [Dow Chemical Company, The; Cook, Kelsey [ORNL; Hettich, Robert {Bob} L [ORNL

    2013-01-01

    Post-translational modifications (PTMs) are known to play a significant role in many biological functions. The focus of this study is to characterize the post-translational modifications of the cellulosome protein complex used by the bacterium Clostridium thermocellum to better understand how this protein machine is tuned for enzymatic cellulose solubilization. To enhance comprehensive characterization, the extracellular cellulosome proteins were analyzed using multiple proteolytic digests (trypsin, Lys-C, Glu-C) and multiple fragmentation techniques (collisionally-activated dissociation, electron transfer dissociation, decision tree). As expected, peptide and protein identifications were increased by utilizing alternate proteases and fragmentation methods, in addition to the increase in protein sequence coverage. The complementarity of these experiments also allowed for a global exploration of PTMs associated with the cellulosome based upon a set of defined PTMs that included methylation, oxidation, acetylation, phosphorylation, and signal peptide cleavage. In these experiments, 85 modified peptides corresponding to 28 cellulosome proteins were identified. Many of these modifications were located in active cellulolytic or structural domains of the cellulosome proteins, suggesting a level of possible regulatory control of protein function in various cellulotyic conditions. The use of multiple enzymes and fragmentation technologies allowed for independent verification of PTMs in different experiments, thus leading to increased confidence in PTM identifications.

  5. Posttranslational nitro-glycative modifications of albumin in Alzheimer's disease: implications in cytotoxicity and amyloid-β peptide aggregation.

    Science.gov (United States)

    Ramos-Fernández, Eva; Tajes, Marta; Palomer, Ernest; Ill-Raga, Gerard; Bosch-Morató, Mònica; Guivernau, Biuse; Román-Dégano, Irene; Eraso-Pichot, Abel; Alcolea, Daniel; Fortea, Juan; Nuñez, Laura; Paez, Antonio; Alameda, Francesc; Fernández-Busquets, Xavier; Lleó, Alberto; Elosúa, Roberto; Boada, Mercé; Valverde, Miguel A; Muñoz, Francisco J

    2014-01-01

    Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

  6. Tax-1 and Tax-2 similarities and differences: Focus on post-translational modifications and NF-кB activation

    Directory of Open Access Journals (Sweden)

    Margret eShirinian

    2013-08-01

    Full Text Available ABSTRACTAlthough human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2 share similar genetic organization, they have major differences in their pathogenesis and disease manifestation. HTLV-1 is capable of transforming T lymphocytes in infected patients and subsequently leads to adult T cell leukemia/lymphoma (ATL whereas HTLV-2 is not clearly associated with lymphoproliferative diseases. Numerous studies have provided accumulating evidence on the involvement of the viral transactivators Tax-1 versus Tax-2 in T cell transformation. Tax-1 is a potent transcriptional activator of both viral and cellular genes. Tax-1 posttranslational modifications and specifically ubiquitylation and SUMOylation have been implicated in NF-кB activation and may contribute to its transformation capacity. Although Tax-2 has similar protein structure compared to Tax-1, the two proteins display differences both in their protein-protein interaction and activation of signal transduction pathways. Recent studies on Tax-2 have suggested ubiquitylation and SUMOylation independent mechanisms of NF-кB activation. In this present review, structural and functional differences between Tax-1 and Tax- 2 will be summarized. Specifically, we will address their subcellular localization, nuclear trafficking and their effect on cellular regulatory proteins. A special attention will be given to Tax-1/Tax-2 post-translational modification such as ubiquitylation, SUMOylation, phosphorylation, acetylation, NF-кB activation and protein-protein interactions involved in oncogenecity both in vivo and in vitro.

  7. Tax-1 and Tax-2 similarities and differences: focus on post-translational modifications and NF-κB activation

    Science.gov (United States)

    Shirinian, Margret; Kfoury, Youmna; Dassouki, Zeina; El-Hajj, Hiba; Bazarbachi, Ali

    2013-01-01

    Although human T cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) share similar genetic organization, they have major differences in their pathogenesis and disease manifestation. HTLV-1 is capable of transforming T lymphocytes in infected patients resulting in adult T cell leukemia/lymphoma whereas HTLV-2 is not clearly associated with lymphoproliferative diseases. Numerous studies have provided accumulating evidence on the involvement of the viral transactivators Tax-1 versus Tax-2 in T cell transformation. Tax-1 is a potent transcriptional activator of both viral and cellular genes. Tax-1 post-translational modifications and specifically ubiquitylation and SUMOylation have been implicated in nuclear factor-kappaB (NF-κB) activation and may contribute to its transformation capacity. Although Tax-2 has similar protein structure compared to Tax-1, the two proteins display differences both in their protein–protein interaction and activation of signal transduction pathways. Recent studies on Tax-2 have suggested ubiquitylation and SUMOylation independent mechanisms of NF-κB activation. In this present review, structural and functional differences between Tax-1 and Tax-2 will be summarized. Specifically, we will address their subcellular localization, nuclear trafficking and their effect on cellular regulatory proteins. A special attention will be given to Tax-1/Tax-2 post-translational modification such as ubiquitylation, SUMOylation, phosphorylation, acetylation, NF-κB activation, and protein–protein interactions involved in oncogenecity both in vivo and in vitro. PMID:23966989

  8. Post-translational modifications near the quinone binding site of mammalian complex I.

    Science.gov (United States)

    Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2013-08-23

    Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.

  9. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence

    DEFF Research Database (Denmark)

    Blom, Nikolaj; Sicheritz-Pontén, Thomas; Gupta, Ramneek

    2004-01-01

    Post-translational modifications (PTMs) occur on almost all proteins analyzed to date. The function of a modified protein is often strongly affected by these modifications and therefore increased knowledge about the potential PTMs of a target protein may increase our understanding of the molecular...... steps by integrating computational approaches into the validation procedures. Many advanced methods for the prediction of PTMs exist and many are made publicly available. We describe our experiences with the development of prediction methods for phosphorylation and glycosylation sites...... and the development of PTM-specific databases. In addition, we discuss novel ideas for PTM visualization (exemplified by kinase landscapes) and improvements for prediction specificity (by using ESS-evolutionary stable sites). As an example, we present a new method for kinase-specific prediction of phosphorylation...

  10. Functional O-GlcNAc modifications: Implications in molecular regulation and pathophysiology

    Science.gov (United States)

    Wells, Lance

    2016-01-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of intracellular proteins. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in response to UDP-GlcNAc levels in the hexosamine biosynthetic pathway (HBP). Due to its reliance on glucose flux and substrate availability, a major focus in the field has been on how O-GlcNAc contributes to metabolic disease. For years this post-translational modification has been known to modify thousands of proteins implicated in various disorders, but direct functional connections have until recently remained elusive. New research is beginning to reveal the specific mechanisms through which O-GlcNAc influences cell dynamics and disease pathology including clear examples of O-GlcNAc modification at a specific site on a given protein altering its biological functions. The following review intends to focus primarily on studies in the last half decade linking O-GlcNAc modification of proteins with chromatin-directed gene regulation, developmental processes, and several metabolically related disorders including Alzheimer’s, heart disease and cancer. These studies illustrate the emerging importance of this post-translational modification in biological processes and multiple pathophysiologies. PMID:24524620

  11. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

    International Nuclear Information System (INIS)

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

    1988-01-01

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

  12. Differential effects of cocaine on histone posttranslational modifications in identified populations of striatal neurons.

    Science.gov (United States)

    Jordi, Emmanuelle; Heiman, Myriam; Marion-Poll, Lucile; Guermonprez, Pierre; Cheng, Shuk Kei; Nairn, Angus C; Greengard, Paul; Girault, Jean-Antoine

    2013-06-04

    Drugs of abuse, such as cocaine, induce changes in gene expression and epigenetic marks including alterations in histone posttranslational modifications in striatal neurons. These changes are thought to participate in physiological memory mechanisms and to be critical for long-term behavioral alterations. However, the striatum is composed of multiple cell types, including two distinct populations of medium-sized spiny neurons, and little is known concerning the cell-type specificity of epigenetic modifications. To address this question we used bacterial artificial chromosome transgenic mice, which express EGFP fused to the N-terminus of the large subunit ribosomal protein L10a driven by the D1 or D2 dopamine receptor (D1R, D2R) promoter, respectively. Fluorescence in nucleoli was used to sort nuclei from D1R- or D2R-expressing neurons and to quantify by flow cytometry the cocaine-induced changes in histone acetylation and methylation specifically in these two types of nuclei. The two populations of medium-sized spiny neurons displayed different patterns of histone modifications 15 min or 24 h after a single injection of cocaine or 24 h after seven daily injections. In particular, acetylation of histone 3 on Lys 14 and of histone 4 on Lys 5 and 12, and methylation of histone 3 on Lys 9 exhibited distinct and persistent changes in the two cell types. Our data provide insights into the differential epigenetic responses to cocaine in D1R- and D2R-positive neurons and their potential regulation, which may participate in the persistent effects of cocaine in these neurons. The method described should have general utility for studying nuclear modifications in different types of neuronal or nonneuronal cell types.

  13. An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum

    KAUST Repository

    Veluchamy, Alaguraj

    2015-05-20

    Background: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. Results: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. Conclusions: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments. © 2015 Veluchamy et al.

  14. Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Akiko Hashiguchi

    2016-12-01

    Full Text Available The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs can regulate protein activity and localization as well as protein–protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.

  15. Artificial Metalloenzymes through Chemical Modification of Engineered Host Proteins

    KAUST Repository

    Zernickel, Anna

    2014-10-01

    With a few exceptions, all organisms are restricted to the 20 canonical amino acids for ribosomal protein biosynthesis. Addition of new amino acids to the genetic code can introduce novel functionalities to proteins, broadening the diversity of biochemical as well as chemical reactions and providing new tools to study protein structure, reactivity, dynamics and protein-protein-interactions. The site directed in vivo incorporation developed by P. G. SCHULTZ and coworkers, using an archeal orthogonal tRNA/aaRS (aminoacyl-tRNA synthase) pair, allows site-specifically insertion of a synthetic unnatural amino acid (UAA) by reprogramming the amber TAG stop codon. A variety of over 80 different UAAs can be introduced by this technique. However by now a very limited number can form kinetically stable bonds to late transition metals. This thesis aims to develop new catalytically active unnatural amino acids or strategies for a posttranslational modification of site-specific amino acids in order to achieve highly enantioselective metallorganic enzyme hybrids (MOEH). As a requirement a stable protein host has to be established, surviving the conditions for incorporation, posttranslational modification and the final catalytic reactions. mTFP* a fluorescent protein was genetically modified by excluding any exposed Cys, His and Met forming a variant mTFP*, which fulfills the required specifications. Posttranslational chemical modification of mTFP* allow the introduction of single site metal chelating moieties. For modification on exposed cysteines different maleiimid containing ligand structures were synthesized. In order to perform copper catalyzed click reactions, suitable unnatural amino acids (para-azido-(L)-phenylalanine, para-ethynyl-(L)-phenylalanine) were synthesized and a non-cytotoxic protocol was established. The triazole ring formed during this reaction may contribute as a moderate σ-donor/π-acceptor ligand to the metal binding site. Since the cell limits the

  16. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    Science.gov (United States)

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  17. Unrestrictive identification of post-translational modifications in the urine proteome without enrichment

    Science.gov (United States)

    2013-01-01

    Background Research on the human urine proteome may lay the foundation for the discovery of relevant disease biomarkers. Post-translational modifications (PTMs) have important effects on the functions of protein biomarkers. Identifying PTMs without enrichment adds no extra steps to conventional identification procedures for urine proteomics. The only difference is that this method requires software that can conduct unrestrictive identifications of PTMs. In this study, routine urine proteomics techniques were used to identify urine proteins. Unspecified PTMs were searched by MODa and PEAKS 6 automated software, followed by a manual search to screen out in vivo PTMs by removing all in vitro PTMs and amino acid substitutions. Results There were 75 peptides with 6 in vivo PTMs that were found by both MODa and PEAKS 6. Of these, 34 peptides in 18 proteins have novel in vivo PTMs compared with the annotation information of these proteins on the Universal Protein Resource website. These new in vivo PTMs had undergone methylation, dehydration, oxidation, hydroxylation, phosphorylation, or dihydroxylation. Conclusions In this study, we identified PTMs of urine proteins without the need for enrichment. Our investigation may provide a useful reference for biomarker discovery in the future. PMID:23317149

  18. Antioxidant systems are regulated by nitric oxide-mediated post-translational modifications (NO-PTMs

    Directory of Open Access Journals (Sweden)

    Juan Carlos Begara-Morales

    2016-02-01

    Full Text Available Nitric oxide (NO is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS, such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs peroxiredoxins (Prx and all the enzymatic components of the ascorbate-glutathione (Asa-GSH cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance.

  19. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    Science.gov (United States)

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  20. Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

    Directory of Open Access Journals (Sweden)

    P. Roncada

    2010-04-01

    Full Text Available Alpha1-acid glycoprotein (AGP is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations. In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far.

  1. Posttranslational Modifications in Spermatozoa and Effects on Male Fertility and Sperm Viability.

    Science.gov (United States)

    Brohi, Rahim Dad; Huo, Li-Jun

    2017-05-01

    Spermatogenesis is a complex and highly regulated process. The ability of spermatozoa to perform its function depends on multiple physiological and genetic factors that are not fully understood. Notably, due to lack of transcriptional and translational activity in spermatozoa, posttranslational modifications (PTMs) play key roles in determining their viability. PTMs not only confer structural changes in the proteome of the spermatozoa cells, but also increase the diversity of the proteome and introduce specific modifications that could be translated into functional changes in the affected spermatozoa. Multiple PTMs of active proteins have been identified in the developing spermatogonia. This review summarizes a diverse range of PTMs taking place in the developing spermatozoa, and analyzes their effects on male fertility and sperm viability. In particular, we discuss how SUMOylation, ubiquitination, phosphorylation, acetylation, glycosylation, and disulphide bond formation in proteins play a role in spermatogenesis, sperm maturation, movement of maturing spermatozoa to epididymis, capacitation, hyperactivation, spermatozoa motility, subversion of immune detection by spermatozoa, sperm to egg recognition and fusion, and the fertilization process. When possible, the specific proteins involved in these processes are highlighted. We point to existing knowledge gaps in the field of proteomics, and provide suggestions for future research on sperm viability and male fertility. We discuss briefly, as an example, the observations in water buffalo, Bubalus bubalis, which provides both meat and milk, and therefore is a reliable source for energy and protein needs of human populations. In conclusions, understanding the ways in which PTMs impact mammalian fertility and reproduction is important to make significant strides for diagnostic and therapeutic strategies in the near future.

  2. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

    Science.gov (United States)

    Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

    2012-04-10

    Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

  3. Pdx1 is post-translationally modified in vivo and serine 61 is the principal site of phosphorylation

    DEFF Research Database (Denmark)

    Frogne, Thomas; Sylvestersen, Kathrine Beck; Kubicek, Stefan

    2012-01-01

    signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We...... alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis...

  4. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    Energy Technology Data Exchange (ETDEWEB)

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  5. Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

    Science.gov (United States)

    Manconi, Barbara; Liori, Barbara; Cabras, Tiziana; Vincenzoni, Federica; Iavarone, Federica; Castagnola, Massimo; Messana, Irene; Olianas, Alessandra

    2017-11-03

    Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.

  6. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

    Science.gov (United States)

    Maréchal, Alexandre; Zou, Lee

    2015-01-01

    The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

  7. Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

    Directory of Open Access Journals (Sweden)

    Allan Olspert

    2016-06-01

    Full Text Available Members of the Caliciviridae family of positive sense RNA viruses cause a wide range of diseases in both humans and animals. The detailed characterization of the calicivirus life cycle had been hampered due to the lack of robust cell culture systems and experimental tools for many of the members of the family. However, a number of caliciviruses replicate efficiently in cell culture and have robust reverse genetics systems available, most notably feline calicivirus (FCV and murine norovirus (MNV. These are therefore widely used as representative members with which to examine the mechanistic details of calicivirus genome translation and replication. The replication of the calicivirus RNA genome occurs via a double-stranded RNA intermediate that is then used as a template for the production of new positive sense viral RNA, which is covalently linked to the virus-encoded protein VPg. The covalent linkage to VPg occurs during genome replication via the nucleotidylylation activity of the viral RNA-dependent RNA polymerase. Using FCV and MNV, we used mass spectrometry-based approach to identify the specific amino acid linked to the 5′ end of the viral nucleic acid. We observed that both VPg proteins are covalently linked to guanosine diphosphate (GDP moieties via tyrosine positions 24 and 26 for FCV and MNV respectively. These data fit with previous observations indicating that mutations introduced into these specific amino acids are deleterious for viral replication and fail to produce infectious virus. In addition, we also detected serine phosphorylation sites within the FCV VPg protein with positions 80 and 107 found consistently phosphorylated on VPg-linked viral RNA isolated from infected cells. This work provides the first direct experimental characterization of the linkage of infectious calicivirus viral RNA to the VPg protein and highlights that post-translational modifications of VPg may also occur during the viral life cycle.

  8. Experimental annotation of post-translational features and translated coding regions in the pathogen Salmonella Typhimurium

    Energy Technology Data Exchange (ETDEWEB)

    Ansong, Charles; Tolic, Nikola; Purvine, Samuel O.; Porwollik, Steffen; Jones, Marcus B.; Yoon, Hyunjin; Payne, Samuel H.; Martin, Jessica L.; Burnet, Meagan C.; Monroe, Matthew E.; Venepally, Pratap; Smith, Richard D.; Peterson, Scott; Heffron, Fred; Mcclelland, Michael; Adkins, Joshua N.

    2011-08-25

    Complete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. For example systems biology-oriented genome scale modeling efforts greatly benefit from accurate annotation of protein-coding genes to develop proper functioning models. However, determining protein-coding genes for most new genomes is almost completely performed by inference, using computational predictions with significant documented error rates (> 15%). Furthermore, gene prediction programs provide no information on biologically important post-translational processing events critical for protein function. With the ability to directly measure peptides arising from expressed proteins, mass spectrometry-based proteomics approaches can be used to augment and verify coding regions of a genomic sequence and importantly detect post-translational processing events. In this study we utilized “shotgun” proteomics to guide accurate primary genome annotation of the bacterial pathogen Salmonella Typhimurium 14028 to facilitate a systems-level understanding of Salmonella biology. The data provides protein-level experimental confirmation for 44% of predicted protein-coding genes, suggests revisions to 48 genes assigned incorrect translational start sites, and uncovers 13 non-annotated genes missed by gene prediction programs. We also present a comprehensive analysis of post-translational processing events in Salmonella, revealing a wide range of complex chemical modifications (70 distinct modifications) and confirming more than 130 signal peptide and N-terminal methionine cleavage events in Salmonella. This study highlights several ways in which proteomics data applied during the primary stages of annotation can improve the quality of genome annotations, especially with regards to the annotation of mature protein products.

  9. Incorporation of post-translational modified amino acids as an approach to increase both chemical and biological diversity of conotoxins and conopeptides.

    Science.gov (United States)

    Espiritu, Michael J; Cabalteja, Chino C; Sugai, Christopher K; Bingham, Jon-Paul

    2014-01-01

    Bioactive peptides from Conus venom contain a natural abundance of post-translational modifications that affect their chemical diversity, structural stability, and neuroactive properties. These modifications have continually presented hurdles in their identification and characterization. Early endeavors in their analysis relied on classical biochemical techniques that have led to the progressive development and use of novel proteomic-based approaches. The critical importance of these post-translationally modified amino acids and their specific assignment cannot be understated, having impact on their folding, pharmacological selectivity, and potency. Such modifications at an amino acid level may also provide additional insight into the advancement of conopeptide drugs in the quest for precise pharmacological targeting. To achieve this end, a concerted effort between the classical and novel approaches is needed to completely elucidate the role of post-translational modifications in conopeptide structure and dynamics. This paper provides a reflection in the advancements observed in dealing with numerous and multiple post-translationally modified amino acids within conotoxins and conopeptides and provides a summary of the current techniques used in their identification.

  10. Post-translational modifications are key players of the Legionella pneumophila infection strategy

    Science.gov (United States)

    Michard, Céline; Doublet, Patricia

    2015-01-01

    Post-translational modifications (PTMs) are widely used by eukaryotes to control the enzymatic activity, localization or stability of their proteins. Traditionally, it was believed that the broad biochemical diversity of the PTMs is restricted to eukaryotic cells, which exploit it in extensive networks to fine-tune various and complex cellular functions. During the last decade, the advanced detection methods of PTMs and functional studies of the host–pathogen relationships highlight that bacteria have also developed a large arsenal of PTMs, particularly to subvert host cell pathways to their benefit. Legionella pneumophila, the etiological agent of the severe pneumonia legionellosis, is the paradigm of highly adapted intravacuolar pathogens that have set up sophisticated biochemical strategies. Among them, L. pneumophila has evolved eukaryotic-like and rare/novel PTMs to hijack host cell processes. Here, we review recent progress about the diversity of PTMs catalyzed by Legionella: ubiquitination, prenylation, phosphorylation, glycosylation, methylation, AMPylation, and de-AMPylation, phosphocholination, and de-phosphocholination. We focus on the host cell pathways targeted by the bacteria catalyzed PTMs and we stress the importance of the PTMs in the Legionella infection strategy. Finally, we highlight that the discovery of these PTMs undoubtedly made significant breakthroughs on the molecular basis of Legionella pathogenesis but also lead the way in improving our knowledge of the eukaryotic PTMs and complex cellular processes that are associated to. PMID:25713573

  11. Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs

    Directory of Open Access Journals (Sweden)

    Alhosna Benjdia

    2017-11-01

    Full Text Available Ribosomally-synthesized and post-translationally modified peptides (RiPPs are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota.

  12. AMS 4.0: consensus prediction of post-translational modifications in protein sequences.

    Science.gov (United States)

    Plewczynski, Dariusz; Basu, Subhadip; Saha, Indrajit

    2012-08-01

    We present here the 2011 update of the AutoMotif Service (AMS 4.0) that predicts the wide selection of 88 different types of the single amino acid post-translational modifications (PTM) in protein sequences. The selection of experimentally confirmed modifications is acquired from the latest UniProt and Phospho.ELM databases for training. The sequence vicinity of each modified residue is represented using amino acids physico-chemical features encoded using high quality indices (HQI) obtaining by automatic clustering of known indices extracted from AAindex database. For each type of the numerical representation, the method builds the ensemble of Multi-Layer Perceptron (MLP) pattern classifiers, each optimising different objectives during the training (for example the recall, precision or area under the ROC curve (AUC)). The consensus is built using brainstorming technology, which combines multi-objective instances of machine learning algorithm, and the data fusion of different training objects representations, in order to boost the overall prediction accuracy of conserved short sequence motifs. The performance of AMS 4.0 is compared with the accuracy of previous versions, which were constructed using single machine learning methods (artificial neural networks, support vector machine). Our software improves the average AUC score of the earlier version by close to 7 % as calculated on the test datasets of all 88 PTM types. Moreover, for the selected most-difficult sequence motifs types it is able to improve the prediction performance by almost 32 %, when compared with previously used single machine learning methods. Summarising, the brainstorming consensus meta-learning methodology on the average boosts the AUC score up to around 89 %, averaged over all 88 PTM types. Detailed results for single machine learning methods and the consensus methodology are also provided, together with the comparison to previously published methods and state-of-the-art software tools. The

  13. Posttranslational modifications of proteins : tools for functional proteomics [Methods in molecular biology, v. 194

    National Research Council Canada - National Science Library

    Kannicht, Christoph

    2002-01-01

    ... single glycosylation sites in a protein. Additional powerful techniques facilitate the analysis of glycosylphosphatidylinositols, lipid modifications, protein phosphorylation and sulfation, protein methylation and acetylation, a-amidation...

  14. Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Motoi Kanagawa

    2016-03-01

    Full Text Available Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies. Here we identify the glycan unit ribitol 5-phosphate (Rbo5P, a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD is cytidine diphosphate ribitol (CDP-Rbo synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

  15. B-Type Natriuretic Peptide: From Posttranslational Processing to Clinical Measurement

    DEFF Research Database (Denmark)

    Goetze, Jens P

    2012-01-01

    BACKGROUND:Plasma cardiac natriuretic peptides and peptide fragments from their molecular precursors are markers of heart disease. Clinical studies have defined the current diagnostic utility of these markers, whereas biochemical elucidation of peptide structure and posttranslational processing has...... revealed new plasma peptide forms of potential clinical use.CONTENT:Natriuretic propeptide structures undergo variable degrees of endo- and exoproteolytic cleavages as well as amino acid modifications, which leave the plasma phase of the peptides highly heterogeneous and dependent on cardiac......-atrial natriuretic peptide and pro-B-type natriuretic peptide are useful plasma markers in heart failure. New data have defined cardiac myocytes as competent endocrine cells in posttranslational processing and cellular secretion....

  16. The Human Thioredoxin System: Modifications and Clinical Applications

    Directory of Open Access Journals (Sweden)

    Seyed Isaac Hashemy

    2011-03-01

    Full Text Available The thioredoxin system, comprising thioredoxin (Trx, thioredoxin reductase (TrxR and NADPH, is one of the major cellular antioxidant systems, implicated in a large and growing number of biological functions. Trx acts as an oxidoreductase via a highly conserved dithiol/disulfide motif located in the active site (-Trp-Cys-Gly-Pro-Cys-Lys-. Different factors are involved in the regulation of Trx activity, including its expression level, localization, protein-protein interactions, post-translational modifications and some chemical inhibitors. Mammalian TrxRs are selenoproteins which have a –Cys-Val-Asn-Val-Gly-Cys- N-terminal active site, as well as a C-terminal selenium-containing active site. Besides two Cys-residues in the redox-regulatory domain of cytosolic Trx (Trx1, human Trx1 has three additional Cys-residues. Post-translational modifications of human Trx1 which are involved in the regulation of its activity can happen via modification of Cys-residues including thiol oxidation, glutathionylation and S-nitrosylation or via modification of other amino acid residues such as nitration of Tyr-49. Because of the numerous functions of the thioredoxin system, its inhibition (mainly happens via the targeting TrxR can result in major cellular consequences, which are potentially pro-oxidant in nature, leading to cell death via necrosis or apoptosis if overexpression of Trx and other antioxidative enzymes can not recuperate cell response. Considering this feature, several anticancer drugs have been used which can inhibit TrxR. Elevated levels of Trx and/or TrxR have been reported in many different human malignancies, positively correlated with aggressive tumor growth and poor prognosis. Moreover, anti-oxidative and anti-apoptotic effects of Trx are reasons to study its clinical application as a drug.

  17. Towards Liquid Chromatography Time-Scale Peptide Sequencing and Characterization of Post-Translational Modifications in the Negative-Ion Mode Using Electron Detachment Dissociation Tandem Mass Spectrometry

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Hørning, Ole B; Jensen, Søren S

    2008-01-01

    Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented...... coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research....

  18. Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

    Science.gov (United States)

    Noberini, Roberta; Osti, Daniela; Miccolo, Claudia; Richichi, Cristina; Lupia, Michela; Corleone, Giacomo; Hong, Sung-Pil; Colombo, Piergiuseppe; Pollo, Bianca; Fornasari, Lorenzo; Pruneri, Giancarlo; Magnani, Luca; Cavallaro, Ugo; Chiocca, Susanna; Minucci, Saverio; Pelicci, Giuliana; Bonaldi, Tiziana

    2018-05-04

    Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations.

  19. Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules

    Directory of Open Access Journals (Sweden)

    Teresa Mendes Maia

    2014-01-01

    Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.

  20. HiHiMap: single-cell quantitation of histones and histone posttranslational modifications across the cell cycle by high-throughput imaging.

    Science.gov (United States)

    Zane, Linda; Chapus, Fleur; Pegoraro, Gianluca; Misteli, Tom

    2017-08-15

    We describe Hi gh-throughput Hi stone Map ping (HiHiMap), a high-throughput imaging method to measure histones and histone posttranslational modifications (PTMs) in single cells. HiHiMap uses imaging-based quantification of DNA and cyclin A to stage individual cells in the cell cycle to determine the levels of histones or histone PTMs in each stage of the cell cycle. As proof of principle, we apply HiHiMap to measure the level of 21 core histones, histone variants, and PTMs in primary, immortalized, and transformed cells. We identify several histone modifications associated with oncogenic transformation. HiHiMap allows the rapid, high-throughput study of histones and histone PTMs across the cell cycle and the study of subpopulations of cells. © 2017 Zane et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  1. ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

    Science.gov (United States)

    Tong, Qing-He; Tao, Tao; Xie, Li-Qi; Lu, Hao-Jie

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity.

    Science.gov (United States)

    Zhang, Yiguo; Hayes, John D

    2013-01-01

    The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.

  3. Albumin modification and fragmentation in renal disease.

    Science.gov (United States)

    Donadio, Carlo; Tognotti, Danika; Donadio, Elena

    2012-02-18

    Albumin is the most important antioxidant substance in plasma and performs many physiological functions. Furthermore, albumin is the major carrier of endogenous molecules and exogenous ligands. This paper reviews the importance of post-translational modifications of albumin and fragments thereof in patients with renal disease. First, current views and controversies on renal handling of proteins, mainly albumin, will be discussed. Post-translational modifications, namely the fragmentation of albumin found with proteomic techniques in nephrotic patients, diabetics, and ESRD patients will be presented and discussed. It is reasonable to hypothesize that proteolytic fragmentation of serum albumin is due to a higher susceptibility to proteases, induced by oxidative stress. The clinical relevance of the fragmentation of albumin has not yet been established. These modifications could affect some physiological functions of albumin and have a patho-physiological role in uremic syndrome. Proteomic analysis of serum allows the identification of over-expressed proteins and can detect post-translational modifications of serum proteins, hitherto hidden, using standard laboratory techniques. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Directory of Open Access Journals (Sweden)

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  5. Monoaminylation of Fibrinogen and Glia-Derived Proteins: Indication for Similar Mechanisms in Posttranslational Protein Modification in Blood and Brain.

    Science.gov (United States)

    Hummerich, René; Costina, Victor; Findeisen, Peter; Schloss, Patrick

    2015-07-15

    Distinct proteins have been demonstrated to be posttranslationally modified by covalent transamidation of serotonin (5-hydropxytryptamin) to glutamine residues of the target proteins. This process is mediated by transglutaminase (TGase) and has been termed "serotonylation." It has also been shown that other biogenic amines, including the neurotransmitters dopamine and norepinephrine, can substitute for serotonin, implying a more general mechanism of "monoaminylation" for this kind of protein modification. Here we transamidated the autofluorescent monoamine monodansylcadaverine (MDC) to purified plasma fibrinogen and to proteins from a primary glia cell culture. Electrophoretic separation of MDC-conjugated proteins followed by mass spectrometry identified three fibrinogen subunits (Aα, Bβ, γ), a homomeric Aα2 dimer, and adducts of >250 kDa molecular weight, as well as several glial proteins. TGase-mediated MDC incorporation was strongly reduced by serotonin, underlining the general mechanism of monoaminylation.

  6. The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

    DEFF Research Database (Denmark)

    Beld, Joris; Sonnenschein, Eva; Vickery, Christopher R.

    2013-01-01

    Covering: up to 2013 Although holo-acyl carrier protein synthase, AcpS, a phosphopantetheinyl transferase (PPTase), was characterized in the 1960s, it was not until the publication of the landmark paper by Lambalot et al. in 1996 that PPTases garnered wide-spread attention being classified...... knowledge on this class of enzymes that post-translationally install a 4′-phosphopantetheine arm on various carrier proteins....

  7. Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

    Directory of Open Access Journals (Sweden)

    Warrilow David

    2008-12-01

    Full Text Available Abstract Background HIV-1 reverse transcriptase (RT is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells. Results Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p668.44 and p518.31. The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs, and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p668.53 and p518.60, suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p668.44 and p518.31 differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay. Conclusion The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.

  8. Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

    Science.gov (United States)

    Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

    2018-01-01

    Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example.

    Science.gov (United States)

    Cheng, Chia-Yang; Chu, Chia-Han; Hsu, Hung-Wei; Hsu, Fang-Rong; Tang, Chung Yi; Wang, Wen-Ching; Kung, Hsing-Jien; Chang, Pei-Ching

    2014-01-01

    Post-translational modification (PTM) of transcriptional factors and chromatin remodelling proteins is recognized as a major mechanism by which transcriptional regulation occurs. Chromatin immunoprecipitation (ChIP) in combination with high-throughput sequencing (ChIP-seq) is being applied as a gold standard when studying the genome-wide binding sites of transcription factor (TFs). This has greatly improved our understanding of protein-DNA interactions on a genomic-wide scale. However, current ChIP-seq peak calling tools are not sufficiently sensitive and are unable to simultaneously identify post-translational modified TFs based on ChIP-seq analysis; this is largely due to the wide-spread presence of multiple modified TFs. Using SUMO-1 modification as an example; we describe here an improved approach that allows the simultaneous identification of the particular genomic binding regions of all TFs with SUMO-1 modification. Traditional peak calling methods are inadequate when identifying multiple TF binding sites that involve long genomic regions and therefore we designed a ChIP-seq processing pipeline for the detection of peaks via a combinatorial fusion method. Then, we annotate the peaks with known transcription factor binding sites (TFBS) using the Transfac Matrix Database (v7.0), which predicts potential SUMOylated TFs. Next, the peak calling result was further analyzed based on the promoter proximity, TFBS annotation, a literature review, and was validated by ChIP-real-time quantitative PCR (qPCR) and ChIP-reChIP real-time qPCR. The results show clearly that SUMOylated TFs are able to be pinpointed using our pipeline. A methodology is presented that analyzes SUMO-1 ChIP-seq patterns and predicts related TFs. Our analysis uses three peak calling tools. The fusion of these different tools increases the precision of the peak calling results. TFBS annotation method is able to predict potential SUMOylated TFs. Here, we offer a new approach that enhances Ch

  10. In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: Implications for biological function

    DEFF Research Database (Denmark)

    Ahmad, I.; Hoessli, D.C.; Gupta, Ramneek

    2007-01-01

    Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence...... both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes...... of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should...

  11. Site specific modification of the human plasma proteome by methylglyoxal

    International Nuclear Information System (INIS)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig S.; Monks, Terrence J.; Lau, Serrine S.

    2015-01-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  12. Site specific modification of the human plasma proteome by methylglyoxal

    Energy Technology Data Exchange (ETDEWEB)

    Kimzey, Michael J.; Kinsky, Owen R. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Yassine, Hussein N. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Tsaprailis, George [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Stump, Craig S. [Department of Medicine, The University of Arizona, Tucson, AZ 85721 (United States); Southern Arizona VA Health Care System, Tucson, AZ 85723 (United States); Monks, Terrence J. [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States); Lau, Serrine S., E-mail: lau@pharmacy.arizona.edu [Southwest Environmental Health Sciences Center, Department of Pharmacology & Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721 (United States)

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC–MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R + 72) and hydroimidazolone (R + 54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan–HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. - Highlights: • Methylglyoxal (MG) selectively modifies arginine sites in human plasma proteome. • Dihydroxyimidazolidine and hydroimidazolone adducts on serum albumin identified • MG modification on albumin R257 associated with loss of drug site I binding capacity • MRM-tandem mass spectrometry enables sensitive detection of albumin MG-R257. • Site-specific MG modification may

  13. Stimulatory effects of curcumin and quercetin on posttranslational modifications of p53 during lung carcinogenesis.

    Science.gov (United States)

    Zhang, P; Zhang, Xy

    2018-06-01

    Lung cancer is responsible for increase in mortality due to cancer-related deaths, and new approaches are being explored for the betterment of the situation. In the present study, chemopreventive efficacy of curcumin and quercetin was investigated against benzo(a)pyrene (BP)-induced lung carcinogenesis. The mice were segregated into five groups, which included normal control, BP-treated, BP + curcumin-treated, BP + quercetin-treated, and BP + curcumin + quercetin-treated groups. The morphological and histological analyses of tumor nodules confirmed lung carcinogenesis22 weeks after weeks single intraperitoneal injection of BP at a dose of 100 mg/kg body weight to mice. Curcumin and quercetin when administered individually as well as in combination significantly elevated the expression of acetylated-p53, which was otherwise depressed due to BP treatment. Also, both the phytochemicals significantly reduced the BP-inflicted increased levels of phosphorylated-p53. Furthermore, observed increase in the number of apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), assay and increased activities of caspase 3 and 9 confirmed the induction of apoptosis by curcumin and quercetin. Moreover, the histological slides also showed noticeable improvement in the histoarchitecture of lungs by phytochemicals. The present study concludes that prophylactic treatment with curcumin and quercetin induces apoptosis in the lungs by modulation of p53 posttranslational modifications.

  14. Biosynthetic Tailoring of Microcin E492m: Post-Translational Modification Affords an Antibacterial Siderophore-Peptide Conjugate

    Science.gov (United States)

    Nolan, Elizabeth M.; Fischbach, Michael A.; Koglin, Alexander; Walsh, Christopher T.

    2008-01-01

    The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of Microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl) serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both aC10 model peptide and full-length MccE492. In the enzymatic product, the terminal serine residue is covalently attached to the C4′ oxygen of the glucose moiety. Non-enzymatic and base-catalyzed migration of the peptide to the C6′ position affords the C6′ glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures. PMID:17973380

  15. Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Maroschik, Belinda; Gürtler, Anne; Krämer, Anne; Rößler, Ute; Gomolka, Maria; Hornhardt, Sabine; Mörtl, Simone; Friedl, Anna A

    2014-01-01

    Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels

  16. Chemical Posttranslational Modification with Designed Rhodium(II) Catalysts.

    Science.gov (United States)

    Martin, S C; Minus, M B; Ball, Z T

    2016-01-01

    Natural enzymes use molecular recognition to perform exquisitely selective transformations on nucleic acids, proteins, and natural products. Rhodium(II) catalysts mimic this selectivity, using molecular recognition to allow selective modification of proteins with a variety of functionalized diazo reagents. The rhodium catalysts and the diazo reactivity have been successfully applied to a variety of protein folds, the chemistry succeeds in complex environments such as cell lysate, and a simple protein blot method accurately assesses modification efficiency. The studies with rhodium catalysts provide a new tool to study and probe protein-binding events, as well as a new synthetic approach to protein conjugates for medical, biochemical, or materials applications. © 2016 Elsevier Inc. All rights reserved.

  17. Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

    Directory of Open Access Journals (Sweden)

    Lam Hon-Ming

    2009-07-01

    Full Text Available Abstract Background Histone modifications and histone variants are of importance in many biological processes. To understand the biological functions of the global dynamics of histone modifications and histone variants in higher plants, we elucidated the variants and post-translational modifications of histones in soybean, a legume plant with a much bigger genome than that of Arabidopsis thaliana. Results In soybean leaves, mono-, di- and tri-methylation at Lysine 4, Lysine 27 and Lysine 36, and acetylation at Lysine 14, 18 and 23 were detected in HISTONE H3. Lysine 27 was prone to being mono-methylated, while tri-methylation was predominant at Lysine 36. We also observed that Lysine 27 methylation and Lysine 36 methylation usually excluded each other in HISTONE H3. Although methylation at HISTONE H3 Lysine 79 was not reported in A. thaliana, mono- and di-methylated HISTONE H3 Lysine 79 were detected in soybean. Besides, acetylation at Lysine 8 and 12 of HISTONE H4 in soybean were identified. Using a combination of mass spectrometry and nano-liquid chromatography, two variants of HISTONE H3 were detected and their modifications were determined. They were different at positions of A31F41S87S90 (HISTONE variant H3.1 and T31Y41H87L90 (HISTONE variant H3.2, respectively. The methylation patterns in these two HISTONE H3 variants also exhibited differences. Lysine 4 and Lysine 36 methylation were only detected in HISTONE H3.2, suggesting that HISTONE variant H3.2 might be associated with actively transcribing genes. In addition, two variants of histone H4 (H4.1 and H4.2 were also detected, which were missing in other organisms. In the histone variant H4.1 and H4.2, the amino acid 60 was isoleucine and valine, respectively. Conclusion This work revealed several distinct variants of soybean histone and their modifications that were different from A. thaliana, thus providing important biological information toward further understanding of the histone

  18. Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins.

    Science.gov (United States)

    Xie, Hongbo; Vucetic, Slobodan; Iakoucheva, Lilia M; Oldfield, Christopher J; Dunker, A Keith; Obradovic, Zoran; Uversky, Vladimir N

    2007-05-01

    devoted to the presentation of 87 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions (Vucetic, S.; Xie, H.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J. Proteome Res. 2007, 5, 1899-1916). Protein structure and functionality can be modulated by various post-translational modifications or/and as a result of binding of specific ligands. Numerous human diseases are associated with protein misfolding/misassembly/misfunctioning. This work concludes the series of papers dedicated to the functional anthology of intrinsic disorder and describes approximately 80 Swiss-Prot functional keywords that are related to ligands, post-translational modifications, and diseases possessing strong positive or negative correlation with the predicted long disordered regions in proteins.

  19. Post-translational modification of osteopontin: Effects on in vitro hydroxyapatite formation and growth

    International Nuclear Information System (INIS)

    Boskey, Adele L.; Christensen, Brian; Taleb, Hayat; Sørensen, Esben S.

    2012-01-01

    Highlights: ► Thrombin-cleaved fragments of milk-osteopontin effect hydroxyapatite formation differently. ► N- and C-terminal fragments promoted hydroxyapatite formation and growth. ► A central fragment inhibited hydroxyapatite formation and growth. ► Binding to collagen or hydroxyapatite seed crystals modified these effects. -- Abstract: The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1–147), a central fragment (aa 148–204) denoted SKK-fragment and a C-terminal fragment (aa 205–262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.

  20. Posttranslational modifications of Forkhead box O transcription factors

    NARCIS (Netherlands)

    Horst, Aart Arno van der

    2006-01-01

    FOXO transcription factors play an important role in essential biological processes such as differentiation, proliferation, apoptosis, DNA repair, metabolism and stress resistance. Phosphorylation is the modification that was first found on FOXOs and much of the subsequent studies focused on this

  1. Glossogyne Tenuifolia Enhances Posttranslational S-Nitrosylation of Proteins in Vascular Endothelial Cells

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    Chao-Ping Wang

    2011-06-01

    Full Text Available Glossogyne tenuifolia (GT is a traditional Chinese herb that possesses strong antioxidant activity and protects against endothelial cell (EC injury by inhibition of free reactive oxygen species (ROS. The aim of this study was to elucidate the mechanisms by which GT prevents endothelial injury using a proteomics approach. We used a sensitive method to analyze the S- nitrosoproteins utilizing a modified biotin-switch method in order to detect the possible effects of GT on protein posttranslational modification. After treatment of vascular ECs with GT, two proteins HspA9 (IS1, beta-actin (IS2 were observed to have increased posttranslational S-nitrosylation, whereas seven proteins, vimentin (DS2, DS3 and DS5, tropomyosin 3, 4 (DS6 and DS7 and oxidative phosphorylation protein such as ATP synthase, F1 complex (DS1 and 80K-H protein (DS4, were found to have decreased posttranslational S-nitrosylation. Due to S-nitrosylation of HspA9 causing the reduction of intracellular ROS and S-nitrosylation of ATP synthase interfering with ATP production and ROS formation, our study may indicate a novel mechanism in which GT protects EC injury by the inhibition of oxidative reaction.

  2. Protein Modifications as Manifestations of Hyperglycemic Glucotoxicity in Diabetes and Its Complications

    Directory of Open Access Journals (Sweden)

    Hong Zheng

    2016-01-01

    Full Text Available Diabetes and its complications are hyperglycemic toxicity diseases. Many metabolic pathways in this array of diseases become aberrant, which is accompanied with a variety of posttranslational protein modifications that in turn reflect diabetic glucotoxicity. In this review, we summarize some of the most widely studied protein modifications in diabetes and its complications. These modifications include glycation, carbonylation, nitration, cysteine S-nitrosylation, acetylation, sumoylation, ADP-ribosylation, O-GlcNAcylation, and succination. All these posttranslational modifications can be significantly attributed to oxidative stress and/or carbon stress induced by diabetic redox imbalance that is driven by activation of pathways, such as the polyol pathway and the ADP-ribosylation pathway. Exploring the nature of these modifications should facilitate our understanding of the pathological mechanisms of diabetes and its associated complications.

  3. An update on post-translational modifications of hydroxyproline-rich glycoproteins: Towards a model highlighting their contribution to plant cell wall architecture

    Directory of Open Access Journals (Sweden)

    May eHijazi

    2014-08-01

    Full Text Available Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp-rich O-glycoproteins (HRGPs were classified into three categories: (i moderately glycosylated extensins (EXTs able to form covalent scaffolds; (ii hyperglycosylated arabinogalactan proteins (AGPs; and (iii Hyp/proline (Pro-Rich proteins (H/PRPs that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs, biosynthesis, structure and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture.

  4. Posttranslational processing of progastrin

    DEFF Research Database (Denmark)

    Bundgaard, Jens René; Rehfeld, Jens F.

    2010-01-01

    as a major neurotransmitter in the central and peripheral nervous systems. The tissue-specific expression of the hormones is regulated at the transcriptional level, but the posttranslational phase is also decisive and is highly complex in order to ensure accurate maturation of the prohormones in a cell...... specific manner. Despite the structural similarities of gastrin and CCK, there are decisive differences in the posttranslational processing and secretion schemes, suggesting that specific features in the processing may have evolved to serve specific purposes. For instance, CCK peptides circulate in low...... picomolar concentrations, whereas the cellular expression of gastrin is expressed at higher levels, and accordingly gastrin circulates in 10-20-fold higher concentrations. Both common cancers and the less frequent neuroendocrine tumors express the gastrin gene and prohormone. But the posttranslational...

  5. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one that ...

  6. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

    Science.gov (United States)

    Nally, Jarlath E; Grassmann, Andre A; Planchon, Sébastien; Sergeant, Kjell; Renaut, Jenny; Seshu, Janakiram; McBride, Alan J; Caimano, Melissa J

    2017-01-01

    Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p 1.25 or expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with

  7. Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

    Directory of Open Access Journals (Sweden)

    Jarlath E. Nally

    2017-08-01

    Full Text Available Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE. Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < −1.25 across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30

  8. A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

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    Patrizia Marinelli

    2018-04-01

    Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

  9. Proteomic analysis reveals APC-dependent post-translational modifications and identifies a novel regulator of β-catenin.

    Science.gov (United States)

    Blundon, Malachi A; Schlesinger, Danielle R; Parthasarathy, Amritha; Smith, Samantha L; Kolev, Hannah M; Vinson, David A; Kunttas-Tatli, Ezgi; McCartney, Brooke M; Minden, Jonathan S

    2016-07-15

    Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called two-dimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development. © 2016. Published by The Company of Biologists Ltd.

  10. Beta-actin deficiency with oxidative posttranslational modifications in Rett syndrome erythrocytes: insights into an altered cytoskeletal organization.

    Directory of Open Access Journals (Sweden)

    Alessio Cortelazzo

    Full Text Available Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT and methyl CpG binding protein 2 (MECP2 gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs, drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: -1.82±0.15, -2.15±0.06, and -2.59±0.48, respectively in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE. Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

  11. Identification of nuclear protein targets for six leukemogenic tyrosine kinases governed by post-translational regulation.

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    Andrew Pierce

    Full Text Available Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

  12. Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

    Science.gov (United States)

    Wu, Zhengliang L.; Lech, Miroslaw

    2005-01-01

    Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

  13. Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

    Science.gov (United States)

    Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

    2018-05-23

    Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

  14. Chemical mechanisms of histone lysine and arginine modifications

    OpenAIRE

    Smith, Brian C.; Denu, John M.

    2008-01-01

    Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, an...

  15. Post-translationally modified muscle-specific ubiquitin ligases as circulating biomarkers in experimental cancer cachexia

    Science.gov (United States)

    Mota, Roberto; Rodríguez, Jessica E; Bonetto, Andrea; O’Connell, Thomas M; Asher, Scott A; Parry, Traci L; Lockyer, Pamela; McCudden, Christopher R; Couch, Marion E; Willis, Monte S

    2017-01-01

    Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Up to 80% of cancer patients experience cachexia, with 20-30% of cancer-related deaths directly linked to cachexia. Despite efforts to identify early cachexia and cancer relapse, clinically useful markers are lacking. Recently, we identified the role of muscle-specific ubiquitin ligases Atrogin-1 (MAFbx, FBXO32) and Muscle Ring Finger-1 in the pathogenesis of cardiac atrophy and hypertrophy. We hypothesized that during cachexia, the Atrogin-1 and MuRF1 ubiquitin ligases are released from muscle and migrate to the circulation where they could be detected and serve as a cachexia biomarker. To test this, we induced cachexia in mice using the C26 adenocarcinoma cells or vehicle (control). Body weight, tumor volume, and food consumption were measured from inoculation until ~day 14 to document cachexia. Western blot analysis of serum identified the presence of Atrogin-1 and MuRF1 with unique post-translational modifications consistent with mono- and poly- ubiquitination of Atrogin-1 and MuRF1 found only in cachectic serum. These findings suggest that both increased Atrogin-1 and the presence of unique post-translational modifications may serve as a surrogate marker specific for cachexia. PMID:28979816

  16. Glycoproteins and Glycosylation Site Assignments in Cereal seed Proteomes

    DEFF Research Database (Denmark)

    Dedvisitsakul, Plaipol

    The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosy......The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications...

  17. NetAcet: prediction of N-terminal acetylation sites

    DEFF Research Database (Denmark)

    Kiemer, Lars; Bendtsen, Jannick Dyrløv; Blom, Nikolaj

    2005-01-01

    Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N-acetylation for ......Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N...

  18. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

    Science.gov (United States)

    Cabral, Wayne A; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R; Chang, Weizhong; Perosky, Joseph E; Makareeva, Elena N; Mertz, Edward L; Leikin, Sergey; Tomer, Kenneth B; Kozloff, Kenneth M; Eyre, David R; Yamauchi, Mitsuo; Marini, Joan C

    2014-06-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink

  19. Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

    International Nuclear Information System (INIS)

    Koch, J.A.; Waxman, D.J.

    1989-01-01

    Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [ 32 P]orthophosphate in the presence of N 6 , O 2' -dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-induced P-450 form PB-4 (P-450 gene IIB1). Two-dimensional gel electrophoresis revealed that these 32 P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 μM. Phosphoamino acid analysis of the 32 P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2. In vitro analyses revealed that phosphorylation of P-450 PB-4 leads to a loss of monooxygenase activity, suggesting that the posttranslational modification of this P-450 enzyme by cAMP-dependent protein kinase may play a role in the modulation of P-450-dependent monooxygenase activity in vivo

  20. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

    Directory of Open Access Journals (Sweden)

    Sudharsana R Ande

    Full Text Available Prohibitin (PHB or PHB1 is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114 and tyrosine 259 (Tyr259 in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121 and threonine 258 (Thr258 respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s or known tyrosine phosphorylation site(s revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

  1. Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

    Science.gov (United States)

    Khan, Aliyya; Eikani, Carlo K; Khan, Hana; Iavarone, Anthony T; Pesavento, James J

    2018-01-05

    The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas' versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.

  2. A systematic identification of species-specific protein succinylation sites using joint element features information

    Directory of Open Access Journals (Sweden)

    Hasan MM

    2017-08-01

    Full Text Available Md Mehedi Hasan,1 Mst Shamima Khatun,2 Md Nurul Haque Mollah,2 Cao Yong,3 Dianjing Guo1 1School of Life Sciences and the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, People’s Republic of China; 2Laboratory of Bioinformatics, Department of Statistics, University of Rajshahi, Rajshahi, Bangladesh; 3Department of Mechanical Engineering and Automation, Harbin Institute of Technology, Shenzhen Graduate School, Shenzhen, People’s Republic of China Abstract: Lysine succinylation, an important type of protein posttranslational modification, plays significant roles in many cellular processes. Accurate identification of succinylation sites can facilitate our understanding about the molecular mechanism and potential roles of lysine succinylation. However, even in well-studied systems, a majority of the succinylation sites remain undetected because the traditional experimental approaches to succinylation site identification are often costly, time-consuming, and laborious. In silico approach, on the other hand, is potentially an alternative strategy to predict succinylation substrates. In this paper, a novel computational predictor SuccinSite2.0 was developed for predicting generic and species-specific protein succinylation sites. This predictor takes the composition of profile-based amino acid and orthogonal binary features, which were used to train a random forest classifier. We demonstrated that the proposed SuccinSite2.0 predictor outperformed other currently existing implementations on a complementarily independent dataset. Furthermore, the important features that make visible contributions to species-specific and cross-species-specific prediction of protein succinylation site were analyzed. The proposed predictor is anticipated to be a useful computational resource for lysine succinylation site prediction. The integrated species-specific online tool of SuccinSite2.0 is publicly

  3. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

    2013-01-01

    phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

  4. Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry

    Science.gov (United States)

    Souda, Puneet; Ryan, Christopher M.; Cramer, William A.; Whitelegge, Julian

    2011-01-01

    Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein’s native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electroncapture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. PMID:21982782

  5. Posttranslational processing of progastrin

    DEFF Research Database (Denmark)

    Bundgaard, Jens René; Rehfeld, Jens F.

    2010-01-01

    as a major neurotransmitter in the central and peripheral nervous systems. The tissue-specific expression of the hormones is regulated at the transcriptional level, but the posttranslational phase is also decisive and is highly complex in order to ensure accurate maturation of the prohormones in a cell...... picomolar concentrations, whereas the cellular expression of gastrin is expressed at higher levels, and accordingly gastrin circulates in 10-20-fold higher concentrations. Both common cancers and the less frequent neuroendocrine tumors express the gastrin gene and prohormone. But the posttranslational......, as are structural features of progastrin that are involved in the precursor activation process. Thus, the review describes how the processing depends on the cell-specific expression of the processing enzymes and kinetics in the secretory pathway....

  6. Chromatin proteins and modifications as drug targets

    DEFF Research Database (Denmark)

    Helin, Kristian; Dhanak, Dashyant

    2013-01-01

    A plethora of groundbreaking studies have demonstrated the importance of chromatin-associated proteins and post-translational modifications of histones, proteins and DNA (so-called epigenetic modifications) for transcriptional control and normal development. Disruption of epigenetic control...... is a frequent event in disease, and the first epigenetic-based therapies for cancer treatment have been approved. A generation of new classes of potent and specific inhibitors for several chromatin-associated proteins have shown promise in preclinical trials. Although the biology of epigenetic regulation...

  7. Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.

    Science.gov (United States)

    Yang, Jing; Tallman, Keri A; Porter, Ned A; Liebler, Daniel C

    2015-03-03

    Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner.

  8. Proteomic profiling and post-translational modifications in human keratinocytes treated with Mucuna pruriens leaf extract.

    Science.gov (United States)

    Cortelazzo, Alessio; Lampariello, Raffaella L; Sticozzi, Claudia; Guerranti, Roberto; Mirasole, Cristiana; Zolla, Lello; Sacchetti, Gianni; Hajek, Joussef; Valacchi, Giuseppe

    2014-02-03

    Mucuna pruriens (Mp) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure. The effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS). Eighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9. Our findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs). © 2013 Published by Elsevier Ireland Ltd.

  9. Identification of SUMO conjugation sites in the budding yeast proteome

    Directory of Open Access Journals (Sweden)

    Miguel Esteras

    2017-10-01

    Full Text Available Post-translational modification by the small ubiquitin-like modifier (SUMO is an important mechanism regulating protein function. Identification of SUMO conjugation sites on substrates is a challenging task. Here we employed a proteomic method to map SUMO acceptor lysines in budding yeast proteins. We report the identification of 257 lysine residues where SUMO is potentially attached. Amongst the hits, we identified already known SUMO substrates and sites, confirming the success of the approach. In addition, we tested several of the novel substrates using SUMO immunoprecipitation analysis and confirmed that the SUMO acceptor lysines identified in these proteins are indeed bona fide SUMOylation sites. We believe that the collection of SUMO sites presented here is an important resource for future functional studies of SUMOylation in yeast.

  10. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  11. SIGNALING TO THE P53 TUMOR SUPPRESSOR THROUGH PATHWAYS ACTIVATED BY GENOTOXIC AND NON-GENOTOXIC STRESSES.

    Energy Technology Data Exchange (ETDEWEB)

    ANDERSON,C.W.APPELLA,E.

    2002-07-01

    The p53 tumor suppressor is a tetrameric transcription factor that is post-translational modified at {approx}18 different sites by phosphorylation, acetylation, or sumoylation in response to various cellular stress conditions. Specific posttranslational modifications, or groups of modifications, that result from the activation of different stress-induced signaling pathways are thought to modulate p53 activity to regulate cell fate by inducing cell cycle arrest, apoptosis, or cellular senescence. Here we review the posttranslational modifications to p53 and the pathways that produce them in response to both genotoxic and non-genotoxic stresses.

  12. Modifications to the remedial action plan and site design for stabilization of the inactive Uranium Mill Tailings Site at Green River, Utah

    International Nuclear Information System (INIS)

    1994-09-01

    Modifications to the water resources protection strategy detailed in the remedial action plan for the Green River, Utah, disposal site are presented. The modifications are based on new information, including ground water quality data collected after remedial action was completed and on a revised assessment of disposal cell design features, surface conditions, and site hydrogeology. The modifications will result in compliance with the U.S. EPA proposed ground water standards (52 FR 36000 (1987))

  13. Abnormal type I collagen post-translational modification and crosslinking in a cyclophilin B KO mouse model of recessive osteogenesis imperfecta.

    Directory of Open Access Journals (Sweden)

    Wayne A Cabral

    2014-06-01

    Full Text Available Cyclophilin B (CyPB, encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO mice are small, with reduced femoral areal bone mineral density (aBMD, bone volume per total volume (BV/TV and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11% collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1 activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

  14. Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

    Science.gov (United States)

    Cabral, Wayne A.; Perdivara, Irina; Weis, MaryAnn; Terajima, Masahiko; Blissett, Angela R.; Chang, Weizhong; Perosky, Joseph E.; Makareeva, Elena N.; Mertz, Edward L.; Leikin, Sergey; Tomer, Kenneth B.; Kozloff, Kenneth M.; Eyre, David R.; Yamauchi, Mitsuo; Marini, Joan C.

    2014-01-01

    Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered

  15. The roles of MHC class II genes and post-translational modification in celiac disease.

    Science.gov (United States)

    Sollid, Ludvig M

    2017-08-01

    Our increasing understanding of the etiology of celiac disease, previously considered a simple food hypersensitivity disorder caused by an immune response to cereal gluten proteins, challenges established concepts of autoimmunity. HLA is a chief genetic determinant, and certain HLA-DQ allotypes predispose to the disease by presenting posttranslationally modified (deamidated) gluten peptides to CD4 + T cells. The deamidation of gluten peptides is mediated by transglutaminase 2. Strikingly, celiac disease patients generate highly disease-specific autoantibodies to the transglutaminase 2 enzyme. The dual role of transglutaminase 2 in celiac disease is hardly coincidental. This paper reviews the genetic mapping and involvement of MHC class II genes in disease pathogenesis, and discusses the evidence that MHC class II genes, via the involvement of transglutaminase 2, influence the generation of celiac disease-specific autoantibodies.

  16. Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer's disease.

    Science.gov (United States)

    Kovacech, B; Novak, M

    2010-12-01

    Deposits of the misfolded neuronal protein tau are major hallmarks of neurodegeneration in Alzheimer's disease (AD) and other tauopathies. The etiology of the transformation process of the intrinsically disordered soluble protein tau into the insoluble misordered aggregate has attracted much attention. Tau undergoes multiple modifications in AD, most notably hyperphosphorylation and truncation. Hyperphosphorylation is widely regarded as the hottest candidate for the inducer of the neurofibrillary pathology. However, the true nature of the impetus that initiates the whole process in the human brains remains unknown. In AD, several site-specific tau cleavages were identified and became connected to the progression of the disease. In addition, western blot analyses of tau species in AD brains reveal multitudes of various truncated forms. In this review we summarize evidence showing that tau truncation alone is sufficient to induce the complete cascade of neurofibrillary pathology, including hyperphosphorylation and accumulation of misfolded insoluble forms of tau. Therefore, proteolytical abnormalities in the stressed neurons and production of aberrant tau cleavage products deserve closer attention and should be considered as early therapeutic targets for Alzheimer's disease.

  17. Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids.

    Science.gov (United States)

    Ho, Ya-Yeh; Huang, Yen-Hua; Huang, Cheng-Yang

    2013-04-01

    Bacterial allantoinase (ALLase) and dihydroorotase (DHOase) are members of the cyclic amidohydrolase family. ALLase and DHOase possess similar binuclear metal centers in the active site in which two metals are bridged by a post-translationally carboxylated lysine. In this study, we determined the effects of carboxylated lysine and metal binding on the activities of ALLase and DHOase. Although DHOase is a metalloenzyme, purified DHOase showed high activity without additional metal supplementation in a reaction mixture or bacterial culture. However, unlike DHOase, ALLase had no activity unless some specific metal ions were added to the reaction mixture or culture. Substituting the metal binding sites H59, H61, K146, H186, H242, or D315 with alanine completely abolished the activity of ALLase. However, the K146C, K146D and K146E mutants of ALLase were still active with about 1-6% activity of the wild-type enzyme. These ALLase K146 mutants were found to have 1.4-1.7 mol metal per mole enzyme subunit, which may indicate that they still contained the binuclear metal center in the active site. The activity of the K146A mutant of the ALLase and the K103A mutant of DHOase can be chemically rescued by short-chain carboxylic acids, such as acetic, propionic, and butyric acids, but not by ethanol, propan-1-ol, and imidazole, in the presence of Co2+ or Mn2+ ions. However, the activity was still ~10-fold less than that of wild-type ALLase. Overall, these results indicated that the 20 natural basic amino acid residues were not sufficiently able to play the role of lysine. Accordingly, we proposed that during evolution, the post-translational modification of carboxylated lysine in the cyclic amidohydrolase family was selected for promoting binuclear metal center self-assembly and increasing the nucleophilicity of the hydroxide at the active site for enzyme catalysis. This kind of chemical rescue combined with site-directed mutagenesis may also be used to identify a binuclear metal

  18. Overexpression of Exportin-5 Overrides the Inhibitory Effect of miRNAs Regulation Control and Stabilize Proteins via Posttranslation Modifications in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Naseruddin Höti

    2017-10-01

    Full Text Available Although XPO5 has been characterized to have tumor-suppressor features in the miRNA biogenesis pathway, the impact of altered expression of XPO5 in cancers is unexplored. Here we report a novel “oncogenic” role of XPO5 in advanced prostate cancer. Using prostate cancer models, we found that excess levels of XPO5 override the inhibitory effect of the canoncial miRNA-mRNA regulation, resulting in a global increase in proteins expression. Importantly, we found that decreased expression of XPO5 could promote an increase in proteasome degradation, whereas overexpression of XPO5 leads to altered protein posttranslational modification via hyperglycosylation, resulting in cellular protein stability. We evaluated the therapeutic advantage of targeting XPO5 in prostate cancer and found that knocking down XPO5 in prostate cancer cells suppressed cellular proliferation and tumor development without significantly impacting normal fibroblast cells survival. To our knowledge, this is the first report describing the oncogenic role of XPO5 in overriding the miRNAs regulation control. Furthermore, we believe that these findings will provide an explanation as to why, in some cancers that express higher abundance of mature miRNAs, fail to suppress their potential protein targets.

  19. NF-Y dependent epigenetic modifications discriminate between proliferating and postmitotic tissue

    DEFF Research Database (Denmark)

    Gurtner, Aymone; Fuschi, Paola; Magi, Fiorenza

    2008-01-01

    The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferatio...

  20. Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products: New Insights Into the Role of Leader and Core Peptides During Biosynthesis

    Science.gov (United States)

    Yang, Xiao; van der Donk, Wilfred A.

    2013-01-01

    Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this review, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the posttranslational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution. PMID:23666908

  1. Post-translational Control of Intracellular Pathogen Sensing Pathways.

    Science.gov (United States)

    Chiang, Cindy; Gack, Michaela U

    2017-01-01

    Mammalian cells recognize virus-derived nucleic acids using a defined set of intracellular sensors including the DNA sensors cyclic GMP-AMP (cGAMP) synthase (cGAS) and interferon gamma (IFNγ)-inducible protein 16 (IFI16) as well as viral RNA receptors of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family. Following innate immune recognition, these sensors launch an immune response that is characterized by the transcriptional upregulation of many antiviral molecules, including proinflammatory cytokines, chemokines, and IFN-stimulated genes. Recent studies have demonstrated that the signal transduction initiated by these sensors is sophisticatedly regulated by post-translational modifications (PTMs) resulting in a robust yet 'tunable' cytokine response to maintain immune homeostasis. Here we summarize recent advances in our understanding of how PTMs and regulatory enzymes control the signaling activity of RLRs, cGAS, and IFI16 as well as their proximal adaptor proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. O-GlcNAc modification: why so intimately associated with phosphorylation?

    Directory of Open Access Journals (Sweden)

    Ande Sudharsana R

    2011-01-01

    Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

  3. Contribution of Post-translational Phosphorylation to Sarcomere-linked Cardiomyopathy Phenotypes

    Directory of Open Access Journals (Sweden)

    Margaret V Westfall

    2016-09-01

    Full Text Available Secondary shifts develop in post-translational phosphorylation of sarcomeric proteins in multi¬ple animal models of inherited cardiomyopathy. These signaling alterations together with the primary mutation are predicted to contribute to the overall cardiac phenotype. As a result, identification and integration of post-translational myofilament signaling responses are identified as priorities for gaining insights into sarcomeric cardiomyopathies. However, significant questions remain about the nature and contribution of post-translational phosphorylation to structural remodeling and cardiac dysfunction in animal models and human patients. This perspective essay discusses specific goals for filling critical gaps about post-translational signaling in response to these inherited mutations, especially within sarcomeric proteins. The discussion focuses primarily on pre-clinical analysis of animal models and defines challenges and future directions in this field.

  4. Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by sup 31 P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation

    Energy Technology Data Exchange (ETDEWEB)

    Thorneley, R.N.F.; Ashby, G.A.; Drummond, M.H.; Eady, R.R.; Huff, S.; Macdonald, C.J. (Univ. of Sussex, Brighton (United Kingdom)); Abell, C.; Schneier, A. (Univ. Chemical Lab., Cambridge (United Kingdom))

    1992-02-04

    A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. {sup 31}P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O{sub 2} and the carbon status of diazotrophic cultures.

  5. Muc1 based breast cancer vaccines: role of post translational modifications

    International Nuclear Information System (INIS)

    Begum, M.; Khurshid, R.; Nagra, S.A.

    2008-01-01

    Vaccine development is one of the most promising fields in cancer research. After autologous transplantation, due to low tumour burden, patients are more likely to respond immunologically to a cancer vaccine. MUC1 with its adhesive and anti adhesive functions, immunostimulatory and immunosuppressive activities, is therefore a good candidate for breast cancer vaccine. A structure-based insight into the immunogenicity of natural MUC1 glyco forms, of its sub-domains, motifs and post translational modification like glycosylation and myriostoylation may aid the design of tumour vaccines. Primary sequences of human MUC1 were retrieved from the SWISSPROT data bank. Protein pattern search: The primary sequence of Human MUC1 was searched at PROSITE (a dictionary of protein sites and patterns) database. Our study observes that post-translational modifications play an important role in presenting MUC1 as a candidate for breast cancer vaccine. It is found that the phosphorylation and glycosylation of important functional motifs of MUC1 may take part in the production of cytokines that may provide immunization. (author)

  6. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    KAUST Repository

    Fiorillo, Annarita

    2014-03-21

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  7. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    Directory of Open Access Journals (Sweden)

    Annarita Fiorillo

    Full Text Available The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3, unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  8. The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.

    KAUST Repository

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

  9. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

    Science.gov (United States)

    Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

    2017-01-01

    ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  10. Positive-Unlabeled Learning for Pupylation Sites Prediction

    Directory of Open Access Journals (Sweden)

    Ming Jiang

    2016-01-01

    Full Text Available Pupylation plays a key role in regulating various protein functions as a crucial posttranslational modification of prokaryotes. In order to understand the molecular mechanism of pupylation, it is important to identify pupylation substrates and sites accurately. Several computational methods have been developed to identify pupylation sites because the traditional experimental methods are time-consuming and labor-sensitive. With the existing computational methods, the experimentally annotated pupylation sites are used as the positive training set and the remaining nonannotated lysine residues as the negative training set to build classifiers to predict new pupylation sites from the unknown proteins. However, the remaining nonannotated lysine residues may contain pupylation sites which have not been experimentally validated yet. Unlike previous methods, in this study, the experimentally annotated pupylation sites were used as the positive training set whereas the remaining nonannotated lysine residues were used as the unlabeled training set. A novel method named PUL-PUP was proposed to predict pupylation sites by using positive-unlabeled learning technique. Our experimental results indicated that PUL-PUP outperforms the other methods significantly for the prediction of pupylation sites. As an application, PUL-PUP was also used to predict the most likely pupylation sites in nonannotated lysine sites.

  11. Epigenomic landscape modified by histone modification correlated with activation of IGF2 gene

    Science.gov (United States)

    The links of histone post-translational modifications and chromatin structure to cell cycle progression, DNA replication, and overall chromosome functions are very clear. The modulation of genome expression as a consequence of chromatin structural changes is most likely a basic mechanism. The epige...

  12. Post-translational regulation enables robust p53 regulation.

    Science.gov (United States)

    Shin, Yong-Jun; Chen, Kai-Yuan; Sayed, Ali H; Hencey, Brandon; Shen, Xiling

    2013-08-30

    The tumor suppressor protein p53 plays important roles in DNA damage repair, cell cycle arrest and apoptosis. Due to its critical functions, the level of p53 is tightly regulated by a negative feedback mechanism to increase its tolerance towards fluctuations and disturbances. Interestingly, the p53 level is controlled by post-translational regulation rather than transcriptional regulation in this feedback mechanism. We analyzed the dynamics of this feedback to understand whether post-translational regulation provides any advantages over transcriptional regulation in regard to disturbance rejection. When a disturbance happens, even though negative feedback reduces the steady-state error, it can cause a system to become less stable and transiently overshoots, which may erroneously trigger downstream reactions. Therefore, the system needs to balance the trade-off between steady-state and transient errors. Feedback control and adaptive estimation theories revealed that post-translational regulation achieves a better trade-off than transcriptional regulation, contributing to a more steady level of p53 under the influence of noise and disturbances. Furthermore, post-translational regulation enables cells to respond more promptly to stress conditions with consistent amplitude. However, for better disturbance rejection, the p53- Mdm2 negative feedback has to pay a price of higher stochastic noise. Our analyses suggest that the p53-Mdm2 feedback favors regulatory mechanisms that provide the optimal trade-offs for dynamic control.

  13. Drug delivery from hydrophobic-modified mesoporous silicas: Control via modification level and site-selective modification

    International Nuclear Information System (INIS)

    Tang Qunli; Chen Yuxi; Chen Jianghua; Li Jin; Xu Yao; Wu Dong; Sun Yuhan

    2010-01-01

    Dimethylsilyl (DMS) modified mesoporous silicas were successfully prepared via co-condensation and post-grafting modification methods. The post-grafting modification was carried out by the reaction of the as-synthesized MCM-41 material (before CTAB removal) with diethoxydimethylsinale (DEDMS). N 2 adsorption-desorption and 29 Si MAS NMR characterization demonstrated that different amount of DMS groups were successfully incorporated into the co-condensation modified samples, and the functional DMS groups were placed selectively on the pore openings and external pore surfaces in the post-grafting modified samples. Subsequently, the controlled drug delivery properties from the resulting DMS-modified mesoporous silicas were investigated in detail. The drug adsorption experiments showed that the adsorption capacities were mainly depended on the content of silanol group (CSG) in the corresponding carriers. The in vitro tests exhibited that the incorporation of DMS groups greatly retarded the ibuprofen release rate. Moreover, the ibuprofen release profiles could be well modulated by varying DMS modification levels and site-selective distribution of functional groups in mesoporous carriers. - The distribution of DMS groups on the pore surfaces of the mesostructures strongly affects the drug release rate. The P-M41-1 and the P-M41-2 possess the close DMS modification levels as the C-M41-10, but the ibuprofen release rates from the P-M41-1 and P-M41-2 are much slower than that from the C-M41-10.

  14. Analysis of Changes in SUMO-2/3 Modification during Breast Cancer Progression and Metastasis

    DEFF Research Database (Denmark)

    Subramonian, Divya; Raghunayakula, Sarita; Olsen, Jesper V

    2014-01-01

    SUMOylation is an essential posttranslational modification and regulates many cellular processes. Dysregulation of SUMOylation plays a critical role in metastasis, yet how its perturbation affects this lethal process of cancer is not well understood. We found that SUMO-2/3 modification is greatly...... in metastatic cells. Targets with altered SUMOylation are involved in cell cycle, migration, inflammation, glycolysis, gene expression, and SUMO/ubiquitin pathways, suggesting that perturbations of SUMO-2/3 modification might contribute to metastasis by affecting these processes. Consistent with this, up...... progression and metastasis....

  15. Effect of the UV modification of α-crystallin on its ability to suppress nonspecific aggregation

    International Nuclear Information System (INIS)

    Ellozy, A.R.; Ceger, Patricia; Wang, R.H.; Dillon, James

    1996-01-01

    Recent studies have shown that structural modifications of α-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these post-translational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of α-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and γ-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295nm) for various time intervals. Thermal denaturation of γ-crystallin with or without α-crystallin was carried out at 70 o C and increases in light scattering were measured at 360 nm. We found that (1) irradiation of γ-crystallin increased its susceptibility to heat-induced scattering. The addition of α-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer γ-crystallin is irradiated and (2) irradiation of α-crystallin decreases its ability to suppress nonspecific aggregation and the presence of 3-HK during irradiation decreases its further. Our results indicate that post-translational modifications of α-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with γ-crystallin. The kinetics of γ-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applied for nonirradiated γ-crystallin. This model does not hold when γ-crystallin is irradiated in the prescence or absence of α-crystallin. In these cases, two step or multistep mechanisms are more likely. (Author)

  16. Exploring oxidative modifications of tyrosine

    DEFF Research Database (Denmark)

    Houée-Lévin, C; Bobrowski, K; Horakova, L

    2015-01-01

    residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different...... effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor...... residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation...

  17. Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis

    Science.gov (United States)

    Benjdia, Alhosna; Guillot, Alain; Ruffié, Pauline; Leprince, Jérôme; Berteau, Olivier

    2017-07-01

    Ribosomally synthesized peptides are built out of L-amino acids, whereas D-amino acids are generally the hallmark of non-ribosomal synthetic processes. Here we show that the model bacterium Bacillus subtilis is able to produce a novel type of ribosomally synthesized and post-translationally modified peptide that contains D-amino acids, and which we propose to call epipeptides. We demonstrate that a two [4Fe-4S]-cluster radical S-adenosyl-L-methionine (SAM) enzyme converts L-amino acids into their D-counterparts by catalysing Cα-hydrogen-atom abstraction and using a critical cysteine residue as the hydrogen-atom donor. Unexpectedly, these D-amino acid residues proved to be essential for the activity of a peptide that induces the expression of LiaRS, a major component of the bacterial cell envelope stress-response system. Present in B. subtilis and in several members of the human microbiome, these epipeptides and radical SAM epimerases broaden the landscape of peptidyl structures accessible to living organisms.

  18. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein

    Directory of Open Access Journals (Sweden)

    Deeksha Vishwamitra

    2015-09-01

    Full Text Available Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+ T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5(p23;q35 that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  19. Site-Specific Modification Using the 2′-Methoxyethyl Group Improves the Specificity and Activity of siRNAs

    Directory of Open Access Journals (Sweden)

    Xinyun Song

    2017-12-01

    Full Text Available Rapid progress has been made toward small interfering RNA (siRNA-based therapy for human disorders, but rationally optimizing siRNAs for high specificity and potent silencing remains a challenge. In this study, we explored the effect of chemical modification at the cleavage site of siRNAs. We found that modifications at positions 9 and 10 markedly reduced the silencing potency of the unmodified strand of siRNAs but were well tolerated by the modified strand. Intriguingly, addition of the 2′-methoxyethyl (MOE group at the cleavage site improved both the specificity and silencing activity of siRNAs by facilitating the oriented RNA-induced silencing complex (RISC loading of the modified strand. Furthermore, we combined MOE modifications at positions 9 and 10 of one strand together with 2′-O-methylation (OMe at position 14 of the other strand and found a synergistic effect that improved the specificity of siRNAs. The surprisingly beneficial effect of the combined modification was validated using siRNA-targeting endogenous gene intercellular adhesion molecule 1 (ICAM1. We found that the combined modifications eliminated its off-target effects. In conclusion, we established effective strategies to optimize siRNAs using site-specific MOE modifications. The findings may allow the creation of superior siRNAs for therapy in terms of activity and specificity.

  20. Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants.

    Science.gov (United States)

    Nemie-Feyissa, Dugassa; Królicka, Adriana; Førland, Nina; Hansen, Margarita; Heidari, Behzad; Lillo, Cathrine

    2013-05-01

    Regulation of nitrate reductase (NR) by reversible phosphorylation at a conserved motif is well established in higher plants, and enables regulation of NR in response to rapid fluctuations in light intensity. This regulation is not conserved in algae NR, and we wished to test the evolutionary origin of the regulatory mechanism by physiological examination of ancient land plants. Especially a member of the lycophytes is of interest since their NR is candidate for regulation by reversible phosphorylation based on sequence analysis. We compared Selaginella kraussiana, a member of the lycophytes and earliest vascular plants, with the angiosperm Arabidopsis thaliana, and also tested the moss Physcomitrella patens. Interestingly, optimization of assay conditions revealed that S. kraussiana NR used NADH as an electron donor like A. thaliana, whereas P. patens NR activity depended on NADPH. Examination of light/darkness effects showed that S. kraussiana NR was rapidly regulated similar to A. thaliana NR when a differential (Mg(2+) contra EDTA) assay was used to reveal activity state of NR. This implies that already existing NR enzyme was post-translationally activated by light in both species. Light had a positive effect also on de novo synthesis of NR in S. kraussiana, which could be shown after the plants had been exposed to a prolonged dark period (7 days). Daily variations in NR activity were mainly caused by post-translational modifications. As for angiosperms, the post-translational light activation of NR in S. kraussiana was inhibited by 3-(3,4-dichlorophenyl)-1*1-dimethylurea (DCMU), an inhibitor of photosynthesis and stomata opening. Evolutionary, a post-translational control mechanism for NR have occurred before or in parallel with development of vascular tissue in land plants, and appears to be part of a complex mechanisms for coordination of CO2 and nitrogen metabolism in these plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  1. Creating diversity by site-selective peptide modification: a customizable unit affords amino acids with high optical purity.

    Science.gov (United States)

    Romero-Estudillo, Ivan; Boto, Alicia

    2013-11-15

    The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes, but still is a difficult synthetic challenge. Herein natural hydroxyproline is introduced as a "convertible" unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, and to achieve the mild, efficient, and site-selective modification of peptides.

  2. Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

    Science.gov (United States)

    Boggild, A K; Sundermann, C A; Estridge, B H

    2002-01-01

    Glutamylated and tyrosinated tubulin were localized in Giardia intestinalis and selected trichomonads of the Tritrichomonadinae subfamily, using specific monoclonal antibodies directed at each of the post-translational modifications. Analysis was carried out using indirect immunofluorescence microscopy. Although trichomonad tubulins remained unlabeled by anti-tyrosine tubulin (TUB-1A2), the presence of the glutamylation motif (GT 335) was confirmed and found to differ in distribution among tritrichomonads. Tritrichomonas muris was most heavily labeled with GT 335, while T. foetus was the least so. Like trichomonads, Giardia was unreactive to anti-tyrosine tubulin; however, the GT 335 antibody produced marked fluorescence in Giardia trophozoites. This study is the first to report immunofluorescent localization of tubulin glutamylation in Giardia and confirms previously reported mass spectrometry data.

  3. Analyzing the evolution of beta-endorphin post-translational processing events: studies on reptiles.

    Science.gov (United States)

    Shoureshi, Pezhman; Baron, Andrea; Szynskie, Laura; Dores, Robert M

    2007-01-01

    In many cartilaginous fishes, most ray-finned fishes, lungfishes, and amphibians, the post-translational processing of POMC includes the monobasic cleavage of beta-endorphin to yield an opioid that is eight to ten amino acids in length. The amino acid motif within the beta-endorphin sequence required for a monobasic cleavage event is -E-R-(S/G)-Q-. Mammals and birds lack this motif and as a result beta-endorphin(1-8) is a not an end-product in either group. Since both mammals and birds were derived from ancestors with reptilian origins, an analysis of beta-endorphin sequences from extant groups of reptiles should provide insights into the manner in which beta-endorphin post-translational processing mechanisms have evolved in amniotes. To this end a POMC cDNA was cloned from the pituitary of the turtle, Chrysemys scripta. The beta-endorphin sequence in this species was compared to other reptile beta-endorphin sequences (i.e., Chinese soft shell turtle and gecko) and to known bird and mammal sequences. This analysis indicated that either the loss of the arginine residue at the cleavage site (the two turtle species, chick, and human) or a substitution at the glutamine position in the consensus sequence (gecko and ostrich) would account for the loss of the monobasic cleavage reaction in that species. Since amphibians are capable of performing the beta-endorphin monobasic reaction, it would appear that the amino acid substitutions that eliminated this post-translational process event in reptilian-related tetrapods must have occurred in the ancestral amniotes.

  4. SUMOylation of the Forkhead transcription factor FOXL2 promotes its stabilization/activation through transient recruitment to PML bodies.

    Directory of Open Access Journals (Sweden)

    Adrien Georges

    Full Text Available BACKGROUND: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications. METHODS AND PRINCIPAL FINDINGS: Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites. CONCLUSIONS: By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity.

  5. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    Science.gov (United States)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  6. Sequence tagging reveals unexpected modifications in toxicoproteomics

    Science.gov (United States)

    Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

    2010-01-01

    Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

  7. Developmental differences in posttranslational calmodulin methylation in pea plants

    International Nuclear Information System (INIS)

    Oh, Sukheung; Roberts, D.M.

    1990-01-01

    A calmodulin-N-methyltransferase was used to analyze the degree of lysine-115 methylation of pea calmodulin. Calmodulin was isolated from segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by the calmodulin methyltransferase in the presence of 3 H-methyl-S-adenosylmethionine. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The methylation of these calmodulin samples occurs specifically at lysine 115 since site-directed mutants of calmodulin with substitutions at this position were not methylated and competitively inhibited methylation. The present findings, combined with previous data showing differences in NAD kinase activation by methylated and unmethylated calmodulins, raise the possibility that posttranslational methylation could affect calmodulin action

  8. Biotinylation is a natural, albeit rare, modification of human histones

    Science.gov (United States)

    Kuroishi, Toshinobu; Rios-Avila, Luisa; Pestinger, Valerie; Wijeratne, Subhashinee S. K.; Zempleni, Janos

    2011-01-01

    Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites. PMID:21930408

  9. Alternate Phosphorylation/O-GlcNAc Modification on Human Insulin IRSs: A Road towards Impaired Insulin Signaling in Alzheimer and Diabetes

    Directory of Open Access Journals (Sweden)

    Zainab Jahangir

    2014-01-01

    Full Text Available Impaired insulin signaling has been thought of as important step in both Alzheimer’s disease (AD and type 2 diabetes mellitus (T2DM. Posttranslational modifications (PTMs regulate functions and interaction of insulin with insulin receptors substrates (IRSs and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR residues on IRSs. Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine (Ser and threonine (Thr residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated; however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize the risk of AD and T2DM.

  10. Identification and Interrogation of Combinatorial Histone Modifications

    Directory of Open Access Journals (Sweden)

    Kelly R Karch

    2013-12-01

    Full Text Available Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs. Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.

  11. A post-translational balancing act: the good and the bad of SUMOylation in pancreatic islets.

    Science.gov (United States)

    MacDonald, Patrick E

    2018-04-01

    Post-translational modification of proteins contributes to the control of cell function and survival. The balance of these in insulin-producing pancreatic beta cells is important for the maintenance of glucose homeostasis. Protection from the damaging effects of reactive oxygen species is required for beta cell survival, but if this happens at the expense of insulin secretory function then the ability of islets to respond to changing metabolic conditions may be compromised. In this issue of Diabetologia, He et al ( https://doi.org/10.1007/s00125-017-4523-9 ) show that post-translational attachment of small ubiquitin-like modifier (SUMO) to target lysine residues (SUMOylation) strikes an important balance between the protection of beta cells from oxidative stress and the maintenance of insulin secretory function. They show that SUMOylation is required to stabilise nuclear factor erythroid 2-related factor 2 (NRF2) and increase antioxidant gene expression. Decreasing SUMOylation in beta cells impairs their antioxidant capacity, causes cell death, hyperglycaemia, and increased sensitivity to streptozotocin-induced diabetes, while increasing SUMOylation is protective. However, this protection from overt diabetes occurs in concert with glucose intolerance due to impaired beta cell function. A possible role for SUMOylation as a key factor balancing beta cell protection vs beta cell responsiveness to metabolic cues is discussed in this Commentary.

  12. Biochemical systems approaches for the analysis of histone modification readout.

    Science.gov (United States)

    Soldi, Monica; Bremang, Michael; Bonaldi, Tiziana

    2014-08-01

    Chromatin is the macromolecular nucleoprotein complex that governs the organization of genetic material in the nucleus of eukaryotic cells. In chromatin, DNA is packed with histone proteins into nucleosomes. Core histones are prototypes of hyper-modified proteins, being decorated by a large number of site-specific reversible and irreversible post-translational modifications (PTMs), which contribute to the maintenance and modulation of chromatin plasticity, gene activation, and a variety of other biological processes and disease states. The observations of the variety, frequency and co-occurrence of histone modifications in distinct patterns at specific genomic loci have led to the idea that hPTMs can create a molecular barcode, read by effector proteins that translate it into a specific transcriptional state, or process, on the underlying DNA. However, despite the fact that this histone-code hypothesis was proposed more than 10 years ago, the molecular details of its working mechanisms are only partially characterized. In particular, two questions deserve specific investigation: how the different modifications associate and synergize into patterns and how these PTM configurations are read and translated by multi-protein complexes into a specific functional outcome on the genome. Mass spectrometry (MS) has emerged as a versatile tool to investigate chromatin biology, useful for both identifying and validating hPTMs, and to dissect the molecular determinants of histone modification readout systems. We review here the MS techniques and the proteomics methods that have been developed to address these fundamental questions in epigenetics research, emphasizing approaches based on the proteomic dissection of distinct native chromatin regions, with a critical evaluation of their present challenges and future potential. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Gains of ubiquitylation sites in highly conserved proteins in the human lineage

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Post-translational modification of lysine residues of specific proteins by ubiquitin modulates the degradation, localization, and activity of these target proteins. Here, we identified gains of ubiquitylation sites in highly conserved regions of human proteins that occurred during human evolution. Results We analyzed human ubiquitylation site data and multiple alignments of orthologous mammalian proteins including those from humans, primates, other placental mammals, opossum, and platypus. In our analysis, we identified 281 ubiquitylation sites in 252 proteins that first appeared along the human lineage during primate evolution: one protein had four novel sites; four proteins had three sites each; 18 proteins had two sites each; and the remaining 229 proteins had one site each. PML, which is involved in neurodevelopment and neurodegeneration, acquired three sites, two of which have been reported to be involved in the degradation of PML. Thirteen human proteins, including ERCC2 (also known as XPD and NBR1, gained human-specific ubiquitylated lysines after the human-chimpanzee divergence. ERCC2 has a Lys/Gln polymorphism, the derived (major allele of which confers enhanced DNA repair capacity and reduced cancer risk compared with the ancestral (minor allele. NBR1 and eight other proteins that are involved in the human autophagy protein interaction network gained a novel ubiquitylation site. Conclusions The gain of novel ubiquitylation sites could be involved in the evolution of protein degradation and other regulatory networks. Although gains of ubiquitylation sites do not necessarily equate to adaptive evolution, they are useful candidates for molecular functional analyses to identify novel advantageous genetic modifications and innovative phenotypes acquired during human evolution.

  14. Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Lerche, Mathilde H.; Poulsen, Flemming Martin

    2001-01-01

    in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found...

  15. Histones and their modifications in ovarian cancer - drivers of disease and therapeutic targets.

    Science.gov (United States)

    Marsh, Deborah J; Shah, Jaynish S; Cole, Alexander J

    2014-01-01

    Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC) is the most common subtype, with the majority of women presenting with advanced disease where 5-year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation, and monoubiquitination, with involvement of enzymes including histone methyltransferases, histone acetyltransferases/deacetylases, and ubiquitin ligases/deubiquitinases, respectively. Complexes such as the Polycomb repressive complex also play roles in the control of histone modifications and more recently roles for long non-coding RNA and microRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here, we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for women with ovarian cancer.

  16. Mining Proteomic Data to Expose Protein Modifications in Methanosarcina mazei strain Gö1

    Directory of Open Access Journals (Sweden)

    Deborah eLeon

    2015-03-01

    Full Text Available Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to mine information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154 proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling. Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more.This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.

  17. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins

    DEFF Research Database (Denmark)

    Diella, F.; Cameron, S.; Gemund, C.

    2004-01-01

    Background: Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a gener...

  18. Age-related carbonylation of fibrocartilage structural proteins drives tissue degenerative modification.

    Science.gov (United States)

    Scharf, Brian; Clement, Cristina C; Yodmuang, Supansa; Urbanska, Aleksandra M; Suadicani, Sylvia O; Aphkhazava, David; Thi, Mia M; Perino, Giorgio; Hardin, John A; Cobelli, Neil; Vunjak-Novakovic, Gordana; Santambrogio, Laura

    2013-07-25

    Aging-related oxidative stress has been linked to degenerative modifications in different organs and tissues. Using redox proteomic analysis and illustrative tandem mass spectrometry mapping, we demonstrate oxidative posttranslational modifications in structural proteins of intervertebral discs (IVDs) isolated from aging mice. Increased protein carbonylation was associated with protein fragmentation and aggregation. Complementing these findings, a significant loss of elasticity and increased stiffness was measured in fibrocartilage from aging mice. Studies using circular dichroism and intrinsic tryptophan fluorescence revealed a significant loss of secondary and tertiary structures of purified collagens following oxidation. Collagen unfolding and oxidation promoted both nonenzymatic and enzymatic degradation. Importantly, induction of oxidative modification in healthy fibrocartilage recapitulated the biochemical and biophysical modifications observed in the aging IVD. Together, these results suggest that protein carbonylation, glycation, and lipoxidation could be early events in promoting IVD degenerative changes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Sidoli, Simone; Haldbo, Simon

    2013-01-01

    Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histo...

  20. Predicting protein amidation sites by orchestrating amino acid sequence features

    Science.gov (United States)

    Zhao, Shuqiu; Yu, Hua; Gong, Xiujun

    2017-08-01

    Amidation is the fourth major category of post-translational modifications, which plays an important role in physiological and pathological processes. Identifying amidation sites can help us understanding the amidation and recognizing the original reason of many kinds of diseases. But the traditional experimental methods for predicting amidation sites are often time-consuming and expensive. In this study, we propose a computational method for predicting amidation sites by orchestrating amino acid sequence features. Three kinds of feature extraction methods are used to build a feature vector enabling to capture not only the physicochemical properties but also position related information of the amino acids. An extremely randomized trees algorithm is applied to choose the optimal features to remove redundancy and dependence among components of the feature vector by a supervised fashion. Finally the support vector machine classifier is used to label the amidation sites. When tested on an independent data set, it shows that the proposed method performs better than all the previous ones with the prediction accuracy of 0.962 at the Matthew's correlation coefficient of 0.89 and area under curve of 0.964.

  1. Histones and their modifications in ovarian cancer – drivers of disease and therapeutic targets

    Directory of Open Access Journals (Sweden)

    Deborah Joy Marsh

    2014-06-01

    Full Text Available Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC is the most common subtype, with the majority of women presenting with advanced disease where 5 year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation and monoubiquitination, with involvement of enzymes including histone methyl transferases (HMTases, histone acetyltransferases/deacetylases and ubiquitin ligases/deubiquitinases respectively. Complexes such as the Polycomb Repressive Complex also play roles in the control of histone modifications and more recently roles for long non-coding (lnc RNA and microRNAs (miRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for

  2. Functional significance of O-GlcNAc modification in regulating neuronal properties.

    Science.gov (United States)

    Hwang, Hongik; Rhim, Hyewhon

    2018-03-01

    Post-translational modifications (PTMs) covalently modify proteins and diversify protein functions. Along with protein phosphorylation, another common PTM is the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine and/or threonine residues. O-GlcNAc modification is similar to phosphorylation in that it occurs to serine and threonine residues and cycles on and off with a similar time scale. However, a striking difference is that the addition and removal of the O-GlcNAc moiety on all substrates are mediated by the two enzymes regardless of proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation can interact or potentially compete with phosphorylation on serine and threonine residues, and thus serves as an important molecular mechanism to modulate protein functions and activation. However, it has been challenging to address the role of O-GlcNAc modification in regulating protein functions at the molecular level due to the lack of convenient tools to determine the sites and degrees of O-GlcNAcylation. Studies in this field have only begun to expand significantly thanks to the recent advances in detection and manipulation methods such as quantitative proteomics and highly selective small-molecule inhibitors for OGT and OGA. Interestingly, multiple brain regions, especially hippocampus, express high levels of both OGT and OGA, and a number of neuron-specific proteins have been reported to undergo O-GlcNAcylation. This review aims to discuss the recent updates concerning the impacts of O-GlcNAc modification on neuronal functions at multiple levels ranging from intrinsic neuronal properties to synaptic plasticity and animal behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

    Science.gov (United States)

    Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Sloan, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth W; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

    2018-01-05

    Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a

  4. An Engineered Version of Human PON2 Opens the Way to Understand the Role of Its Post-Translational Modifications in Modulating Catalytic Activity.

    Directory of Open Access Journals (Sweden)

    Luigi Mandrich

    Full Text Available The human paraoxonase 2 (PON2 has been described as a highly specific lactonase hydrolysing the quorum sensing molecule N-(3-oxododecanoyl-L-homoserine lactone (3OC12-HSL and having secondary esterase but not phosphotriesterase activity, in contrast with the related enzymes PON1 and PON3. It has been suggested that PON2 enzyme activity is dependent on glycosylation and its N-terminal region has been recently demonstrated to be a transmembrane domain mediating association to membranes. In the present study we describe a mutated form of PON2, lacking the above N-terminal region, which has been further stabilized by the insertion of six amino acidic substitutions. The engineered version, hence forth called rPON2, has been over-expressed in E.coli, refolded from inclusion bodies and purified, yielding an enzyme with the same characteristics as the full length enzyme. Therefore the first conclusion of this work was that the catalytic activity is independent from the N-terminus and protein glycosylation. The kinetic characterization confirmed the primary activity on 3OC12-HSL; accordingly, in vitro experiments of inhibition of the biofilm formed by Pseudomonas aeruginosa (PAO1 have demonstrated that rPON2 is more effective than PON1. In addition, we observed small but significant activity against organophosphorothiotes pesticides, m-parathion, coumaphos and malathion.The availability of fair amount of active protein allowed to pinpoint, by mass-spectrometry, ubiquitination of Lys 168 induced in rPON2 by HeLa extract and to correlate such post-translational modification to the modulation of catalytic activity. A mutational analysis of the modified residue confirmed the result.

  5. Tissue-specific expression and post-translational modifications of plant- and bacterial-type phosphoenolpyruvate carboxylase isozymes of the castor oil plant, Ricinus communis L.

    Science.gov (United States)

    O’Leary, Brendan; Fedosejevs, Eric T.; Hill, Allyson T.; Bettridge, James; Park, Joonho; Rao, Srinath K.; Leach, Craig A.; Plaxton, William C.

    2011-01-01

    This study employs transcript profiling together with immunoblotting and co-immunopurification to assess the tissue-specific expression, protein:protein interactions, and post-translational modifications (PTMs) of plant- and bacterial-type phosphoenolpyruvate carboxylase (PEPC) isozymes (PTPC and BTPC, respectively) in the castor plant, Ricinus communis. Previous studies established that the Class-1 PEPC (PTPC homotetramer) of castor oil seeds (COS) is activated by phosphorylation at Ser-11 and inhibited by monoubiquitination at Lys-628 during endosperm development and germination, respectively. Elimination of photosynthate supply to developing COS by depodding caused the PTPC of the endosperm and cotyledon to be dephosphorylated, and then subsequently monoubiquitinated in vivo. PTPC monoubiquitination rather than phosphorylation is widespread throughout the castor plant and appears to be the predominant PTM of Class-1 PEPC that occurs in planta. The distinctive developmental patterns of PTPC phosphorylation versus monoubiquitination indicates that these two PTMs are mutually exclusive. By contrast, the BTPC: (i) is abundant in the inner integument, cotyledon, and endosperm of developing COS, but occurs at low levels in roots and cotyledons of germinated COS, (ii) shows a unique developmental pattern in leaves such that it is present in leaf buds and young expanding leaves, but undetectable in fully expanded leaves, and (iii) tightly interacts with co-expressed PTPC to form the novel and allosterically-desensitized Class-2 PEPC heteromeric complex. BTPC and thus Class-2 PEPC up-regulation appears to be a distinctive feature of rapidly growing and/or biosynthetically active tissues that require a large anaplerotic flux from phosphoenolpyruvate to replenish tricarboxylic acid cycle C-skeletons being withdrawn for anabolism. PMID:21841182

  6. Designing and Producing Modified, New-to-Nature Peptides with Antimicrobial Activity by Use of a Combination of Various Lantibiotic Modification Enzymes

    NARCIS (Netherlands)

    van Heel, Auke J.; Mu, Dongdong; Montalban-Lopez, Manuel; Hendriks, Djoke; Kuipers, Oscar P.

    Lanthipeptides are peptides that contain several post-translationally modified amino acid residues and commonly show considerable antimicrobial activity. After translation, the amino acid residues of these peptides are modified by a distinct set of modification enzymes. This process results in

  7. A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles

    DEFF Research Database (Denmark)

    Wagner, Sebastian Alexander; Beli, Petra; Weinert, Brian Tate

    2011-01-01

    Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single...

  8. Fluorescent polymer-based post-translational differentiation and subtyping of breast cancer cells.

    Science.gov (United States)

    Scott, Michael D; Dutta, Rinku; Haldar, Manas K; Wagh, Anil; Gustad, Thomas R; Law, Benedict; Friesner, Daniel L; Mallik, Sanku

    2012-12-07

    Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were modulated differently in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions.

  9. Managing the complexity of communication: regulation of gap junctions by post-translational modification

    DEFF Research Database (Denmark)

    Axelsen, Lene Nygaard; Callø, Kirstine; von Holstein-Rathlou, Niels-Henrik

    2013-01-01

    Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein...... probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated...

  10. GPS 2.1: enhanced prediction of kinase-specific phosphorylation sites with an algorithm of motif length selection.

    Science.gov (United States)

    Xue, Yu; Liu, Zexian; Cao, Jun; Ma, Qian; Gao, Xinjiao; Wang, Qingqi; Jin, Changjiang; Zhou, Yanhong; Wen, Longping; Ren, Jian

    2011-03-01

    As the most important post-translational modification of proteins, phosphorylation plays essential roles in all aspects of biological processes. Besides experimental approaches, computational prediction of phosphorylated proteins with their kinase-specific phosphorylation sites has also emerged as a popular strategy, for its low-cost, fast-speed and convenience. In this work, we developed a kinase-specific phosphorylation sites predictor of GPS 2.1 (Group-based Prediction System), with a novel but simple approach of motif length selection (MLS). By this approach, the robustness of the prediction system was greatly improved. All algorithms in GPS old versions were also reserved and integrated in GPS 2.1. The online service and local packages of GPS 2.1 were implemented in JAVA 1.5 (J2SE 5.0) and freely available for academic researches at: http://gps.biocuckoo.org.

  11. Identification of sumoylation sites in CCDC6, the first identified RET partner gene in papillary thyroid carcinoma, uncovers a mode of regulating CCDC6 function on CREB1 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Chiara Luise

    Full Text Available CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424 which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.

  12. Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

    Science.gov (United States)

    Medina, Fernando; Quintremil, Sebastian; Alberti, Carolina; Barriga, Andres; Cartier, Luis; Puente, Javier; Ramírez, Eugenio; Ferreira, Arturo; Tanaka, Yuetsu; Valenzuela, Maria Antonieta

    2014-04-01

    The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

  13. Fast Multi-blind Modification Search through Tandem Mass Spectrometry*

    Science.gov (United States)

    Na, Seungjin; Bandeira, Nuno; Paek, Eunok

    2012-01-01

    With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel “multi-blind” spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data. PMID:22186716

  14. Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Tacchi, Jessica L; Raymond, Benjamin B A; Haynes, Paul A; Berry, Iain J; Widjaja, Michael; Bogema, Daniel R; Woolley, Lauren K; Jenkins, Cheryl; Minion, F Chris; Padula, Matthew P; Djordjevic, Steven P

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. © 2016 The Authors.

  15. Chromatin modifications and the DNA damage response to ionizing radiation

    International Nuclear Information System (INIS)

    Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

  16. Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications

    Science.gov (United States)

    2015-01-01

    Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118. PMID:25167464

  17. Evaluation of proteomic search engines for the analysis of histone modifications.

    Science.gov (United States)

    Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C; Garcia, Benjamin A

    2014-10-03

    Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.

  18. Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Christian Gu

    Full Text Available RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.

  19. MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome

    NARCIS (Netherlands)

    Ruprecht, Benjamin; Roesli, Christoph; Lemeer, Simone; Kuster, Bernhard

    2016-01-01

    Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows.

  20. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    DEFF Research Database (Denmark)

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh

    2015-01-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically...

  1. Infection Reveals a Modification of SIRT2 Critical for Chromatin Association

    Directory of Open Access Journals (Sweden)

    Jorge M. Pereira

    2018-04-01

    Full Text Available Summary: Sirtuin 2 is a nicotinamide-adenine-dinucleotide-dependent deacetylase that regulates cell processes such as carcinogenesis, cell cycle, DNA damage, and infection. Subcellular localization of SIRT2 is crucial for its function but is poorly understood. Infection with the bacterial pathogen Listeria monocytogenes, which relocalizes SIRT2 from the cytoplasm to the chromatin, provides an ideal stimulus for the molecular study of this process. In this report, we provide a map of SIRT2 post-translational modification sites and focus on serine 25 phosphorylation. We show that infection specifically induces dephosphorylation of S25, an event essential for SIRT2 chromatin association. Furthermore, we identify a nuclear complex formed by the phosphatases PPM1A and PPM1B, with SIRT2 essential for controlling H3K18 deacetylation and SIRT2-mediated gene repression during infection and necessary for a productive Listeria infection. This study reveals a molecular mechanism regulating SIRT2 function and localization, paving the way for understanding other SIRT2-regulated cellular processes. : Sirtuins are enzymes critical for various processes, including genomic stability, metabolism, and aging. Through study of Listeria monocytogenes, a bacterial pathogen that exploits SIRT2 for productive infection, Pereira et al. uncover a SIRT2 modification necessary for chromatin association and function. Keywords: chromatin, sirtuin, Listeria monocytogenes, phosphorylation, PPM1, histone acetylation, H3K18, infection, subcellular localization

  2. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  3. JASSA: a comprehensive tool for prediction of SUMOylation sites and SIMs.

    Science.gov (United States)

    Beauclair, Guillaume; Bridier-Nahmias, Antoine; Zagury, Jean-François; Saïb, Ali; Zamborlini, Alessia

    2015-11-01

    Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) proteins, a process termed SUMOylation, is involved in many fundamental cellular processes. SUMO proteins are conjugated to a protein substrate, creating an interface for the recruitment of cofactors harboring SUMO-interacting motifs (SIMs). Mapping both SUMO-conjugation sites and SIMs is required to study the functional consequence of SUMOylation. To define the best candidate sites for experimental validation we designed JASSA, a Joint Analyzer of SUMOylation site and SIMs. JASSA is a predictor that uses a scoring system based on a Position Frequency Matrix derived from the alignment of experimental SUMOylation sites or SIMs. Compared with existing web-tools, JASSA displays on par or better performances. Novel features were implemented towards a better evaluation of the prediction, including identification of database hits matching the query sequence and representation of candidate sites within the secondary structural elements and/or the 3D fold of the protein of interest, retrievable from deposited PDB files. JASSA is freely accessible at http://www.jassa.fr/. Website is implemented in PHP and MySQL, with all major browsers supported. guillaume.beauclair@inserm.fr Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  5. Tyrosine sulfation, a post-translational modification of microvillar enzymes in the small intestinal enterocyte

    DEFF Research Database (Denmark)

    Danielsen, E M

    1987-01-01

    Protein sulfation in small intestinal epithelial cells was studied by labelling of organ cultured mucosal explants with [35S]-sulfate. Six bands in SDS-PAGE became selectively labelled; four, of 250, 200, 166 and 130 kd, were membrane-bound and two, of 75 and 60 kd, were soluble. The sulfated mem...... sulfated. Most if not all the sulfate was bound to tyrosine residues rather than to the carbohydrate of the microvillar enzymes, showing that this type of modification can occur on plasma membrane proteins as well as on secretory proteins....

  6. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Lyon, David; Young, Clifford

    2017-01-01

    that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

  7. A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry

    Science.gov (United States)

    Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has b...

  8. Modification and characterization of sites giving acid cracking on aluminium oxide supports; Modification et caracterisation des sites responsables du craquage acide sur des supports a base d`alumine

    Energy Technology Data Exchange (ETDEWEB)

    Guillaume, D

    1997-10-23

    The aim of this work is to characterize the surface acid properties of {gamma} alumina, especially the nature, the amount, the strength and the environment of acid sites. The techniques that have been used are: the thermo-desorption of trimethyl-phosphine followed by {sup 31}P MAS NMR, the infrared spectroscopy, the n-heptane cracking, the modification of {gamma} alumina by the addition of chlorine, silicon and alkali (K{sup +}, Na{sup +}), alkaline-earths (Mg{sup 2+}, Ca{sup 2+}), lanthanum (La{sup 3+}) cations. The combination of these techniques has allowed to identify the surface acid properties of {gamma} alumina through the n-heptane transformation reactions under reforming conditions. We have shown that Lewis acidity is responsible for cracking reactions leading to C{sub 1}-C{sub 6} and C{sub 2}-C{sub 5} whereas the cracking of n-heptane producing C{sub 3}-C{sub 4} is due to Broensted acid sites. The isomerization and cyclization reactions both require weaker Broensted acid sites than cracking leading to C{sub 3}-C{sub 4}. The mechanisms involved in the formation of products of these reactions have been identified. Despite the complexity of surface acid properties of {gamma} alumina, this study has determined the environment of catalytically active sites, considering the presence of cation vacancies. (author) 206 refs.

  9. Detection of site specific glycosylation in proteins using flow cytometry†

    Science.gov (United States)

    Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

    2009-01-01

    We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

  10. Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

    Science.gov (United States)

    Strader, Michael Brad; Hicks, Wayne A; Kassa, Tigist; Singleton, Eileen; Soman, Jayashree; Olson, John S; Weiss, Mitchell J; Mollan, Todd L; Wilson, Michael T; Alayash, Abdu I

    2014-08-08

    A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

    2015-05-15

    High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

  12. Proteomic analysis of the cyanobacterium of the Azolla symbiosis: identity, adaptation, and NifH modification.

    Science.gov (United States)

    Ekman, Martin; Tollbäck, Petter; Bergman, Birgitta

    2008-01-01

    Cyanobacteria are able to form stable nitrogen-fixing symbioses with diverse eukaryotes. To extend our understanding of adaptations imposed by plant hosts, two-dimensional gel electrophoresis and mass spectrometry (MS) were used for comparative protein expression profiling of a cyanobacterium (cyanobiont) dwelling in leaf cavities of the water-fern Azolla filiculoides. Homology-based protein identification using peptide mass fingerprinting [matrix-assisted laser desorption ionization-time of flight (MALDI-TOF-MS)], tandem MS analyses, and sequence homology searches resulted in an identification success rate of 79% of proteins analysed in the unsequenced cyanobiont. Compared with a free-living strain, processes related to energy production, nitrogen and carbon metabolism, and stress-related functions were up-regulated in the cyanobiont while photosynthesis and metabolic turnover rates were down-regulated, stressing a slow heterotrophic mode of growth, as well as high heterocyst frequencies and nitrogen-fixing capacities. The first molecular data set on the nature of the NifH post-translational modification in cyanobacteria was also obtained: peptide mass spectra of the protein demonstrated the presence of a 300-400 Da protein modification localized to a specific 13 amino acid sequence, within the part of the protein that is ADP-ribosylated in other bacteria and close to the active site of nitrogenase. Furthermore, the distribution of the highest scoring database hits for the identified proteins points to the possibility of using proteomic data in taxonomy.

  13. Identification of histone modifications in biomedical text for supporting epigenomic research.

    Science.gov (United States)

    Kolárik, Corinna; Klinger, Roman; Hofmann-Apitius, Martin

    2009-01-30

    Posttranslational modifications of histones influence the structure of chromatine and in such a way take part in the regulation of gene expression. Certain histone modification patterns, distributed over the genome, are connected to cell as well as tissue differentiation and to the adaption of organisms to their environment. Abnormal changes instead influence the development of disease states like cancer. The regulation mechanisms for modifying histones and its functionalities are the subject of epigenomics investigation and are still not completely understood. Text provides a rich resource of knowledge on epigenomics and modifications of histones in particular. It contains information about experimental studies, the conditions used, and results. To our knowledge, no approach has been published so far for identifying histone modifications in text. We have developed an approach for identifying histone modifications in biomedical literature with Conditional Random Fields (CRF) and for resolving the recognized histone modification term variants by term standardization. For the term identification F1 measures of 0.84 by 10-fold cross-validation on the training corpus and 0.81 on an independent test corpus have been obtained. The standardization enabled the correct transformation of 96% of the terms from training and 98% from test the corpus. Due to the lack of terminologies exhaustively covering specific histone modification types, we developed a histone modification term hierarchy for use in a semantic text retrieval system. The developed approach highly improves the retrieval of articles describing histone modifications. Since text contains context information about performed studies and experiments, the identification of histone modifications is the basis for supporting literature-based knowledge discovery and hypothesis generation to accelerate epigenomic research.

  14. Application of the MIDAS approach for analysis of lysine acetylation sites.

    Science.gov (United States)

    Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

    2013-01-01

    Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation.

  15. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation

    Directory of Open Access Journals (Sweden)

    Cody Caba

    2018-02-01

    Full Text Available Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI, the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys57 and Lys401 of human PDI in vitro. Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys57 and Lys401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin. A total of 28 acetyllysine residues were identified, including acLys57 and acLys401. The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  16. Conserved Residues Lys57 and Lys401 of Protein Disulfide Isomerase Maintain an Active Site Conformation for Optimal Activity: Implications for Post-Translational Regulation.

    Science.gov (United States)

    Caba, Cody; Ali Khan, Hyder; Auld, Janeen; Ushioda, Ryo; Araki, Kazutaka; Nagata, Kazuhiro; Mutus, Bulent

    2018-01-01

    Despite its study since the 1960's, very little is known about the post-translational regulation of the multiple catalytic activities performed by protein disulfide isomerase (PDI), the primary protein folding catalyst of the cell. This work identifies a functional role for the highly conserved CxxC-flanking residues Lys 57 and Lys 401 of human PDI in vitro . Mutagenesis studies have revealed these residues as modulating the oxidoreductase activity of PDI in a pH-dependent manner. Non-conservative amino acid substitutions resulted in enzyme variants upwards of 7-fold less efficient. This attenuated activity was found to translate into a 2-fold reduction of the rate of electron shuttling between PDI and the intraluminal endoplasmic reticulum oxidase, ERO1α, suggesting a functional significance to oxidative protein folding. In light of this, the possibility of lysine acetylation at residues Lys 57 and Lys 401 was assessed by in vitro treatment using acetylsalicylic acid (aspirin). A total of 28 acetyllysine residues were identified, including acLys 57 and acLys 401 . The kinetic behavior of the acetylated protein form nearly mimicked that obtained with a K57/401Q double substitution variant providing an indication that acetylation of the active site-flanking lysine residues can act to reversibly modulate PDI activity.

  17. Covalent protein modification with ISG15 via a conserved cysteine in the hinge region.

    Directory of Open Access Journals (Sweden)

    Veronika N Bade

    Full Text Available The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls, ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation. ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no

  18. Regulation of multispanning membrane protein topology via post-translational annealing.

    Science.gov (United States)

    Van Lehn, Reid C; Zhang, Bin; Miller, Thomas F

    2015-09-26

    The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

  19. Extracellular and Intracellular Cyclophilin A, Native and Post-Translationally Modified, Show Diverse and Specific Pathological Roles in Diseases.

    Science.gov (United States)

    Xue, Chao; Sowden, Mark P; Berk, Bradford C

    2018-05-01

    CypA (cyclophilin A) is a ubiquitous and highly conserved protein with peptidyl prolyl isomerase activity. Because of its highly abundant level in the cytoplasm, most studies have focused on the roles of CypA as an intracellular protein. However, emerging evidence suggests an important role for extracellular CypA in the pathogenesis of several diseases through receptor (CD147 or other)-mediated autocrine and paracrine signaling pathways. In this review, we will discuss the shared and unique pathological roles of extracellular and intracellular CypA in human cardiovascular diseases. In addition, the evolving role of post-translational modifications of CypA in the pathogenesis of disease is discussed. Finally, recent studies with drugs specific for extracellular CypA show its importance in disease pathogenesis in several animal models and make extracellular CypA a new therapeutic target. © 2018 American Heart Association, Inc.

  20. Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

    Science.gov (United States)

    Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

    2008-05-30

    Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

  1. Success: evolutionary and structural properties of amino acids prove effective for succinylation site prediction.

    Science.gov (United States)

    López, Yosvany; Sharma, Alok; Dehzangi, Abdollah; Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Sattar, Abdul; Tsunoda, Tatsuhiko

    2018-01-19

    Post-translational modification is considered an important biological mechanism with critical impact on the diversification of the proteome. Although a long list of such modifications has been studied, succinylation of lysine residues has recently attracted the interest of the scientific community. The experimental detection of succinylation sites is an expensive process, which consumes a lot of time and resources. Therefore, computational predictors of this covalent modification have emerged as a last resort to tackling lysine succinylation. In this paper, we propose a novel computational predictor called 'Success', which efficiently uses the structural and evolutionary information of amino acids for predicting succinylation sites. To do this, each lysine was described as a vector that combined the above information of surrounding amino acids. We then designed a support vector machine with a radial basis function kernel for discriminating between succinylated and non-succinylated residues. We finally compared the Success predictor with three state-of-the-art predictors in the literature. As a result, our proposed predictor showed a significant improvement over the compared predictors in statistical metrics, such as sensitivity (0.866), accuracy (0.838) and Matthews correlation coefficient (0.677) on a benchmark dataset. The proposed predictor effectively uses the structural and evolutionary information of the amino acids surrounding a lysine. The bigram feature extraction approach, while retaining the same number of features, facilitates a better description of lysines. A support vector machine with a radial basis function kernel was used to discriminate between modified and unmodified lysines. The aforementioned aspects make the Success predictor outperform three state-of-the-art predictors in succinylation detection.

  2. Prediction of citrullination sites by incorporating k-spaced amino acid pairs into Chou's general pseudo amino acid composition.

    Science.gov (United States)

    Ju, Zhe; Wang, Shi-Yun

    2018-04-22

    As one of the most important and common protein post-translational modifications, citrullination plays a key role in regulating various biological processes and is associated with several human diseases. The accurate identification of citrullination sites is crucial for elucidating the underlying molecular mechanisms of citrullination and designing drugs for related human diseases. In this study, a novel bioinformatics tool named CKSAAP_CitrSite is developed for the prediction of citrullination sites. With the assistance of support vector machine algorithm, the highlight of CKSAAP_CitrSite is to adopt the composition of k-spaced amino acid pairs surrounding a query site as input. As illustrated by 10-fold cross-validation, CKSAAP_CitrSite achieves a satisfactory performance with a Sensitivity of 77.59%, a Specificity of 95.26%, an Accuracy of 89.37% and a Matthew's correlation coefficient of 0.7566, which is much better than those of the existing prediction method. Feature analysis shows that the N-terminal space containing pairs may play an important role in the prediction of citrullination sites, and the arginines close to N-terminus tend to be citrullinated. The conclusions derived from this study could offer useful information for elucidating the molecular mechanisms of citrullination and related experimental validations. A user-friendly web-server for CKSAAP_CitrSite is available at 123.206.31.171/CKSAAP_CitrSite/. Copyright © 2017. Published by Elsevier B.V.

  3. Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the O-linked N-acetylglucosamine transferase (OGT).

    Science.gov (United States)

    Bauer, Christina; Göbel, Klaus; Nagaraj, Nagarjuna; Colantuoni, Christian; Wang, Mengxi; Müller, Udo; Kremmer, Elisabeth; Rottach, Andrea; Leonhardt, Heinrich

    2015-02-20

    TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Identification of Protein Pupylation Sites Using Bi-Profile Bayes Feature Extraction and Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Xiaowei Zhao

    2013-01-01

    Full Text Available Pupylation, one of the most important posttranslational modifications of proteins, typically takes place when prokaryotic ubiquitin-like protein (Pup is attached to specific lysine residues on a target protein. Identification of pupylation substrates and their corresponding sites will facilitate the understanding of the molecular mechanism of pupylation. Comparing with the labor-intensive and time-consuming experiment approaches, computational prediction of pupylation sites is much desirable for their convenience and fast speed. In this study, a new bioinformatics tool named EnsemblePup was developed that used an ensemble of support vector machine classifiers to predict pupylation sites. The highlight of EnsemblePup was to utilize the Bi-profile Bayes feature extraction as the encoding scheme. The performance of EnsemblePup was measured with a sensitivity of 79.49%, a specificity of 82.35%, an accuracy of 85.43%, and a Matthews correlation coefficient of 0.617 using the 5-fold cross validation on the training dataset. When compared with other existing methods on a benchmark dataset, the EnsemblePup provided better predictive performance, with a sensitivity of 80.00%, a specificity of 83.33%, an accuracy of 82.00%, and a Matthews correlation coefficient of 0.629. The experimental results suggested that EnsemblePup presented here might be useful to identify and annotate potential pupylation sites in proteins of interest. A web server for predicting pupylation sites was developed.

  5. Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Wagner, Sebastian A; Horn, Heiko

    2011-01-01

    Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines....... With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites...... between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines...

  6. ICRPfinder: a fast pattern design algorithm for coding sequences and its application in finding potential restriction enzyme recognition sites

    Directory of Open Access Journals (Sweden)

    Stafford Phillip

    2009-09-01

    Full Text Available Abstract Background Restriction enzymes can produce easily definable segments from DNA sequences by using a variety of cut patterns. There are, however, no software tools that can aid in gene building -- that is, modifying wild-type DNA sequences to express the same wild-type amino acid sequences but with enhanced codons, specific cut sites, unique post-translational modifications, and other engineered-in components for recombinant applications. A fast DNA pattern design algorithm, ICRPfinder, is provided in this paper and applied to find or create potential recognition sites in target coding sequences. Results ICRPfinder is applied to find or create restriction enzyme recognition sites by introducing silent mutations. The algorithm is shown capable of mapping existing cut-sites but importantly it also can generate specified new unique cut-sites within a specified region that are guaranteed not to be present elsewhere in the DNA sequence. Conclusion ICRPfinder is a powerful tool for finding or creating specific DNA patterns in a given target coding sequence. ICRPfinder finds or creates patterns, which can include restriction enzyme recognition sites, without changing the translated protein sequence. ICRPfinder is a browser-based JavaScript application and it can run on any platform, in on-line or off-line mode.

  7. Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling

    Czech Academy of Sciences Publication Activity Database

    Doubravská, Lenka; Krausová, Michaela; Gradl, D.; Vojtěchová, Martina; Tůmová, Lucie; Lukáš, Jan; Valenta, Tomáš; Pospíchalová, Vendula; Fafílek, Bohumil; Plachý, Jiří; Sebesta, O.; Kořínek, Vladimír

    2011-01-01

    Roč. 23, č. 5 (2011), s. 837-848 ISSN 0898-6568 R&D Projects: GA ČR(CZ) GA204/07/1567; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50520514 Keywords : Wnt signaling * post-translational modification * acylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.058, year: 2011

  8. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. PLMD: An updated data resource of protein lysine modifications.

    Science.gov (United States)

    Xu, Haodong; Zhou, Jiaqi; Lin, Shaofeng; Deng, Wankun; Zhang, Ying; Xue, Yu

    2017-05-20

    Post-translational modifications (PTMs) occurring at protein lysine residues, or protein lysine modifications (PLMs), play critical roles in regulating biological processes. Due to the explosive expansion of the amount of PLM substrates and the discovery of novel PLM types, here we greatly updated our previous studies, and presented a much more integrative resource of protein lysine modification database (PLMD). In PLMD, we totally collected and integrated 284,780 modification events in 53,501 proteins across 176 eukaryotes and prokaryotes for up to 20 types of PLMs, including ubiquitination, acetylation, sumoylation, methylation, succinylation, malonylation, glutarylation, glycation, formylation, hydroxylation, butyrylation, propionylation, crotonylation, pupylation, neddylation, 2-hydroxyisobutyrylation, phosphoglycerylation, carboxylation, lipoylation and biotinylation. Using the data set, a motif-based analysis was performed for each PLM type, and the results demonstrated that different PLM types preferentially recognize distinct sequence motifs for the modifications. Moreover, various PLMs synergistically orchestrate specific cellular biological processes by mutual crosstalks with each other, and we totally found 65,297 PLM events involved in 90 types of PLM co-occurrences on the same lysine residues. Finally, various options were provided for accessing the data, while original references and other annotations were also present for each PLM substrate. Taken together, we anticipated the PLMD database can serve as a useful resource for further researches of PLMs. PLMD 3.0 was implemented in PHP + MySQL and freely available at http://plmd.biocuckoo.org. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  10. Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

    Directory of Open Access Journals (Sweden)

    Tobias D Schneider

    Full Text Available Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.

  11. Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha

    International Nuclear Information System (INIS)

    Whiteheart, S.W.; Shenbagamurthi, P.; Chen, L.; Cotter, R.J.; Hart, G.W.

    1989-01-01

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with [ 3 H] ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major [ 3 H]ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release [ 3 H]ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues

  12. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    Science.gov (United States)

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  13. Permit application modifications

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-11-01

    This document contains the Permit Application Modifications for the Y-12 Industrial Landfill V site on the Oak Ridge Reservation. These modifications include the assessment of stability of the proposed Landfill V under static and loading conditions. Analyses performed include the general slope stability, veneer stability of the bottom liner and cover system, and a liquefaction potential assessment of the foundation soils.

  14. Permit application modifications

    International Nuclear Information System (INIS)

    1995-11-01

    This document contains the Permit Application Modifications for the Y-12 Industrial Landfill V site on the Oak Ridge Reservation. These modifications include the assessment of stability of the proposed Landfill V under static and loading conditions. Analyses performed include the general slope stability, veneer stability of the bottom liner and cover system, and a liquefaction potential assessment of the foundation soils

  15. Enzymes Involved in AMPylation and deAMPylation.

    Science.gov (United States)

    Casey, Amanda K; Orth, Kim

    2018-02-14

    Posttranslational modifications are covalent changes made to proteins that typically alter the function or location of the protein. AMPylation is an emerging posttranslational modification that involves the addition of adenosine monophosphate (AMP) to a protein. Like other, more well-studied posttranslational modifications, AMPylation is predicted to regulate the activity of the modified target proteins. However, the scope of this modification both in bacteria and in eukaryotes remains to be fully determined. In this review, we provide an up to date overview of the known AMPylating enzymes, the regulation of these enzymes, and the effect of this modification on target proteins.

  16. In silico modeling of lipase H | Jabeen | African Journal of ...

    African Journals Online (AJOL)

    LAH 2 is a type of autosomal recessive hypotrichosis that affect hairs, eyebrows, scalp and eyelashes. Mutations in Lipase H gene result in LAH 2. Changes that result from mutation on physiochemical properties, post-translational modifications, functional sites, secondary structure and tertiary structure lipase H gene (LIPH) ...

  17. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation

    DEFF Research Database (Denmark)

    Weinert, Brian T; Schölz, Christian; Wagner, Sebastian A

    2013-01-01

    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), hu...

  18. Savannah River Site reactor hardware design modification study

    International Nuclear Information System (INIS)

    Fisher, J.E.

    1990-01-01

    A study was undertaken to assess the merits of proposed design modifications to the Savannah River Site (SRS) reactors. The evaluation was based on the responses calculated by the RELAP5 systems code to double-ended guillotine break loss-of-coolant-accidents (DEGB LOCAs). The three concepts evaluated were (a) elevated plenum inlet piping with a guard vessel and clamshell enclosures, (b) closure of both rotovalves in the affected loop, and (c) closure of the pump suction valve in the affected loop. Each concept included a fast reactor shutdown (to 65% power in 100 ms) and a 2-s ac pump trip. System recovery potential was evaluated for break locations at the pump suction, the pump discharge, and the plenum inlet. The code version used was RELAP5/MOD2.5 version 3d3, a preliminary version of RELAP5/MOD3. The model was a three-dimensional representation of the K-Reactor water plenum and moderator tank. It included explicit representations of all six loops, which were based on the configuration of L-Reactor. A combination of features is recommended to ensure liquid inventory recovery for all break locations. Valve closure design performance for a break location in the short section of piping between the reactor concrete shield and the pump suction valve would benefit from the clamshell enclosing that section of piping. 7 refs., 10 figs., 2 tabs

  19. Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

    Science.gov (United States)

    Zimnik, Susan; Gaestel, Matthias; Niedenthal, Rainer

    2009-03-01

    Post-translational modifications control the physiological activity of the signal transducer and activator of transcription STAT1. While phosphorylation at tyrosine Y701 is a prerequisite for STAT1 dimerization, its SUMOylation represses the transcriptional activity. Recently, we have demonstrated that SUMOylation at lysine K703 inhibits the phosphorylation of nearby localized Y701 of STAT1. Here, we analysed the influence of phosphorylation of Y701 on SUMOylation of K703 in vivo. For that reason, an Ubc9/substrate dimerization-dependent SUMOylation (USDDS) system was developed, which consists of fusions of the SUMOylation substrate and of the SUMO-conjugating enzyme Ubc9 to the chemically activatable heterodimerization domains FKBP and FRB, respectively. When FKBP fusion proteins of STAT1, p53, CRSP9, FOS, CSNK2B, HES1, TCF21 and MYF6 are coexpressed with Ubc9-FRB, treatment of HEK293 cells with the rapamycin-related dimerizer compound AP21967 induces SUMOylation of these proteins in vivo. For STAT1-FKBP and p53-FKBP we show that this SUMOylation takes place at their specific SUMOylation sites in vivo. Using USDDS, we then demonstrate that STAT1 phosphorylation at Y701 induced by interferon-beta treatment inhibits SUMOylation of K703 in vivo. Thus, pY701 and SUMO-K703 of STAT1 represent mutually exclusive modifications, which prevent signal integration at this molecule and probably ensure the existence of differentially modified subpopulations of STAT1 necessary for its regulated nuclear cytoplasmic activation/inactivation cycle.

  20. Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Mowei; Paša-Tolić, Ljiljana; Stenoien, David L.

    2016-12-21

    Histones play central roles in most chromosomal functions and both their basic biology and roles in disease have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here, we used state of the art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as search engine, and LcMsSpectator as a data visualization tool. ProMex sums across retention time to maximize sensitivity and accuracy for low abundance species in MS1deconvolution. MSPathFinder searches the MS2 data against protein sequence databases with user-defined modifications. LcMsSpectator presents the results from ProMex and MSPathFinder in a format that allows quick manual evaluation of critical attributes for high-confidence identifications. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

  1. Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

    Science.gov (United States)

    2015-01-01

    Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

  2. Epigenetic and Posttranslational Modifications in Light Signal Transduction and the Circadian Clock in Neurospora crassa

    Directory of Open Access Journals (Sweden)

    Marco Proietto

    2015-07-01

    Full Text Available Blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. One of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. Circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. Neurospora crassa provides an excellent model for studying the molecular mechanisms involved in these phenomena. The White Collar Complex (WCC, a blue-light receptor and transcription factor of the circadian oscillator, and Frequency (FRQ, the circadian clock pacemaker, are at the core of the Neurospora circadian system. The eukaryotic circadian clock relies on transcriptional/translational feedback loops: some proteins rhythmically repress their own synthesis by inhibiting the activity of their transcriptional factors, generating self-sustained oscillations over a period of about 24 h. One of the basic mechanisms that perpetuate self-sustained oscillations is post translation modification (PTM. The acronym PTM generically indicates the addition of acetyl, methyl, sumoyl, or phosphoric groups to various types of proteins. The protein can be regulatory or enzymatic or a component of the chromatin. PTMs influence protein stability, interaction, localization, activity, and chromatin packaging. Chromatin modification and PTMs have been implicated in regulating circadian clock function in Neurospora. Research into the epigenetic control of transcription factors such as WCC has yielded new insights into the temporal modulation of light-dependent gene transcription. Here we report on epigenetic and protein PTMs in the regulation of the Neurospora crassa circadian clock. We also present a model that illustrates the molecular mechanisms at the basis of the blue light control of the circadian clock.

  3. Modifications to the Remedial Action Plan and site design for stabilization of the inactive uranium mill tailings site at Green River, Utah

    International Nuclear Information System (INIS)

    1991-10-01

    This modification to the Green River Final Remedial Action Plan (FRAP) represents the changes made to the document in accordance with a joint agreement between the US Department of Energy (DOE) and the US Nuclear Regulatory Commission (NRC) outlined in a letter dated August 7, 1991. As specified in this letter, methylene chloride will no longer be analyzed in groundwater samples collected from on-site monitor wells. All references to methylene chloride sampling have been deleted from the FRAP, as indicated by the pages in Section 2.0 of this document

  4. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    OpenAIRE

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

  5. Computational Identification of Protein Pupylation Sites by Using Profile-Based Composition of k-Spaced Amino Acid Pairs.

    Directory of Open Access Journals (Sweden)

    Md Mehedi Hasan

    Full Text Available Prokaryotic proteins are regulated by pupylation, a type of post-translational modification that contributes to cellular function in bacterial organisms. In pupylation process, the prokaryotic ubiquitin-like protein (Pup tagging is functionally analogous to ubiquitination in order to tag target proteins for proteasomal degradation. To date, several experimental methods have been developed to identify pupylated proteins and their pupylation sites, but these experimental methods are generally laborious and costly. Therefore, computational methods that can accurately predict potential pupylation sites based on protein sequence information are highly desirable. In this paper, a novel predictor termed as pbPUP has been developed for accurate prediction of pupylation sites. In particular, a sophisticated sequence encoding scheme [i.e. the profile-based composition of k-spaced amino acid pairs (pbCKSAAP] is used to represent the sequence patterns and evolutionary information of the sequence fragments surrounding pupylation sites. Then, a Support Vector Machine (SVM classifier is trained using the pbCKSAAP encoding scheme. The final pbPUP predictor achieves an AUC value of 0.849 in 10-fold cross-validation tests and outperforms other existing predictors on a comprehensive independent test dataset. The proposed method is anticipated to be a helpful computational resource for the prediction of pupylation sites. The web server and curated datasets in this study are freely available at http://protein.cau.edu.cn/pbPUP/.

  6. Measuring site occupancy

    DEFF Research Database (Denmark)

    Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James

    2014-01-01

    occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass...... peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise...... and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response....

  7. FeatureMap3D - a tool to map protein features and sequence conservation onto homologous structures in the PDB

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Rapacki, Krzysztof; Stærfeldt, Hans Henrik

    2006-01-01

    FeatureMap3D is a web-based tool that maps protein features onto 3D structures. The user provides sequences annotated with any feature of interest, such as post-translational modifications, protease cleavage sites or exonic structure and FeatureMap3D will then search the Protein Data Bank (PDB) f...

  8. mRNA Display Selection of a High-Affinity, Modification-Specific Phospho-IκBα-Binding Fibronectin

    Science.gov (United States)

    Olson, C. Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W.

    2009-01-01

    The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IκBα. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IκBα peptide with Kd = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IκBα from mammalian cell extract and stabilizes phospho-IκBα in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IκB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors. PMID:18590330

  9. mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin.

    Science.gov (United States)

    Olson, C Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W

    2008-08-15

    The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IkappaBalpha. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IkappaBalpha peptide with K d = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IkappaBalpha from mammalian cell extract and stabilizes phospho-IkappaBalpha in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IkappaB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.

  10. The impact of chromatin modification on the development of chronic complications in patients with diabetes

    Directory of Open Access Journals (Sweden)

    Małgorzata Wegner

    2015-08-01

    Full Text Available Diabetes is a chronic, metabolic disease. Over 347 million people worldwide have diabetes. Chronic complications (retinopathy, nephropathy or neuropathy are the major dangerous outcome of this disease. Recent studies indicate a significant role of epigenetic regulation in the development of chronic complications in patients with diabetes. Hyperglycemia could cause abnormal regulation of the activity of enzymes participating in the post-translational histone modifications (PTHMs and initiation of changes in patterns of DNA methylation. It leads to modification of chromatin structure. These epigenetic abnormalities result in changes in the expression of genes involved in development of chronic inflammation, such as NF-KAPPAB (nuclear factor kappaB gene, TNFα (tumor necrosis factor a gene, IL6 (interleukin 6 gene or MCP1 (monocyte chemoattractant protein 1 gene. It enhances endothelial cell dysfunction, which plays an important role in development of chronic, diabetic complications. In addition, caused by hyperglycemia epigenetic modifications changes in structure of chromatin explains “metabolic memory”, a phenomenon of presence of pathological pathways related to the prolonged hyperglycemia in the past, despite maintaining good metabolic control later on.

  11. Safe genetic modification of cardiac stem cells using a site-specific integration technique.

    Science.gov (United States)

    Lan, Feng; Liu, Junwei; Narsinh, Kazim H; Hu, Shijun; Han, Leng; Lee, Andrew S; Karow, Marisa; Nguyen, Patricia K; Nag, Divya; Calos, Michele P; Robbins, Robert C; Wu, Joseph C

    2012-09-11

    Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (Pmodification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

  12. Predicting sumoylation sites using support vector machines based on various sequence features, conformational flexibility and disorder.

    Science.gov (United States)

    Yavuz, Ahmet Sinan; Sezerman, Osman Ugur

    2014-01-01

    Sumoylation, which is a reversible and dynamic post-translational modification, is one of the vital processes in a cell. Before a protein matures to perform its function, sumoylation may alter its localization, interactions, and possibly structural conformation. Abberations in protein sumoylation has been linked with a variety of disorders and developmental anomalies. Experimental approaches to identification of sumoylation sites may not be effective due to the dynamic nature of sumoylation, laborsome experiments and their cost. Therefore, computational approaches may guide experimental identification of sumoylation sites and provide insights for further understanding sumoylation mechanism. In this paper, the effectiveness of using various sequence properties in predicting sumoylation sites was investigated with statistical analyses and machine learning approach employing support vector machines. These sequence properties were derived from windows of size 7 including position-specific amino acid composition, hydrophobicity, estimated sub-window volumes, predicted disorder, and conformational flexibility. 5-fold cross-validation results on experimentally identified sumoylation sites revealed that our method successfully predicts sumoylation sites with a Matthew's correlation coefficient, sensitivity, specificity, and accuracy equal to 0.66, 73%, 98%, and 97%, respectively. Additionally, we have showed that our method compares favorably to the existing prediction methods and basic regular expressions scanner. By using support vector machines, a new, robust method for sumoylation site prediction was introduced. Besides, the possible effects of predicted conformational flexibility and disorder on sumoylation site recognition were explored computationally for the first time to our knowledge as an additional parameter that could aid in sumoylation site prediction.

  13. Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.

    Science.gov (United States)

    Arnaudo, Anna M; Link, A James; Garcia, Benjamin A

    2016-03-18

    The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers. Here, we present a method that merges bioorthogonal chemistry with mass spectrometry for the study of modifications on newly synthesized histones in mammalian cells. HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine and heavy (13)C6,(15)N4 isotope labeled arginine. Heavy amino acid labeling marks newly synthesized histones while azidohomoalanine incorporation allows for their isolation using bioorthogonal ligation. Labeled mononucleosomes were covalently linked via a copper catalyzed reaction to a FLAG-GGR-alkyne peptide, immunoprecipitated, and subjected to mass spectrometry for quantitative modification analysis. Mononucleosomes containing new histones were successfully isolated using this approach. Additionally, the development of this method highlights the potential deleterious effects of azidohomoalanine labeling on protein PTMs and cell cycle progression, which should be considered for future studies utilizing bioorthogonal labeling strategies in mammalian cells.

  14. Site-selective modification of peptides: From "customizable units" to novel α-aryl and α-alkyl glycine derivatives, and components of branched peptides.

    Science.gov (United States)

    Romero-Estudillo, Iván; Saavedra, Carlos; Boto, Alicia; Álvarez, Eleuterio

    2015-09-01

    The creation of peptide libraries by site-selective modification of a few peptide substrates would increase the efficiency of discovery processes, but still is a real synthetic challenge. The site-selective modification of small peptides at serine or threonine residues, by using a short scission-addition procedure, allows the preparation of peptides with unnatural α-aryl glycines. In a similar way, the scission of hydroxyproline residues is the key step in the production of optically pure α-alkyl glycines which are precursors or components of branched peptides. With these versatile processes, a single peptide can be transformed into a variety of peptide derivatives. The process takes place under mild conditions, and good global yields are obtained. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 650-662, 2015. © 2015 Wiley Periodicals, Inc.

  15. Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Haselmann, Kim F; Budnik, Bogdan A

    2003-01-01

    the PTM site. Chromatographic peak analysis continues until full sequence coverage is obtained, after which the molecular mass is reconstructed and compared with the measured value. An agreement indicates that the PTM characterization was complete. This procedure applied to the bovine milk PP3 protein...

  16. Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

    Science.gov (United States)

    Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

    2010-12-01

    Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models

  17. Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

    Energy Technology Data Exchange (ETDEWEB)

    Fardini, Yann [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France); Perez-Cervera, Yobana [Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d' Ascq (France); Facultad de Odontología, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca (Mexico); Camoin, Luc [INSERM, U1068, CRCM, Marseille Protéomique IBiSA, Marseille, F-13009 (France); Institut Paoli-Calmettes Team, Cell Polarity, Cell Signaling and Cancer, Marseille, F-13009 (France); Aix-Marseille Université, F-13284, Marseille (France); CNRS, UMR7258, CRCM, Marseille, F-13009 (France); Pagesy, Patrick [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France); Lefebvre, Tony [Structural and Functional Glycobiology Unit, Lille 1 University, CNRS (UMR 8576), IFR 117, Villeneuve d' Ascq (France); Issad, Tarik, E-mail: tarik.issad@inserm.fr [INSERM, U1016, Institut Cochin, Paris (France); CNRS, UMR8104, Paris (France); Université Paris Descartes, Sorbonne Paris Cité, Paris (France)

    2015-06-26

    O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified. - Highlights: • We mutate four previously identified O-GlcNAcylation sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity.

  18. Regulatory O-GlcNAcylation sites on FoxO1 are yet to be identified

    International Nuclear Information System (INIS)

    Fardini, Yann; Perez-Cervera, Yobana; Camoin, Luc; Pagesy, Patrick; Lefebvre, Tony; Issad, Tarik

    2015-01-01

    O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified. - Highlights: • We mutate four previously identified O-GlcNAcylation sites on Foxo1. • Unexpectedly, these mutations do not reduce Foxo1 O-GlcNAcylation. • These mutation do not reduce Foxo1 transcriptional activity. • We identify a new O-GlcNAcylation site on Foxo1 by mass spectrometry. • Mutation of this site increases Foxo1 transcriptional activity

  19. SNOSite: exploiting maximal dependence decomposition to identify cysteine S-nitrosylation with substrate site specificity.

    Directory of Open Access Journals (Sweden)

    Tzong-Yi Lee

    Full Text Available S-nitrosylation, the covalent attachment of a nitric oxide to (NO the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-nitrosylation remains unknown. Based on a total of 586 experimentally identified S-nitrosylation sites from SNAP/L-cysteine-stimulated mouse endothelial cells, this work presents an informatics investigation on S-nitrosylation sites including structural factors such as the flanking amino acids composition, the accessible surface area (ASA and physicochemical properties, i.e. positive charge and side chain interaction parameter. Due to the difficulty to obtain the conserved motifs by conventional motif analysis, maximal dependence decomposition (MDD has been applied to obtain statistically significant conserved motifs. Support vector machine (SVM is applied to generate predictive model for each MDD-clustered motif. According to five-fold cross-validation, the MDD-clustered SVMs could achieve an accuracy of 0.902, and provides a promising performance in an independent test set. The effectiveness of the model was demonstrated on the correct identification of previously reported S-nitrosylation sites of Bos taurus dimethylarginine dimethylaminohydrolase 1 (DDAH1 and human hemoglobin subunit beta (HBB. Finally, the MDD-clustered model was adopted to construct an effective web-based tool, named SNOSite (http://csb.cse.yzu.edu.tw/SNOSite/, for identifying S-nitrosylation sites on the uncharacterized protein sequences.

  20. GSHSite: exploiting an iteratively statistical method to identify s-glutathionylation sites with substrate specificity.

    Directory of Open Access Journals (Sweden)

    Yi-Ju Chen

    Full Text Available S-glutathionylation, the covalent attachment of a glutathione (GSH to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA. TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN and human protein tyrosine phosphatase 1b (PTP1B. Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/, for identifying uncharacterized GSH substrate sites on the protein sequences.

  1. Arginine Methylation Regulates MEIS2 Nuclear Localization to Promote Neuronal Differentiation of Adult SVZ Progenitors

    Directory of Open Access Journals (Sweden)

    Jasmine Kolb

    2018-04-01

    Full Text Available Summary: Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. Here, we report that MEIS2, an essential regulator of adult SVZ neurogenesis, is subject to posttranslational regulation in the SVZ olfactory bulb neurogenic system. Nuclear accumulation of MEIS2 in adult SVZ-derived progenitor cells follows downregulation of EGFR signaling and is modulated by methylation of MEIS2 on a conserved arginine, which lies in close proximity to nested binding sites for the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1. Methylation impairs interaction with CRM1 without affecting PBX1 dimerization and thereby allows MEIS2 nuclear accumulation, a prerequisite for neuronal differentiation. Our results describe a form of posttranscriptional modulation of adult SVZ neurogenesis whereby an extrinsic signal fine-tunes neurogenesis through posttranslational modification of a transcriptional regulator of cell fate. : A hallmark of adult neurogenesis is its strong dependence on physiological stimuli and environmental signals. Schulte and colleagues show that the nuclear localization and activity of a transcriptional regulator of adult neurogenesis is controlled by posttranslational modification. Their results link intrinsic control over neuron production to external signals and help to explain how adult neurogenesis can occur “on demand.” Keywords: subventricular zone, stem cell niche, posttranslational modification, controlled nuclear import, TALE-homdomain protein, MEIS2, PBX1, CRM1, neurogenesis, stem cell niche

  2. Accommodating for plant modifications

    International Nuclear Information System (INIS)

    Weirich, P.H.

    1977-01-01

    Modification to a nuclear power plant may have different causes: 1) new instructions by the authorities; 2) changes of the marginal conditions on the construction site; 3) progress in the technological development. - Examples from different plants are supposed to demonstrate how such changes influence the planning or the construction and how they are integrated in the process of preparation. A distinction can be made between modifications before the completion of the submission of the order, during the phase of preparatory planning and during the construction phase. Of great importance are especially modifications made after the beginning of the construction works, since, in general, there is little scope for technical modifications and since consequences for the time schedule are to be expected. (orig.) [de

  3. Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.

    Directory of Open Access Journals (Sweden)

    Cédric James Laedermann

    2015-11-01

    Full Text Available In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs is a very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential. Navs are composed of 9 different isoforms with distinct biophysical properties. Studying the mutations associated with the increase or absence of pain sensitivity in humans, as well as other expression studies, have highlighted Nav1.7, Nav1.8 and Nav1.9 as being the most important contributors to the control of nociceptive neuronal electrogenesis. Modulating their expression and/or function can impact the shape of the action potential and consequently modify pain transmission, a process that is observed in persistent pain conditions.Post-translational modification (PTM of Navs is a well-known process that modifies their expression and function. In chronic pain syndromes, the release of inflammatory molecules into the direct environment of dorsal root ganglia (DRG sensory neurons leads to an abnormal activation of enzymes that induce Navs PTM. The addition of small molecules, i.e. peptides, phosphoryl groups, ubiquitin moieties and/or carbohydrates, can modify the function of Navs in two different ways: via direct physical interference with the subunit of Nav gating, or via the control of Nav trafficking. Both mechanisms have a profound impact on neuronal excitability. In this review we will discuss the role of Protein Kinase A, B and C, Mitogen Activated Protein Kinases and Ca++/Calmodulin-dependent Kinase II in peripheral chronic pain syndromes. We will also discuss more recent findings that the ubiquitination of Nav1.7 by Nedd4-2 and the effect of methylglyoxal on Nav1.8 are also implicated in the development of experimental neuropathic pain. We will address the potential roles of other PTMs in chronic pain and highlight the need for further investigation of PTMs of Navs in order to develop new pharmacological

  4. Analysis of the state of posttranslational calmodulin methylation in developing pea plants

    International Nuclear Information System (INIS)

    Oh, Sukheung; Roberts, D.M.

    1990-01-01

    A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of [ 3 H]methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated and green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity

  5. Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

    Science.gov (United States)

    Sverdlov, Aaron L; Elezaby, Aly; Qin, Fuzhong; Behring, Jessica B; Luptak, Ivan; Calamaras, Timothy D; Siwik, Deborah A; Miller, Edward J; Liesa, Marc; Shirihai, Orian S; Pimentel, David R; Cohen, Richard A; Bachschmid, Markus M; Colucci, Wilson S

    2016-01-11

    Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  6. Accurate in silico identification of protein succinylation sites using an iterative semi-supervised learning technique.

    Science.gov (United States)

    Zhao, Xiaowei; Ning, Qiao; Chai, Haiting; Ma, Zhiqiang

    2015-06-07

    As a widespread type of protein post-translational modifications (PTMs), succinylation plays an important role in regulating protein conformation, function and physicochemical properties. Compared with the labor-intensive and time-consuming experimental approaches, computational predictions of succinylation sites are much desirable due to their convenient and fast speed. Currently, numerous computational models have been developed to identify PTMs sites through various types of two-class machine learning algorithms. These methods require both positive and negative samples for training. However, designation of the negative samples of PTMs was difficult and if it is not properly done can affect the performance of computational models dramatically. So that in this work, we implemented the first application of positive samples only learning (PSoL) algorithm to succinylation sites prediction problem, which was a special class of semi-supervised machine learning that used positive samples and unlabeled samples to train the model. Meanwhile, we proposed a novel succinylation sites computational predictor called SucPred (succinylation site predictor) by using multiple feature encoding schemes. Promising results were obtained by the SucPred predictor with an accuracy of 88.65% using 5-fold cross validation on the training dataset and an accuracy of 84.40% on the independent testing dataset, which demonstrated that the positive samples only learning algorithm presented here was particularly useful for identification of protein succinylation sites. Besides, the positive samples only learning algorithm can be applied to build predictors for other types of PTMs sites with ease. A web server for predicting succinylation sites was developed and was freely accessible at http://59.73.198.144:8088/SucPred/. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    Directory of Open Access Journals (Sweden)

    Jiangning Song

    Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

  8. Role of Transcription Factor Modifications in the Pathogenesis of Insulin Resistance

    Directory of Open Access Journals (Sweden)

    Mi-Young Kim

    2012-01-01

    Full Text Available Non-alcoholic fatty liver disease (NAFLD is characterized by fat accumulation in the liver not due to alcohol abuse. NAFLD is accompanied by variety of symptoms related to metabolic syndrome. Although the metabolic link between NAFLD and insulin resistance is not fully understood, it is clear that NAFLD is one of the main cause of insulin resistance. NAFLD is shown to affect the functions of other organs, including pancreas, adipose tissue, muscle and inflammatory systems. Currently efforts are being made to understand molecular mechanism of interrelationship between NAFLD and insulin resistance at the transcriptional level with specific focus on post-translational modification (PTM of transcription factors. PTM of transcription factors plays a key role in controlling numerous biological events, including cellular energy metabolism, cell-cycle progression, and organ development. Cell type- and tissue-specific reversible modifications include lysine acetylation, methylation, ubiquitination, and SUMOylation. Moreover, phosphorylation and O-GlcNAcylation on serine and threonine residues have been shown to affect protein stability, subcellular distribution, DNA-binding affinity, and transcriptional activity. PTMs of transcription factors involved in insulin-sensitive tissues confer specific adaptive mechanisms in response to internal or external stimuli. Our understanding of the interplay between these modifications and their effects on transcriptional regulation is growing. Here, we summarize the diverse roles of PTMs in insulin-sensitive tissues and their involvement in the pathogenesis of insulin resistance.

  9. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    Science.gov (United States)

    Long, Katherine S.

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms. PMID:22143525

  10. Intact glycopeptide characterization using mass spectrometry

    OpenAIRE

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prykova, Iya; Wu, Si

    2016-01-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to r...

  11. B-Type Natriuretic Peptide: From Posttranslational Processing to Clinical Measurement

    DEFF Research Database (Denmark)

    Goetze, Jens P

    2012-01-01

    BACKGROUND:Plasma cardiac natriuretic peptides and peptide fragments from their molecular precursors are markers of heart disease. Clinical studies have defined the current diagnostic utility of these markers, whereas biochemical elucidation of peptide structure and posttranslational processing has...... pathophysiology and capacity. An ongoing characterization of the molecular heterogeneity may not only help us to appreciate the biosynthetic capacity of the endocrine heart but may also lead to the discovery of new and more disease-specific targets for future molecular diagnosis.SUMMARY:Peptides derived from pro...

  12. Identification of S-glutathionylation sites in species-specific proteins by incorporating five sequence-derived features into the general pseudo-amino acid composition.

    Science.gov (United States)

    Zhao, Xiaowei; Ning, Qiao; Ai, Meiyue; Chai, Haiting; Yang, Guifu

    2016-06-07

    As a selective and reversible protein post-translational modification, S-glutathionylation generates mixed disulfides between glutathione (GSH) and cysteine residues, and plays an important role in regulating protein activity, stability, and redox regulation. To fully understand S-glutathionylation mechanisms, identification of substrates and specific S-Glutathionylated sites is crucial. Experimental identification of S-glutathionylated sites is labor-intensive and time consuming, so establishing an effective computational method is much desirable due to their convenient and fast speed. Therefore, in this study, a new bioinformatics tool named SSGlu (Species-Specific identification of Protein S-glutathionylation Sites) was developed to identify species-specific protein S-glutathionylated sites, utilizing support vector machines that combine multiple sequence-derived features with a two-step feature selection. By 5-fold cross validation, the performance of SSGlu was measured with an AUC of 0.8105 and 0.8041 for Homo sapiens and Mus musculus, respectively. Additionally, SSGlu was compared with the existing methods, and the higher MCC and AUC of SSGlu demonstrated that SSGlu was very promising to predict S-glutathionylated sites. Furthermore, a site-specific analysis showed that S-glutathionylation intimately correlated with the features derived from its surrounding sites. The conclusions derived from this study might help to understand more of the S-glutathionylation mechanism and guide the related experimental validation. For public access, SSGlu is freely accessible at http://59.73.198.144:8080/SSGlu/. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    Science.gov (United States)

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

  14. The NEDD8 modification pathway in plants

    Directory of Open Access Journals (Sweden)

    Claus eSchwechheimer

    2014-03-01

    Full Text Available NEDD8, in plants and yeasts also known as RELATED TO UBIQUITIN (RUB, is an evolutionarily conserved 76 amino acid protein highly related to ubiquitin. Like ubiquitin, NEDD8 can be conjugated to and deconjugated from target proteins, but unlike ubiquitin, NEDD8 has not been reported to form chains similar to the different polymeric ubiquitin chains that have a role in a diverse set of cellular processes. NEDD8-modification is best known as a posttranslational modification of the cullin subunits of cullin-RING E3 ubiquitin ligases. In this context, structural analyses have revealed that neddylation induces a conformation change of the cullin that brings the ubiquitylation substrates into proximity of the interacting E2 conjugating enzyme. In turn, NEDD8 deconjugation destabilizes the cullin RING ligase complex allowing for the exchange of substrate recognition subunits via the exchange factor CAND1. In plants, components of the neddylation and deneddylation pathway were identified based on mutants with defects in auxin and light responses and the characterization of these mutants has been instrumental for the elucidation of the neddylation pathway. More recently, there has been evidence from animal and plant systems that NEDD8 conjugation may also regulate the behavior or fate of non-cullin substrates in a number of ways. Here, the current knowledge on NEDD8 processing, conjugation and deconjugation is presented, where applicable, in the context of specific signaling pathways from plants.

  15. NBA-Palm: prediction of palmitoylation site implemented in Naïve Bayes algorithm.

    Science.gov (United States)

    Xue, Yu; Chen, Hu; Jin, Changjiang; Sun, Zhirong; Yao, Xuebiao

    2006-10-17

    Protein palmitoylation, an essential and reversible post-translational modification (PTM), has been implicated in cellular dynamics and plasticity. Although numerous experimental studies have been performed to explore the molecular mechanisms underlying palmitoylation processes, the intrinsic feature of substrate specificity has remained elusive. Thus, computational approaches for palmitoylation prediction are much desirable for further experimental design. In this work, we present NBA-Palm, a novel computational method based on Naïve Bayes algorithm for prediction of palmitoylation site. The training data is curated from scientific literature (PubMed) and includes 245 palmitoylated sites from 105 distinct proteins after redundancy elimination. The proper window length for a potential palmitoylated peptide is optimized as six. To evaluate the prediction performance of NBA-Palm, 3-fold cross-validation, 8-fold cross-validation and Jack-Knife validation have been carried out. Prediction accuracies reach 85.79% for 3-fold cross-validation, 86.72% for 8-fold cross-validation and 86.74% for Jack-Knife validation. Two more algorithms, RBF network and support vector machine (SVM), also have been employed and compared with NBA-Palm. Taken together, our analyses demonstrate that NBA-Palm is a useful computational program that provides insights for further experimentation. The accuracy of NBA-Palm is comparable with our previously described tool CSS-Palm. The NBA-Palm is freely accessible from: http://www.bioinfo.tsinghua.edu.cn/NBA-Palm.

  16. Functional advantages of dynamic protein disorder.

    Science.gov (United States)

    Berlow, Rebecca B; Dyson, H Jane; Wright, Peter E

    2015-09-14

    Intrinsically disordered proteins participate in many important cellular regulatory processes. The absence of a well-defined structure in the free state of a disordered domain, and even on occasion when it is bound to physiological partners, is fundamental to its function. Disordered domains are frequently the location of multiple sites for post-translational modification, the key element of metabolic control in the cell. When a disordered domain folds upon binding to a partner, the resulting complex buries a far greater surface area than in an interaction of comparably-sized folded proteins, thus maximizing specificity at modest protein size. Disorder also maintains accessibility of sites for post-translational modification. Because of their inherent plasticity, disordered domains frequently adopt entirely different structures when bound to different partners, increasing the repertoire of available interactions without the necessity for expression of many different proteins. This feature also adds to the faithfulness of cellular regulation, as the availability of a given disordered domain depends on competition between various partners relevant to different cellular processes. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Bo [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Fangjun, E-mail: wangfj@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Zou, Hanfa [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China)

    2017-01-15

    Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations. - Highlights: • All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9). • The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved. • The newly expressed or disappeared proteins and protein

  18. Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

    Science.gov (United States)

    Mozhdehi, Davoud; Luginbuhl, Kelli M.; Simon, Joseph R.; Dzuricky, Michael; Berger, Rüdiger; Varol, H. Samet; Huang, Fred C.; Buehne, Kristen L.; Mayne, Nicholas R.; Weitzhandler, Isaac; Bonn, Mischa; Parekh, Sapun H.; Chilkoti, Ashutosh

    2018-05-01

    Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials—fatty-acid-modified elastin-like polypeptides—using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

  19. A central role for ubiquitination within a circadian clock protein modification code

    Directory of Open Access Journals (Sweden)

    Katarina eStojkovic

    2014-08-01

    Full Text Available Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components, but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.

  20. An ion-current mutant of Paramecium tetraurelia with defects in the primary structure and post-translational N-methylation of calmodulin

    International Nuclear Information System (INIS)

    Wallen-Friedman, M.A.

    1988-01-01

    My work on pantophobiac A 2 (pntA 2 ), a behavioral mutant of Paramecium tetraurelia, suggest that the Ca ++ -binding protein calmodulin (CaM), and post-translation N-methylation of CaM, are important for Ca ++ -related ion-current function. Calmodulin from wild-type Paramecium has two sites of lysine-N-methylation. Both of these sites are almost fully methylated in vivo; thus wild-type calmodulin is a poor substrate for N-methylation in vitro. In contrast, pntA/ 2 CaM can be heavily N-methylated in vitro, suggesting that the mutant calmodulin is under-methylated in vivo. Amino-acid composition analysis showed that CaM lysine 115 is undermethylated in pntA 2 . Once pntA 2 CaM is N-methylated, the [methyl- 3 H] group does not turn over in either wild-type or pntA 2 cytoplasmic fractions. The methylating enzymes in pntA 2 high-speed supernatant fractions are active, but may be less robust than those of the wild type, suggesting a possible control of these enzymes by CaM

  1. Design of a novel integration-deficient lentivector technology that incorporates genetic and posttranslational elements to target human dendritic cells.

    Science.gov (United States)

    Tareen, Semih U; Kelley-Clarke, Brenna; Nicolai, Christopher J; Cassiano, Linda A; Nelson, Lisa T; Slough, Megan M; Vin, Chintan D; Odegard, Jared M; Sloan, Derek D; Van Hoeven, Neal; Allen, James M; Dubensky, Thomas W; Robbins, Scott H

    2014-03-01

    As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

  2. Stability and Application of Reactive Nitrogen and Oxygen Species-Induced Hemoglobin Modifications in Dry Blood Spots As Analyzed by Liquid Chromatography Tandem Mass Spectrometry.

    Science.gov (United States)

    Chen, Hauh-Jyun Candy; Fan, Chih-Huang; Yang, Ya-Fen

    2016-12-19

    Dried blood spot (DBS) is an emerging microsampling technique for the bioanalysis of small molecules, including fatty acids, metabolites, drugs, and toxicants. DBS offers many advantages as a sample format including easy sample collection and cheap sample shipment. Hemoglobin adducts have been recognized as a suitable biomarker for monitoring chemical exposure. We previously reported that certain modified peptides in hemoglobin derived from reactive chlorine, nitrogen, and oxygen species are associated with factors including smoking, diabetes mellitus, and aging. However, the stability of these oxidation-induced modifications of hemoglobin remains unknown and whether they can be formed artifactually during storage of DBS. To answer these questions, globin extracted from the DBS cards was analyzed, and the stability of the modifications was evaluated. After storage of the DBS cards at 4 °C or room temperature up to 7 weeks, we isolated globin from a quarter of the spot every week. The extents of 11 sites and types of post-translational modifications (PTMs), including nitration and nitrosylation of tyrosine and oxidation of cysteine and methionine residues, in human hemoglobin were measured in the trypsin digest by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) using selected reaction monitoring. The extents of all these PTMs are stable within 14 days when stored on DBS at room temperature and at 4 °C, while those from direct extraction of fresh blood are stable for at least 8 weeks when stored as an aqueous solution at -20 °C. Extraction of globin from a DBS card is of particular importance for hemolytic blood samples. To our knowledge, this is the first report on the stability of oxidative modifications of hemoglobin on DBSs, which are stable for 14 days under ambient conditions (room temperature, in air). Therefore, it is feasible and convenient to analyze these hemoglobin modifications from DBSs in studies

  3. Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors

    Directory of Open Access Journals (Sweden)

    Sylva Brabencová

    2017-12-01

    Full Text Available Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0 and Wassilewskija (Ws homogenized by two techniques (grinding in a cryomill or with a mortar and pestle. We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants. Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

  4. Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors.

    Science.gov (United States)

    Brabencová, Sylva; Ihnatová, Ivana; Potěšil, David; Fojtová, Miloslava; Fajkus, Jiří; Zdráhal, Zbyněk; Lochmanová, Gabriela

    2017-01-01

    Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

  5. Simultaneous quantification of protein phosphorylation sites using liquid chromatography-tandem mass spectrometry-based targeted proteomics: a linear algebra approach for isobaric phosphopeptides.

    Science.gov (United States)

    Xu, Feifei; Yang, Ting; Sheng, Yuan; Zhong, Ting; Yang, Mi; Chen, Yun

    2014-12-05

    As one of the most studied post-translational modifications (PTM), protein phosphorylation plays an essential role in almost all cellular processes. Current methods are able to predict and determine thousands of phosphorylation sites, whereas stoichiometric quantification of these sites is still challenging. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted proteomics is emerging as a promising technique for site-specific quantification of protein phosphorylation using proteolytic peptides as surrogates of proteins. However, several issues may limit its application, one of which relates to the phosphopeptides with different phosphorylation sites and the same mass (i.e., isobaric phosphopeptides). While employment of site-specific product ions allows for these isobaric phosphopeptides to be distinguished and quantified, site-specific product ions are often absent or weak in tandem mass spectra. In this study, linear algebra algorithms were employed as an add-on to targeted proteomics to retrieve information on individual phosphopeptides from their common spectra. To achieve this simultaneous quantification, a LC-MS/MS-based targeted proteomics assay was first developed and validated for each phosphopeptide. Given the slope and intercept of calibration curves of phosphopeptides in each transition, linear algebraic equations were developed. Using a series of mock mixtures prepared with varying concentrations of each phosphopeptide, the reliability of the approach to quantify isobaric phosphopeptides containing multiple phosphorylation sites (≥ 2) was discussed. Finally, we applied this approach to determine the phosphorylation stoichiometry of heat shock protein 27 (HSP27) at Ser78 and Ser82 in breast cancer cells and tissue samples.

  6. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    Science.gov (United States)

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-07

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  7. Identification of cofactor and herbicide binding domains in acetolactate synthase by bromopyruvate modification

    International Nuclear Information System (INIS)

    Van Dyk, D.E.; Schloss, J.V.

    1987-01-01

    Bromopyruvate is an affinity label for acetolactate synthase isozyme II from Salmonella typhimurium (ALSII). The concentration of bromopyruvate giving half-maximal inactivation is 0.1 mM, and the maximal rate of inactivation is 0.56 hr -1 . Inactivation with [ 14 C]bromopyruvate is associated with the incorporation of 4 molecules of reagent per active site lost. Two cysteinyl residues are modified extremely rapidly, with no loss of enzymatic activity, as judged by quenching the reaction with thiol after its initial phase. Inactivation is a consequence of the additional two moles of reagent incorporated per mole of protomer. The additional incorporation is divided between one major and two minor sites of modification. Substantial protection against inactivation is afforded by FAD, with virtually complete protection provided by a mixture of FAD and thiamine pyrophosphate (TPP). The major site of modification, protected by FAD, is cysteinyl residue number67, based upon amino acid sequence analysis of the purified tryptic peptide that encompasses this site. The remaining site of modification, protected by TPP, is associated with cysteinyl residue number44. Both sites of modification are afforded protection by the sulfonylurea herbicide sulfometuron methyl (SM). Although inactivation by bromopyruvate exhibits rate saturation, indicating binding as a prerequisite to inactivation, neither pyruvate nor α-ketobutyrate prevent modification of the enzyme by bromopyruvate. Thus, it would appear that the bromopyruvate binding site is not the site normally occupied by substrate

  8. N-glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Jørgensen, Stine; Bräuner-Osborne, Hans

    2015-01-01

    Investigation of post-translational modifications of receptor proteins is important for our understanding of receptor pharmacology and disease physiology. However, our knowledge about post-translational modifications of class C G protein-coupled receptors and how these modifications regulate expr...... covalently linked dimers through cysteine disulfide linkage in the extracellular amino-terminal domain and here we show that GPRC6A indeed is a homodimer and that a disulfide bridge between the C131 residues is formed....

  9. Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

    Directory of Open Access Journals (Sweden)

    Laurence Prunetti

    2016-01-01

    Full Text Available Translation initiation factor 5A (IF5A is essential and highly conserved in Eukarya (eIF5A and Archaea (aIF5A. The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958 in agmatine synthesis. The agmatinase-like gene (HVO_2299 was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies.

  10. Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes

    Science.gov (United States)

    2017-01-01

    Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications. PMID:28135077

  11. Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin

    Science.gov (United States)

    André, Pascale; Spertini, Olivier; Guia, Sophie; Rihet, Pascal; Dignat-George, Françoise; Brailly, Hervé; Sampol, José; Anderson, Paul J.; Vivier, Eric

    2000-01-01

    Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-γ and tumor necrosis factor-α) and chemokines (e.g., macrophage inflammatory protein-1α) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR+ NK cells to specific tissues. PMID:10725346

  12. Possible Role of the Transglutaminases in the Pathogenesis of Alzheimer's Disease and Other Neurodegenerative Diseases

    OpenAIRE

    Martin, Antonio; De Vivo, Giulia; Gentile, Vittorio

    2011-01-01

    Transglutaminases are ubiquitous enzymes which catalyze posttranslational modifications of proteins. Recently, transglutaminase-catalyzed post-translational modification of proteins has been shown to be involved in the molecular mechanisms responsible for human diseases. Transglutaminase activity has been hypothesized to be involved also in the pathogenetic mechanisms responsible for several human neurodegenerative diseases. Alzheimer's disease and other neurodegenerative diseases, such as Pa...

  13. Distally based sural neuro-fasciocutaneous perforator flap for foot and ankle reconstruction: Surgical modifications for flap pedicle and donor site closure without skin graft.

    Science.gov (United States)

    Chi, Zhenglin; Chen, Yiheng; Chu, Tinggang; Gao, Weiyang; Li, Zhijie; Yan, Hede; Song, Yonghuan

    2018-02-01

    The conventional procedure of the sural neuro-fasciocutaneous flap enables the supply of blood and venous drainage by increasing the width of the adipofascial tissue and preserving tiny venous return routes. Moreover, skin graft is a common method for donor site closure, which may lead to some complications and influence the aesthetic appearance. We report modifications for a distally based sural neuro-fasciocutaneous perforator flap and a relaying flap for donor site closure without skin graft. Twelve patients undergoing the modified flap for foot and ankle reconstruction were included in this study between 2014 and 2016. A peroneal-based perforator, a superficial vein, and the vascular axis of the sural nerve were included in the pedicle. A Z-shape skin incision was performed to explore the perforator vessels and a relaying island perforator flap was used to close the donor site. All flaps survived completely without necrosis. The area of the flaps ranged from 16 × 8 cm to 30 × 15 cm. The diameter width of the pedicle ranged from 1.0 to 2.0 cm. A relaying perforator island flap was used in 10 cases for donor site closure and no skin graft was performed. There were no serious donor site complications. All patients were satisfied with the aesthetic outcome postoperatively at the final follow-up. The distally based sural neuro-fasciocutaneous perforator flap is considered a reliable method for foot and ankle reconstruction. The modification for flap pedicle and donor site closure method without skin graft should be recommended. Copyright © 2017. Published by Elsevier Ltd.

  14. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

    Directory of Open Access Journals (Sweden)

    Barry M Zee

    Full Text Available Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC, which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state.

  15. NBA-Palm: prediction of palmitoylation site implemented in Naïve Bayes algorithm

    Directory of Open Access Journals (Sweden)

    Jin Changjiang

    2006-10-01

    Full Text Available Abstract Background Protein palmitoylation, an essential and reversible post-translational modification (PTM, has been implicated in cellular dynamics and plasticity. Although numerous experimental studies have been performed to explore the molecular mechanisms underlying palmitoylation processes, the intrinsic feature of substrate specificity has remained elusive. Thus, computational approaches for palmitoylation prediction are much desirable for further experimental design. Results In this work, we present NBA-Palm, a novel computational method based on Naïve Bayes algorithm for prediction of palmitoylation site. The training data is curated from scientific literature (PubMed and includes 245 palmitoylated sites from 105 distinct proteins after redundancy elimination. The proper window length for a potential palmitoylated peptide is optimized as six. To evaluate the prediction performance of NBA-Palm, 3-fold cross-validation, 8-fold cross-validation and Jack-Knife validation have been carried out. Prediction accuracies reach 85.79% for 3-fold cross-validation, 86.72% for 8-fold cross-validation and 86.74% for Jack-Knife validation. Two more algorithms, RBF network and support vector machine (SVM, also have been employed and compared with NBA-Palm. Conclusion Taken together, our analyses demonstrate that NBA-Palm is a useful computational program that provides insights for further experimentation. The accuracy of NBA-Palm is comparable with our previously described tool CSS-Palm. The NBA-Palm is freely accessible from: http://www.bioinfo.tsinghua.edu.cn/NBA-Palm.

  16. Native pyroglutamation of huwentoxin-IV: a post-translational modification that increases the trapping ability to the sodium channel.

    Science.gov (United States)

    Rong, Mingqiang; Duan, Zhigui; Chen, Juliang; Li, Jianglin; Xiao, Yuchen; Liang, Songping

    2013-01-01

    Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.

  17. Native pyroglutamation of huwentoxin-IV: a post-translational modification that increases the trapping ability to the sodium channel.

    Directory of Open Access Journals (Sweden)

    Mingqiang Rong

    Full Text Available Huwentoxin-IV (HWTX-IV, a tetrodotoxin-sensitive (TTX-s sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV, having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC50 nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms, mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms. Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.

  18. Metal-Free, Site-Selective Peptide Modification by Conversion of "Customizable" Units into β-Substituted Dehydroamino Acids.

    Science.gov (United States)

    Saavedra, Carlos J; Hernández, Dácil; Boto, Alicia

    2018-01-12

    Our site-selective modification of serine or threonine units in peptides allows the generation of β-substituted dehydroamino acids, which increase peptide resistance to hydrolysis and may improve their biological properties. Both the terminal and internal positions can be modified, and different customizable units can be activated separately. Remarkably, high Z selectivity is achieved, even at internal positions. The conversion involves a one-pot oxidative radical scission/phosphorylation process by using the low-toxicity (diacetoxyiodo)benzene/iodine system as the scission reagent. The resulting α-amino phosphonates undergo a Horner-Wadsworth-Emmons reaction to produce the dehydroamino acid derivatives (in a Z/E ratio of usually >98:2) under mild and metal-free conditions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Indigenous and acquired modifications in the aminoglycoside binding sites of Pseudomonas aeruginosa rRNAs

    DEFF Research Database (Denmark)

    Gutierrez, Belen; Douthwaite, Stephen Roger; Gonzalez-Zorn, Bruno

    2013-01-01

    (housekeeping) modifications at m (4)Cm1402, m (3)U1498, m (2)G1516, m (6) 2A1518, and m (6) 2A1519; helix 69 is modified at m (3)Ψ1915, with m (5)U1939 and m (5)C1962 modification in adjacent sequences. All modifications were close to stoichiometric, with the exception of m (3)Ψ1915, where about 80% of r...

  20. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Science.gov (United States)

    Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

    2016-01-01

    The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  1. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Directory of Open Access Journals (Sweden)

    Venkatesh Kota

    Full Text Available The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO, but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP elements from the 5' untranslated regions (UTR of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1 mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  2. Recent findings and technological advances in phosphoproteomics for cells and tissues.

    Science.gov (United States)

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

  3. Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

    Science.gov (United States)

    Pellegrino, Stefania; Altmeyer, Matthias

    2016-01-01

    Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

  4. An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

    International Nuclear Information System (INIS)

    Rosenke, Kyle; Samuel, Melanie A.; McDowell, Eric T.; Toerne, Melissa A.; Fortunato, Elizabeth A.

    2006-01-01

    The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection

  5. Predicting Post-Translational Modifications from Local Sequence Fragments Using Machine Learning Algorithms: Overview and Best Practices.

    Science.gov (United States)

    Tatjewski, Marcin; Kierczak, Marcin; Plewczynski, Dariusz

    2017-01-01

    Here, we present two perspectives on the task of predicting post translational modifications (PTMs) from local sequence fragments using machine learning algorithms. The first is the description of the fundamental steps required to construct a PTM predictor from the very beginning. These steps include data gathering, feature extraction, or machine-learning classifier selection. The second part of our work contains the detailed discussion of more advanced problems which are encountered in PTM prediction task. Probably the most challenging issues which we have covered here are: (1) how to address the training data class imbalance problem (we also present statistics describing the problem); (2) how to properly set up cross-validation folds with an approach which takes into account the homology of protein data records, to address this problem we present our folds-over-clusters algorithm; and (3) how to efficiently reach for new sources of learning features. Presented techniques and notes resulted from intense studies in the field, performed by our and other groups, and can be useful both for researchers beginning in the field of PTM prediction and for those who want to extend the repertoire of their research techniques.

  6. Histone modifications influence mediator interactions with chromatin

    Science.gov (United States)

    Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun; Liu, Zhongle; Quan, Amy; Costanzo, Michael; Dávila López, Marcela; Westholm, Jakub Orzechowski; Ronne, Hans; Boone, Charles; Gustafsson, Claes M.; Myers, Lawrence C.

    2011-01-01

    The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator—histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. PMID:21742760

  7. The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Marie Trudel

    2016-01-01

    Full Text Available Polycystin-1 (PC1 plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease’s (PKD Achilles’ heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease.

  8. Post-Translational Modifications of Histones in Human Sperm

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Jana; Stixová, Lenka; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, G.; Zdráhal, Z.; Sehnalová, Petra; Dabravolski, S.; Hejatko, J.; Bártová, Eva

    2015-01-01

    Roč. 116, č. 10 (2015), s. 2195-2209 ISSN 0730-2312 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : HUMAN SPERM * HISTONES * PROTAMINE P2 Subject RIV: BO - Biophysics Impact factor: 3.446, year: 2015

  9. Transcarboxylase (TC): demonstration by site-directed mutagenesis that methionines at the biotin site are essential for catalysis

    International Nuclear Information System (INIS)

    Wood, H.G.; Shenoy, B.C.; Kumar, G.K.; Paranjape, S.; Murtif, V.; Samols, D.

    1987-01-01

    All biotin enzymes that have thus far been sequenced contain a conserved region ALA MET BCT MET. Two possible roles of the conserved region are (i) for recognition of the specific lysine of the enzyme that is to be biotinated posttranslationally by the synthetase or (ii) for activation of the biotin to function as a carboxyl carrier. The BCT of TC is at residue 89 of the 1.3S subunit. By site-directed mutagenesis, single amino acid substitutions have been made giving LEU 88, THR 88 and LEU 90 and these mutant subunits have been expressed in E. coli and isolated. Catalysis by TC involves Partial Reactions: (1) - 00 14 CCH 2 COCOO - + 1.3S biotin pyruvate + 1.3S biotin-COO - catalyzed by the 5S subunit (2) 14 CH 3 CH( 14 COO - )COSCoA + 1.3S biotin CH 3 CH 2 COSCoA + 1.3S biotin- 14 COO - , catalyzed by the 12S subunit. The mutant subunits LEU 88 and THR 88 are inactive in Reaction 1. In Reaction 2, they are 8% as active as the 1.3S wild type. At 10 times the concentration of the wild type, they are 40% as active. The LEU 90 subunit is about 40% as active as wild type in both Reactions 1 and 2. Thus, the two METS are functionally not equivalent. What their catalytic roles are remains to be determined. Shenoy et al. have shown these modifications do not effect the synthetase reaction

  10. The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification.

    Science.gov (United States)

    Hains, Peter G; Robinson, Phillip J

    2017-09-01

    Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2-5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics.

  11. Non-degradative Ubiquitination of Protein Kinases.

    Directory of Open Access Journals (Sweden)

    K Aurelia Ball

    2016-06-01

    Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  12. Postulated weather modification effects of large energy releases

    Energy Technology Data Exchange (ETDEWEB)

    Ramsdell, J.V.; Scott, B.C.; Orgill, M.M.; Renne, D.S.; Hubbard, J.E.; McGinnis, K.A.

    1977-02-01

    Postulated impacts of large energy releases were examined in the light of existing technical information. The magnitudes of direct atmospheric modifications were estimated, and the ecological and economic implications of the modifications were explored. Energy releases from energy centers (10 to 40 power plants at a single site) and individual power plant clusters (1 to 4 power plants) were considered. In the atmosphere the energy will exist initially as increased temperature (sensible heat), moisture (latent heat), and air motion (kinetic energy). Addition of energy could result in increased cloudiness and fog, and changed precipitation patterns. A framework for economic analysis of the impacts of the postulated atmospheric modifications was established on the basis of costs and benefits. Willingness-to-pay was selected as the appropriate measure for valuing each impact. The primary and secondary atmospheric modifications may affect recreation, transportation, and aesthetics as well as agriculture and forestry. Economic values can be placed on some of the effects. However, the willingness of people to pay to gain benefits and avoid damages in many cases can only be determined through extensive surveys. The economic consequences of a given energy release would be highly site specific.

  13. Postulated weather modification effects of large energy releases

    International Nuclear Information System (INIS)

    Ramsdell, J.V.; Scott, B.C.; Orgill, M.M.; Renne, D.S.; Hubbard, J.E.; McGinnis, K.A.

    1977-02-01

    Postulated impacts of large energy releases were examined in the light of existing technical information. The magnitudes of direct atmospheric modifications were estimated, and the ecological and economic implications of the modifications were explored. Energy releases from energy centers (10 to 40 power plants at a single site) and individual power plant clusters (1 to 4 power plants) were considered. In the atmosphere the energy will exist initially as increased temperature (sensible heat), moisture (latent heat), and air motion (kinetic energy). Addition of energy could result in increased cloudiness and fog, and changed precipitation patterns. A framework for economic analysis of the impacts of the postulated atmospheric modifications was established on the basis of costs and benefits. Willingness-to-pay was selected as the appropriate measure for valuing each impact. The primary and secondary atmospheric modifications may affect recreation, transportation, and aesthetics as well as agriculture and forestry. Economic values can be placed on some of the effects. However, the willingness of people to pay to gain benefits and avoid damages in many cases can only be determined through extensive surveys. The economic consequences of a given energy release would be highly site specific

  14. Chemical modification of b-lactoglobulin by quinones

    Directory of Open Access Journals (Sweden)

    DUSAN SLADIC

    2003-05-01

    Full Text Available The avarone/avarol quinone/hydroquinone couple, as well as their derivatives show considerable antitumor activity. In this work, covalent modifications of b-lactoglobulin, isolated from cow milk, by avarone, its model compound 2-tert-butyl-1,4-benzoquinone, and several of their alkylthio derivatives were studied. The techniques applied for assaying the modifications were: UV/VIS spectrophotometry, SDS PAGE and isoelectrofocusing. The results of the SDS PAGE suggest that polymerisation of the protein occurs. The shift of the pI of the protein upon modification toward lower values indicates that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

  15. Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Clausen, Henrik

    2012-01-01

    Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis...... and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3...

  16. Acrolein Modification Impairs Key Functional Features of Rat Apolipoprotein E: Identification of Modified Sites by Mass Spectrometry

    Science.gov (United States)

    Tran, Tuyen N.; Kosaraju, Malathi G.; Tamamizu-Kato, Shiori; Akintunde, Olayemi; Zheng, Ying; Bielicki, John K.; Pinkerton, Kent; Uchida, Koji; Lee, Yuan Yu; Narayanaswami, Vasanthy

    2014-01-01

    Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for low-density lipoprotein receptor (LDLr) family of proteins, in addition to playing a role in promoting macrophage cholesterol efflux in atherosclerotic lesions. The objective of this study is to examine the effect of acrolein modification on the structure and function of rat apoE and to determine sites and nature of modification by mass spectrometry. Acrolein is a highly reactive aldehyde, which is generated endogenously as one of the products of lipid peroxidation and is present in the environment in pollutants such as tobacco smoke and heated oils. In initial studies, acrolein-modified apoE was identified by immunoprecipitation using an acrolein-lysine specific antibody, in the plasma of ten-week old male rats that were exposed to filtered air (FA) or low doses of environmental tobacco smoke (ETS). While both groups displayed acrolein-modified apoE in the lipoprotein fraction, the ETS group had higher levels in lipid-free fraction compared to the FA group. This observation provided the rationale to further investigate the effect of acrolein modification on rat apoE at a molecular level. Treatment of recombinant rat apoE with a 10-fold molar excess of acrolein resulted in: (i) a significant decrease in lipid-binding and cholesterol efflux abilities, (ii) impairment in the LDLr- and heparin-binding capabilities, and (iii) significant alterations in the overall stability of the protein. The disruption in the functional abilities is attributed directly or indirectly to acrolein modification yielding: an aldimine adduct at K149 and K155 (+38); a propanal adduct at K135 and K138 (+56); an Nε-(3-methylpyridinium)lysine (MP-lysine) at K64, K67 and K254 (+76), and Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) derivative at position K68 (+94), as determined by Matrix-Assisted Laser

  17. Response of Elk to Habitat Modification Near Natural Gas Development

    Science.gov (United States)

    Van Dyke, Fred; Fox, Autumn; Harju, Seth M.; Dzialak, Matthew R.; Hayden-Wing, Larry D.; Winstead, Jeffrey B.

    2012-11-01

    Elk (Cervus elaphus) are known to shift habitat use in response to environmental modifications, including those associated with various forms of energy development. The specific behavioral responses underlying these trends, however, have not been effectively studied. To investigate such effects, we examined elk response to habitat alteration near natural gas wells in Las Animas County, Colorado, USA in 2008-2010. We created 10 1-ha openings in forests adjacent to 10 operating natural gas wells by removing standing timber in 2008, with concomitant establishment of 10 1-ha control sites adjacent to the same wells. On each site, we estimated elk use, indexed by pellet density, before and after timber removal. Concurrently, we measured plant production and cover, nutritional quality, species composition and biomass removed by elk and other large herbivores. Species richness and diversity, graminoid and forb cover, and graminoid and forb biomass increased on cut sites following tree removal. Differences were greater in 2010 than in 2009, and elk and deer removed more plant biomass in 2010 than 2009. Elk use of cut sites was 37 % lower than control sites in 2009, but 46 % higher in 2010. The initially lower use of cut sites may be attributable to lack of winter forage on these sites caused by timber removal and associated surface modification. The increased use of cut sites in 2010 suggested that elk possessed the behavioral capacity, over time, to exploit enhanced forage resources in the proximity of habitat modifications and human activity associated with maintenance of operating natural gas wells.

  18. p53 Acetylation: Regulation and Consequences

    International Nuclear Information System (INIS)

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer

  19. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  20. Proteomic analysis of post translational modifications in cyanobacteria.

    Science.gov (United States)

    Xiong, Qian; Chen, Zhuo; Ge, Feng

    2016-02-16

    Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive in various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. The systematic investigation of PTMs could contribute to the comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This review aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation

    OpenAIRE

    Stinemetz, Emily K.; Gao, Peng; Pinkston, Kenneth L.; Montealegre, Maria Camila; Murray, Barbara E.; Harvey, Barrett R.

    2017-01-01

    AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were ...

  2. Chiral reagents in glycosylation and modification of carbohydrates.

    Science.gov (United States)

    Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

    2018-02-05

    Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

  3. Current status of the plant phosphorylation site database PhosPhAt and its use as a resource for molecular plant physiology.

    Science.gov (United States)

    Arsova, Borjana; Schulze, Waltraud X

    2012-01-01

    As the most studied post-translational modification, protein phosphorylation is analyzed in a growing number of proteomic experiments. These high-throughput approaches generate large datasets, from which specific spectrum-based information can be hard to find. In 2007, the PhosPhAt database was launched to collect and present Arabidopsis phosphorylation sites identified by mass spectrometry from and for the scientific community. At present, PhosPhAt 3.0 consolidates phosphoproteomics data from 19 published proteomic studies. Out of 5460 listed unique phosphoproteins, about 25% have been identified in at least two independent experimental setups. This is especially important when considering issues of false positive and false negative identification rates and data quality (Durek etal., 2010). This valuable data set encompasses over 13205 unique phosphopeptides, with unambiguous mapping to serine (77%), threonine (17%), and tyrosine (6%). Sorting the functional annotations of experimentally found phosphorylated proteins in PhosPhAt using Gene Ontology terms shows an over-representation of proteins in regulatory pathways and signaling processes. A similar distribution is found when the PhosPhAt predictor, trained on experimentally obtained plant phosphorylation sites, is used to predict phosphorylation sites for the Arabidopsis genome. Finally, the possibility to insert a protein sequence into the PhosPhAt predictor allows species independent use of the prediction resource. In practice, PhosPhAt also allows easy exploitation of proteomic data for design of further targeted experiments.

  4. The HIVToolbox 2 web system integrates sequence, structure, function and mutation analysis.

    Directory of Open Access Journals (Sweden)

    David P Sargeant

    Full Text Available There is enormous interest in studying HIV pathogenesis for improving the treatment of patients with HIV infection. HIV infection has become one of the best-studied systems for understanding how a virus can hijack a cell. To help facilitate discovery, we previously built HIVToolbox, a web system for visual data mining. The original HIVToolbox integrated information for HIV protein sequence, structure, functional sites, and sequence conservation. This web system has been used for almost 40,000 searches. We report improvements to HIVToolbox including new functions and workflows, data updates, and updates for ease of use. HIVToolbox2, is an improvement over HIVToolbox with new functions. HIVToolbox2 has new functionalities focused on HIV pathogenesis including drug-binding sites, drug-resistance mutations, and immune epitopes. The integrated, interactive view enables visual mining to generate hypotheses that are not readily revealed by other approaches. Most HIV proteins form multimers, and there are posttranslational modification and protein-protein interaction sites at many of these multimerization interfaces. Analysis of protease drug binding sites reveals an anatomy of drug resistance with different types of drug-resistance mutations regionally localized on the surface of protease. Some of these drug-resistance mutations have a high prevalence in specific HIV-1 M subtypes. Finally, consolidation of Tat functional sites reveals a hotspot region where there appear to be 30 interactions or posttranslational modifications. A cursory analysis with HIVToolbox2 has helped to identify several global patterns for HIV proteins. An initial analysis with this tool identifies homomultimerization of almost all HIV proteins, functional sites that overlap with multimerization sites, a global drug resistance anatomy for HIV protease, and specific distributions of some DRMs in specific HIV M subtypes. HIVToolbox2 is an open-access web application available at

  5. Epigenetic modifications by Trithorax group proteins during early embryogenesis: do members of Trx-G function as maternal effect genes?

    Science.gov (United States)

    Andreu-Vieyra, Claudia; Matzuk, Martin M

    2007-02-01

    Maternal effect genes encode transcripts that are expressed during oogenesis. These gene products are stored in the oocyte and become functional during resumption of meiosis and zygote genome activation, and in embryonic stem cells. To date, a few maternal effect genes have been identified in mammals. Epigenetic modifications have been shown to be important during early embryonic development and involve DNA methylation and post-translational modification of core histones. During development, two families of proteins have been shown to be involved in epigenetic changes: Trithorax group (Trx-G) and Polycomb group (Pc-G) proteins. Trx-G proteins function as transcriptional activators and have been shown to accumulate in the oocyte. Deletion of Trx-G members using conventional knockout technology results in embryonic lethality in the majority of the cases analysed to date. Recent studies using conditional knockout mice have revealed that at least one family member is necessary for zygote genome activation. We propose that other Trx-G members may also regulate embryonic genome activation and that the use of oocyte-specific deletor mouse lines will help clarify their roles in this process.

  6. A genome-wide screen in human embryonic stem cells reveals novel sites of allele-specific histone modification associated with known disease loci

    LENUS (Irish Health Repository)

    Prendergast, James G D

    2012-05-19

    AbstractBackgroundChromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs).ResultsUsing a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders.ConclusionThese results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

  7. Active-site modification of mammalian DNA polymerase β with pyridoxal 5'-phosphate: Mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

    International Nuclear Information System (INIS)

    Basu, A.; Kedar, P.; Wilson, S.H.; Modak, M.J.

    1989-01-01

    Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase β. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [ 3 H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate

  8. Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

    Directory of Open Access Journals (Sweden)

    Emily K Stinemetz

    Full Text Available AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE, has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

  9. Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

    Science.gov (United States)

    Stinemetz, Emily K; Gao, Peng; Pinkston, Kenneth L; Montealegre, Maria Camila; Murray, Barbara E; Harvey, Barrett R

    2017-01-01

    AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

  10. Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

    Science.gov (United States)

    Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

    2017-10-01

    Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Dietary fat and fiber interact to uniquely modify global histone post-translational epigenetic programming in a rat colon cancer progression model.

    Science.gov (United States)

    Triff, Karen; McLean, Mathew W; Callaway, Evelyn; Goldsby, Jennifer; Ivanov, Ivan; Chapkin, Robert S

    2018-04-16

    Dietary fermentable fiber generates short-chain fatty acids (SCFA), e.g., butyrate, in the colonic lumen which serves as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFA) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the global dimension of post-translational modification of histones (including H3K4me3 and H3K9ac) and clarify the chemoprotective impact of dietary bioactive compounds on transcriptional control in a preclinical model of colon cancer, we generated high-resolution genome-wide RNA (RNA-Seq) and "chromatin-state" (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed diets containing bioactive (i) fish oil, (ii) fermentable fiber (a rich source of SCFA), (iii) a combination of fish oil plus pectin or (iv) control, devoid of fish oil or pectin. In general, poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels. The combinatorial diet (fish oil + pectin) uniquely affected transcriptional profiles in the intestinal epithelium, e.g., upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFA and were also correlated with the mitochondrial L-carnitine shuttle and the inhibition of lipogenesis. These findings demonstrate that the chemoprotective fish oil + pectin combination diet uniquely induces global histone state modifications linked to the expression of chemoprotective genes. This article is protected by copyright. All rights reserved. © 2018 UICC.

  12. Measuring site occupancy: a new perspective on cysteine oxidation.

    Science.gov (United States)

    Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna; Williamson, James; Roepstorff, Peter

    2014-10-01

    Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass spectrometry. The method relies on differential reduction of "total" cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMT(TM) alkylation. Enrichment of iodoTMT(TM)-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH. We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site

  13. Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    KAUST Repository

    An, Xiaomeng

    2017-06-21

    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

  14. Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

    2015-01-01

    BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn......BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence...

  15. Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

    Science.gov (United States)

    Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

    2017-06-01

    Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

  16. The Role of Protein Modifications of T-Bet in Cytokine Production and Differentiation of T Helper Cells

    Directory of Open Access Journals (Sweden)

    Sera Oh

    2014-01-01

    Full Text Available T-Bet (T-box protein expressed in T cells, also called as TBX21 was originally cloned as a key transcription factor involved in the commitment of T helper (Th cells to the Th1 lineage. T-Bet directly activates IFN-γ gene transcription and enhances development of Th1 cells. T-Bet simultaneously modulates IL-2 and Th2 cytokines in an IFN-γ-independent manner, resulting in an attenuation of Th2 cell development. Numerous studies have demonstrated that T-bet plays multiple roles in many subtypes of immune cells, including B cell, dendritic cells, natural killer (NK cells, NK T cells, and innate lymphoid cells. Therefore, T-bet is crucial for the development and coordination of both innate and adaptive immune responses. To fulfill these multiple roles, T-bet undergoes several posttranslational protein modifications, such as phosphorylation at tyrosine, serine, and threonine residues, and ubiquitination at lysine residues, which affect lineage commitment during Th cell differentiation. This review presents a current overview of the progress made in understanding the roles of various types of T-bet protein modifications in the regulation of cytokine production during Th cell differentiation.

  17. Structural basis for the site-specific incorporation of lysine derivatives into proteins.

    Directory of Open Access Journals (Sweden)

    Veronika Flügel

    Full Text Available Posttranslational modifications (PTMs of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS:tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

  18. Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

    International Nuclear Information System (INIS)

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    2009-01-01

    Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26 tm1Sor , revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

  19. Protein covalent modification by biologically active quinones

    Directory of Open Access Journals (Sweden)

    MIROSLAV J. GASIC

    2004-11-01

    Full Text Available The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4 upon modification toward lower values (from pI 5.0 to 5.3 indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

  20. Direct analysis of site-specific N-glycopeptides of serological proteins in dried blood spot samples.

    Science.gov (United States)

    Choi, Na Young; Hwang, Heeyoun; Ji, Eun Sun; Park, Gun Wook; Lee, Ju Yeon; Lee, Hyun Kyoung; Kim, Jin Young; Yoo, Jong Shin

    2017-08-01

    Dried blood spot (DBS) samples have a number of advantages, especially with respect to ease of collection, transportation, and storage and to reduce biohazard risk. N-glycosylation is a major post-translational modification of proteins in human blood that is related to a variety of biological functions, including metastasis, cell-cell interactions, inflammation, and immunization. Here, we directly analyzed tryptic N-glycopeptides from glycoproteins in DBS samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS) without centrifugation of blood samples, depletion of major proteins, desalting of tryptic peptides, and enrichment of N-glycopeptides. Using this simple method, we identified a total of 41 site-specific N-glycopeptides from 16 glycoproteins in the DBS samples, from immunoglobulin gamma 1 (IgG-1, 10 mg/mL) down to complement component C7 (50 μg/mL). Of these, 32 N-glycopeptides from 14 glycoproteins were consistently quantified over 180 days stored at room temperature. The major abundant glycoproteins in the DBS samples were IgG-1 and IgG-2, which contain nine asialo-fucosylated complex types of 16 different N-glycopeptide isoforms. Sialo-non-fucosylated complex types were primarily detected in the other glycoproteins such as alpha-1-acid glycoprotein 1, 2, alpha-1-antitypsin, alpha-2-macroglobulin, haptoglobin, hemopexin, Ig alpha 1, 2 chain C region, kininogen-1, prothrombin, and serotransferrin. We first report the characterization of site-specific N-glycoproteins in DBS samples by LC-MS/MS with minimal sample preparation.

  1. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  2. Design assurance programme for modifications at the Ringhals Nuclear Power Plant

    International Nuclear Information System (INIS)

    Persson, K.

    1985-01-01

    The Engineering Department within the site organization is responsible for modifications and improvements of plant design. Internal and external specialists are also involved in the design activities. Modifications follow the procedures laid down in the Design Assurance Programme (DAP), according to the overall directions given in 10CFR50 APP B and the more detailed recommendations in American Nuclear Standard 3.2, and the United States Nuclear Regulatory Commission's Regulatory Guides 1.33 and 1.64. The site Quality Assurance Department has adapted these directions to Swedish conditions. All plant modifications are ordered from customers within the site organization. The design work is performed according to the following steps: priority of problems or requirements; overall analysis of problems or requirements; basic design (preliminary and final); detailed design (electrical, mechanical, civil); purchase of equipment; installation in plant; testing of installed equipment. The project engineer is responsible for modifications from the initiation of analysis until testing and documentation are finalized. For each step, procedures and checklists are available. Internal and external specialists as well as the Swedish Nuclear Inspectorate are involved when appropriate. Design engineers and QC/QE engineers are also involved. Special groups within the site organization handle purchase and installation. Testing the installed equipment is performed in co-operation with the operation staff. Deviations from DAP must be reported by all personnel, and corrective actions must be taken. Internal audits have to be done once a year for the entire DAP and the audit, with its findings and the corrective actions taken, must be reported to the QA group. (author)

  3. Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation

    Science.gov (United States)

    Tropberger, Philipp; Mercier, Alexandre; Robinson, Margaret; Zhong, Weidong; Ganem, Don E.; Holdorf, Meghan

    2015-01-01

    Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection. PMID:26438841

  4. SwissPalm: Protein Palmitoylation database.

    Science.gov (United States)

    Blanc, Mathieu; David, Fabrice; Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

    2015-01-01

    Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

  5. iSNO-PseAAC: predict cysteine S-nitrosylation sites in proteins by incorporating position specific amino acid propensity into pseudo amino acid composition.

    Directory of Open Access Journals (Sweden)

    Yan Xu

    Full Text Available Posttranslational modifications (PTMs of proteins are responsible for sensing and transducing signals to regulate various cellular functions and signaling events. S-nitrosylation (SNO is one of the most important and universal PTMs. With the avalanche of protein sequences generated in the post-genomic age, it is highly desired to develop computational methods for timely identifying the exact SNO sites in proteins because this kind of information is very useful for both basic research and drug development. Here, a new predictor, called iSNO-PseAAC, was developed for identifying the SNO sites in proteins by incorporating the position-specific amino acid propensity (PSAAP into the general form of pseudo amino acid composition (PseAAC. The predictor was implemented using the conditional random field (CRF algorithm. As a demonstration, a benchmark dataset was constructed that contains 731 SNO sites and 810 non-SNO sites. To reduce the homology bias, none of these sites were derived from the proteins that had [Formula: see text] pairwise sequence identity to any other. It was observed that the overall cross-validation success rate achieved by iSNO-PseAAC in identifying nitrosylated proteins on an independent dataset was over 90%, indicating that the new predictor is quite promising. Furthermore, a user-friendly web-server for iSNO-PseAAC was established at http://app.aporc.org/iSNO-PseAAC/, by which users can easily obtain the desired results without the need to follow the mathematical equations involved during the process of developing the prediction method. It is anticipated that iSNO-PseAAC may become a useful high throughput tool for identifying the SNO sites, or at the very least play a complementary role to the existing methods in this area.

  6. Nanoparticles for Site Specific Genome Editing

    Science.gov (United States)

    McNeer, Nicole Ali

    Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

  7. Biosynthesis of the Urease Metallocenter*

    Science.gov (United States)

    Farrugia, Mark A.; Macomber, Lee; Hausinger, Robert P.

    2013-01-01

    Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview. PMID:23539618

  8. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    DEFF Research Database (Denmark)

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

  9. p15PAF is an intrinsically disordered protein with nonrandom structural preferences at sites of interaction with other proteins.

    Science.gov (United States)

    De Biasio, Alfredo; Ibáñez de Opakua, Alain; Cordeiro, Tiago N; Villate, Maider; Merino, Nekane; Sibille, Nathalie; Lelli, Moreno; Diercks, Tammo; Bernadó, Pau; Blanco, Francisco J

    2014-02-18

    We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15(PAF) allowed us to measure 86 N-H(N) residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15(PAF) for degradation). In accordance with these findings, analysis of the (15)N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15(PAF) and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15(PAF). The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15(PAF). Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Structural analysis of the endogenous glycoallergen Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils

    Energy Technology Data Exchange (ETDEWEB)

    Rodríguez-Romero, Adela, E-mail: adela@unam.mx; Hernández-Santoyo, Alejandra; Fuentes-Silva, Deyanira [Universidad Nacional Autónoma de México, Circuito Exterior, CU, 04310 Coyoacán, DF (Mexico); Palomares, Laura A. [Universidad Nacional Autónoma de México, Apartado Postal 510-3, 62250 Cuernavaca, MOR (Mexico); Muñoz-Cruz, Samira; Yépez-Mulia, Lilian [Centro Médico Nacional Siglo XXI, IMSS, Avenida Cuauhtémoc 330, Colonia Doctores, Mexico, DF (Mexico); Orozco-Martínez, Socorro [Instituto Nacional de Pediatría, Insurgentes Sur 3700C, 04530 Cuicuilco, DF (Mexico); Universidad Nacional Autónoma de México, Circuito Exterior, CU, 04310 Coyoacán, DF (Mexico)

    2014-02-01

    This study describes the three-dimensional structure of the endogenous glycosylated allergen Hev b 2 (endo-β-1,3-glucanase), which exhibits three post-translational modifications that form a patch on the surface of the molecule that is proposed to be an allergenic IgE epitope. Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.

  11. Post-translational processing of synaptophysin in the rat retina is disrupted by diabetes.

    Directory of Open Access Journals (Sweden)

    Travis S D'Cruz

    Full Text Available Synaptophysin, is an abundant presynaptic protein involved in synaptic vesicle recycling and neurotransmitter release. Previous work shows that its content is significantly reduced in the rat retina by streptozotocin (STZ-diabetes. This study tested the hypothesis that STZ-diabetes alters synaptophysin protein turnover and glycosylation in the rat retina. Whole explant retinas from male Sprague-Dawley rats were used in this study. Rats were made diabetic by a single intraperitoneal STZ injection (65 mg/kg body weight in 10 mM sodium citrate, pH 4.5. mRNA translation was measured using a (35S-methionine labeling assay followed by synaptophysin immunoprecipitation and autoradiography. A pulse-chase study was used to determine the depletion of newly synthesized synaptophysin. Depletion of total synaptophysin was determined after treatment with cycloheximide. Mannose rich N-glycosylated synaptophysin was detected by treating retinal lysates with endoglycosidase H followed by immunoblot analysis. Synaptophysin mRNA translation was significantly increased after 1 month (p<0.001 and 2 months (p<0.05 of STZ-diabetes, compared to age-matched controls. Newly synthesized synaptophysin degradation was significantly accelerated in the retina after 1 and 2 months of diabetes compared to controls (p<0.05. Mannose rich glycosylated synaptophysin was significantly increased after 1 month of STZ-diabetes compared to controls (p<0.05.These data suggest that diabetes increases mRNA translation of synaptophysin in the retina, resulting in an accumulation of mannose rich glycosylated synaptophysin, a transient post-translational state of the protein. This diabetes-induced irregularity in post-translational processing could explain the accelerated degradation of retinal synaptophysin in diabetes.

  12. The etiology of human age-related cataract. Proteins don't last forever.

    Science.gov (United States)

    Truscott, Roger J W; Friedrich, Michael G

    2016-01-01

    It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  14. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  15. SUMO-2 and PIAS1 Modulate Insoluble Mutant Huntingtin Protein Accumulation

    Directory of Open Access Journals (Sweden)

    Jacqueline Gire O’Rourke

    2013-07-01

    Full Text Available A key feature in Huntington disease (HD is the accumulation of mutant Huntingtin (HTT protein, which may be regulated by posttranslational modifications. Here, we define the primary sites of SUMO modification in the amino-terminal domain of HTT, show modification downstream of this domain, and demonstrate that HTT is modified by the stress-inducible SUMO-2. A systematic study of E3 SUMO ligases demonstrates that PIAS1 is an E3 SUMO ligase for both HTT SUMO-1 and SUMO-2 modification and that reduction of dPIAS in a mutant HTT Drosophila model is protective. SUMO-2 modification regulates accumulation of insoluble HTT in HeLa cells in a manner that mimics proteasome inhibition and can be modulated by overexpression and acute knockdown of PIAS1. Finally, the accumulation of SUMO-2-modified proteins in the insoluble fraction of HD postmortem striata implicates SUMO-2 modification in the age-related pathogenic accumulation of mutant HTT and other cellular proteins that occurs during HD progression.

  16. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    Science.gov (United States)

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  17. Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA.

    Science.gov (United States)

    Kim, Yong-Hak; Song, Wooseok; Kim, Jin-Sik; Jiao, Li; Lee, Kangseok; Ha, Nam-Chul

    2015-08-01

    The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

    Directory of Open Access Journals (Sweden)

    Erawan Borkham-Kamphorst

    2018-04-01

    Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

  19. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate

    2012-01-01

    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine...... acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S....... cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved...

  20. Phycourobilin in Trichromatic Phycocyanin from Oceanic Cyanobacteria Is Formed Post-translationally by a Phycoerythrobilin Lyase-Isomerase*S⃞

    Science.gov (United States)

    Blot, Nicolas; Wu, Xian-Jun; Thomas, Jean-Claude; Zhang, Juan; Garczarek, Laurence; Böhm, Stephan; Tu, Jun-Ming; Zhou, Ming; Plöscher, Matthias; Eichacker, Lutz; Partensky, Frédéric; Scheer, Hugo; Zhao, Kai-Hong

    2009-01-01

    Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (λmax ∼ 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its α-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters. PMID:19182270

  1. Proteins from Erwinia asparaginase Erwinase ® and E. coli asparaginase 2 MEDAC ® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear.

    Science.gov (United States)

    Bae, Narkhyun; Pollak, Arnold; Lubec, Gert

    2011-07-01

    L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase(®))) and L-asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac(®))), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC-MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The molecular basis of aminoacylase 1 deficiency

    DEFF Research Database (Denmark)

    Sommer, Anke; Christensen, Ernst; Schwenger, Susanne

    2011-01-01

    deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted...... using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase...

  3. Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

    Science.gov (United States)

    Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

    2013-01-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

  4. Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

    2013-12-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

  5. Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

    Directory of Open Access Journals (Sweden)

    Sheng Zhi-Ya

    2008-02-01

    Full Text Available Abstract Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM. When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%. When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor. Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP

  6. Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

    Science.gov (United States)

    Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

    1999-03-01

    The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

  7. Alteration of human serum albumin binding properties induced by modifications: A review

    Science.gov (United States)

    Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

    2018-01-01

    Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

  8. Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

    Science.gov (United States)

    Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

    2006-03-06

    Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely

  9. Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology

    NARCIS (Netherlands)

    van den Bosch, Thea; Leus, Niek; Timmerman, Tirza; Dekker, Frank J

    2016-01-01

    Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues,

  10. Surface modification of polyester biomaterials for tissue engineering

    International Nuclear Information System (INIS)

    Jiao Yanpeng; Cui Fuzhai

    2007-01-01

    Surfaces play an important role in a biological system for most biological reactions occurring at surfaces and interfaces. The development of biomaterials for tissue engineering is to create perfect surfaces which can provoke specific cellular responses and direct new tissue regeneration. The improvement in biocompatibility of biomaterials for tissue engineering by directed surface modification is an important contribution to biomaterials development. Among many biomaterials used for tissue engineering, polyesters have been well documented for their excellent biodegradability, biocompatibility and nontoxicity. However, poor hydrophilicity and the lack of natural recognition sites on the surface of polyesters have greatly limited their further application in the tissue engineering field. Therefore, how to introduce functional groups or molecules to polyester surfaces, which ideally adjust cell/tissue biological functions, becomes more and more important. In this review, recent advances in polyester surface modification and their applications are reviewed. The development of new technologies or methods used to modify polyester surfaces for developing their biocompatibility is introduced. The results of polyester surface modifications by surface morphological modification, surface chemical group/charge modification, surface biomacromolecule modification and so on are reported in detail. Modified surface properties of polyesters directly related to in vitro/vivo biological performances are presented as well, such as protein adsorption, cell attachment and growth and tissue response. Lastly, the prospect of polyester surface modification is discussed, especially the current conception of biomimetic and molecular recognition. (topical review)

  11. Comparative Genomics Reveals the Diversity of Restriction-Modification Systems and DNA Methylation Sites in Listeria monocytogenes.

    Science.gov (United States)

    Chen, Poyin; den Bakker, Henk C; Korlach, Jonas; Kong, Nguyet; Storey, Dylan B; Paxinos, Ellen E; Ashby, Meredith; Clark, Tyson; Luong, Khai; Wiedmann, Martin; Weimer, Bart C

    2017-02-01

    Listeria monocytogenes is a bacterial pathogen that is found in a wide variety of anthropogenic and natural environments. Genome sequencing technologies are rapidly becoming a powerful tool in facilitating our understanding of how genotype, classification phenotypes, and virulence phenotypes interact to predict the health risks of individual bacterial isolates. Currently, 57 closed L. monocytogenes genomes are publicly available, representing three of the four phylogenetic lineages, and they suggest that L. monocytogenes has high genomic synteny. This study contributes an additional 15 closed L. monocytogenes genomes that were used to determine the associations between the genome and methylome with host invasion magnitude. In contrast to previous findings, large chromosomal inversions and rearrangements were detected in five isolates at the chromosome terminus and within rRNA genes, including a previously undescribed inversion within rRNA-encoding regions. Each isolate's epigenome contained highly diverse methyltransferase recognition sites, even within the same serotype and methylation pattern. Eleven strains contained a single chromosomally encoded methyltransferase, one strain contained two methylation systems (one system on a plasmid), and three strains exhibited no methylation, despite the occurrence of methyltransferase genes. In three isolates a new, unknown DNA modification was observed in addition to diverse methylation patterns, accompanied by a novel methylation system. Neither chromosome rearrangement nor strain-specific patterns of epigenome modification observed within virulence genes were correlated with serotype designation, clonal complex, or in vitro infectivity. These data suggest that genome diversity is larger than previously considered in L. monocytogenes and that as more genomes are sequenced, additional structure and methylation novelty will be observed in this organism. Listeria monocytogenes is the causative agent of listeriosis, a disease

  12. Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

    Science.gov (United States)

    Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

  13. Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07

    NARCIS (Netherlands)

    Marino, Fabio; Mommen, Geert P M; Jeko, Anita; Meiring, Hugo D; van Gaans-van den Brink, Jacqueline A M; Scheltema, Richard A; van Els, Cécile A C M; Heck, Albert J R

    Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA

  14. Progressive methylation of ageing histones by Dot1 functions as a timer

    NARCIS (Netherlands)

    De Vos, Dirk; Frederiks, Floor; Terweij, Marit; van Welsem, Tibor; Verzijlbergen, Kitty F.; Iachina, Ekaterina; de Graaf, Erik L.; Altelaar, A. F. Maarten; Oudgenoeg, Gideon; Heck, Albert J. R.; Krijgsveldz, Jeroen; Bakker, Barbara M.; van Leeuwen, Fred

    Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated

  15. The prepuce free flap in 10 patients : modifications in flap design and surgical technique

    NARCIS (Netherlands)

    Werker, Paul M N

    The prepuce free flap was used in 10 oral and oropharyngeal reconstructions. During the course of this study, various modifications took place. Residual penile skin necrosis and skin island necrosis early in the series led to modification of flap design. This solved the donor-site problem by placing

  16. Methylglyoxal-induced modification causes aggregation of myoglobin

    Science.gov (United States)

    Banerjee, Sauradipta; Maity, Subhajit; Chakraborti, Abhay Sankar

    2016-02-01

    Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with proteins to form advanced glycation end products (AGEs) following Maillard-like reaction. We have investigated the in vitro effect of MG (200 μM) on the monomeric heme protein myoglobin (Mb) (100 μM) in a time-dependent manner (7 to 18 days incubation at 25 °C). MG induces significant structural alterations of the heme protein, including heme loss, changes in tryptophan fluorescence, decrease of α-helicity with increased β-sheet content etc. These changes occur gradually with increased period of incubation. Incubation of Mb with MG for 7 days results in formation of the AGE adducts: carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87 and carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133. On increasing the period of incubation up to 14 days, additional AGEs namely, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139 have been detected. MG also induces aggregation of Mb, which is clearly evident with longer period of incubation (18 days), and appears to have amyloid nature. MG-derived AGEs may thus have an important role as the precursors of protein aggregation, which, in turn, may be associated with physiological complications.

  17. Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria.

    OpenAIRE

    Hansen, M A; Kirpekar, F; Ritterbusch, W; Vester, B

    2002-01-01

    Posttranscriptional modifications were mapped in helices 90-92 of 23S rRNA from the following phylogenetically diverse organisms: Haloarcula marismortui, Sulfolobus acidocaldarius, Bacillus subtilis, and Bacillus stearothermophilus. Helix 92 is a component of the ribosomal A-site, which contacts the aminoacyl-tRNA during protein synthesis, implying that posttranscriptional modifications in helices 90-92 may be important for ribosome function. RNA fragments were isolated from 23S rRNA by site-...

  18. Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages.

    Science.gov (United States)

    Kaur, Inderjeet; Zeeshan, Mohammad; Saini, Ekta; Kaushik, Abhinav; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

    2016-10-20

    Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.

  19. Functional group and stereochemical requirements for substrate binding by ghrelin O-acyltransferase revealed by unnatural amino acid incorporation.

    Science.gov (United States)

    Cleverdon, Elizabeth R; Davis, Tasha R; Hougland, James L

    2018-04-21

    Ghrelin is a small peptide hormone that undergoes a unique posttranslational modification, serine octanoylation, to play its physiological roles in processes including hunger signaling and glucose metabolism. Ghrelin O-acyltransferase (GOAT) catalyzes this posttranslational modification, which is essential for ghrelin to bind and activate its cognate GHS-R1a receptor. Inhibition of GOAT offers a potential avenue for modulating ghrelin signaling for therapeutic effect. Defining the molecular characteristics of ghrelin that lead to binding and recognition by GOAT will facilitate the development and optimization of GOAT inhibitors. We show that small peptide mimics of ghrelin substituted with 2,3-diaminopropanoic acid in place of the serine at the site of octanoylation act as submicromolar inhibitors of GOAT. Using these chemically modified analogs of desacyl ghrelin, we define key functional groups within the N-terminal sequence of ghrelin essential for binding to GOAT and determine GOAT's tolerance to backbone methylations and altered amino acid stereochemistry within ghrelin. Our study provides a structure-activity analysis of ghrelin binding to GOAT that expands upon activity-based investigations of ghrelin recognition and establishes a new class of potent substrate-mimetic GOAT inhibitors for further investigation and therapeutic interventions targeting ghrelin signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Characterization of Chromatin Structure-associated Histone Modifications in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chang Pyo Hong

    2012-09-01

    Full Text Available Chromatin structure and dynamics that are influenced by epigenetic marks, such as histone modification and DNA methylation, play a crucial role in modulating gene transcription. To understand the relationship between histone modifications and regulatory elements in breast cancer cells, we compared our chromatin immunoprecipitation sequencing (ChIP-Seq histone modification patterns for histone H3K4me1, H3K4me3, H3K9/16ac, and H3K27me3 in MCF-7 cells with publicly available formaldehyde-assisted isolation of regulatory elements (FAIRE-chip signals in human chromosomes 8, 11, and 12, identified by a method called FAIRE. Active regulatory elements defined by FAIRE were highly associated with active histone modifications, like H3K4me3 and H3K9/16ac, especially near transcription start sites. The H3K9/16ac-enriched genes that overlapped with FAIRE signals (FAIRE-H3K9/14ac were moderately correlated with gene expression levels. We also identified functional sequence motifs at H3K4me1-enriched FAIRE sites upstream of putative promoters, suggesting that regulatory elements could be associated with H3K4me1 to be regarded as distal regulatory elements. Our results might provide an insight into epigenetic regulatory mechanisms explaining the association of histone modifications with open chromatin structure in breast cancer cells.

  1. Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

    Science.gov (United States)

    Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

    2018-01-20

    PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Production of n-glycoproteins for enzyme assisted glycomodification

    DEFF Research Database (Denmark)

    2017-01-01

    The present invention relates to a cell comprising a gene encoding a polypeptide of interest, wherein the polypeptide of interest is expressed comprisingone or more posttranslational modification patterns. These modifications are useful for example in improvement of pharmacokinetic properties, i....

  3. Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

    DEFF Research Database (Denmark)

    Macek, B.; Gnad, F.; Soufi, Boumediene

    2008-01-01

    Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes...

  4. Regulation of dynamin family proteins by post-translational

    Indian Academy of Sciences (India)

    2017-04-22

    Apr 22, 2017 ... School of Biological Sciences, National Institute of Science Education and Research-. Bhubaneswar .... translational modifications provides the cell a dynamic and ..... Drp1 which confers cellular protection from mitochondrial.

  5. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

    Science.gov (United States)

    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  6. The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response

    International Nuclear Information System (INIS)

    Tiwari, Abhinav; Igoshin, Oleg A; Balázsi, Gábor; Gennaro, Maria Laura

    2010-01-01

    Bacterial persistence is the phenomenon in which a genetically identical fraction of a bacterial population can survive exposure to stress by reduction or cessation of growth. Persistence in mycobacteria has been recently linked to a stress-response network, consisting of the MprA/MprB two-component system and alternative sigma factor σ E . This network contains multiple positive transcriptional feedback loops which may give rise to bistability, making it a good candidate for controlling the mycobacterial persistence switch. To analyze the possibility of bistability, we develop a method that involves decoupling of the network into transcriptional and post-translational interaction modules. As a result we reduce the dimensionality of the dynamical system and independently analyze input–output relations in the two modules to formulate a necessary condition for bistability in terms of their logarithmic gains. We show that neither the positive autoregulation in the MprA/MprB network nor the σ E -mediated transcriptional feedback is sufficient to induce bistability in a biochemically realistic parameter range. Nonetheless, inclusion of the post-translational regulation of σ E by RseA increases the effective cooperativity of the system, resulting in bistability that is robust to parameter variation. We predict that overexpression or deletion of RseA, the key element controlling the ultrasensitive response, can eliminate bistability

  7. NANOG Is Multiply Phosphorylated and Directly Modified by ERK2 and CDK1 In Vitro

    Directory of Open Access Journals (Sweden)

    Justin Brumbaugh

    2014-01-01

    Full Text Available NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS. MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.

  8. Quantitative Analysis and Efficient Surface Modification of Silica Nanoparticles

    Directory of Open Access Journals (Sweden)

    Hak-Sung Jung

    2012-01-01

    Full Text Available Aminofunctional trialkoxysilanes such as aminopropyltrimethoxysilane (APTMS and (3-trimethoxysilylpropyldiethylenetriamine (DETAS were employed as a surface modification molecule for generating monolayer modification on the surface of silica (SiO2 nanoparticles. We were able to quantitatively analyze the number of amine functional groups on the modified SiO2 nanoparticles by acid-base back titration method and determine the effective number of amine functional groups for the successive chemical reaction by absorption measurements after treating with fluorescent rhodamine B isothiocyanate (RITC molecules. The numbers of amine sites measured by back titration were 2.7 and 7.7 ea/nm2 for SiO2-APTMS and SiO2-DETAS, respectively, while the numbers of effective amine sites measured by absorption calibration were about one fifth of the total amine sites, namely, 0.44 and 1.3 ea/nm2 for SiO2-APTMS(RITC and SiO2-DETAS(RITC, respectively. Furthermore, it was confirmed that the reactivity of amino groups on the surface-modified silica nanoparticles could be maintained in ethanol for more than 1.5 months without showing any significant differences in the reactivity.

  9. dbPAF: an integrative database of protein phosphorylation in animals and fungi.

    Science.gov (United States)

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-03-24

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.

  10. Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies

    NARCIS (Netherlands)

    Swatek, Kirby N; Aumayr, Martina; Pruneda, Jonathan N; Visser, Linda J; Berryman, Stephen; Kueck, Anja F; Geurink, Paul P; Ovaa, Huib; van Kuppeveld, Frank J M; Tuthill, Tobias J; Skern, Tim; Komander, David

    2018-01-01

    In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader

  11. Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications.

    Directory of Open Access Journals (Sweden)

    Runar Gjerp Solstad

    Full Text Available The global problem of microbial resistance to antibiotics has resulted in an urgent need to develop new antimicrobial agents. Natural antimicrobial peptides are considered promising candidates for drug development. Echinoderms, which rely on innate immunity factors in the defence against harmful microorganisms, are sources of novel antimicrobial peptides. This study aimed to isolate and characterise antimicrobial peptides from the Edible sea urchin Echinus esculentus. Using bioassay-guided purification and cDNA cloning, three antimicrobial peptides were characterised from the haemocytes of the sea urchin; two heterodimeric peptides and a cysteine-rich peptide. The peptides were named EeCentrocin 1 and 2 and EeStrongylocin 2, respectively, due to their apparent homology to the published centrocins and strongylocins isolated from the green sea urchin Strongylocentrotus droebachiensis. The two centrocin-like peptides EeCentrocin 1 and 2 are intramolecularly connected via a disulphide bond to form a heterodimeric structure, containing a cationic heavy chain of 30 and 32 amino acids and a light chain of 13 amino acids. Additionally, the light chain of EeCentrocin 2 seems to be N-terminally blocked by a pyroglutamic acid residue. The heavy chains of EeCentrocins 1 and 2 were synthesised and shown to be responsible for the antimicrobial activity of the natural peptides. EeStrongylocin 2 contains 6 cysteines engaged in 3 disulphide bonds. A fourth peptide (Ee4635 was also discovered but not fully characterised. Using mass spectrometric and NMR analyses, EeCentrocins 1 and 2, EeStrongylocin 2 and Ee4635 were all shown to contain post-translationally brominated Trp residues in the 6 position of the indole ring.

  12. BBD Optimization of K-ZnO Catalyst Modification Process for Heterogeneous Transesterification of Rice Bran Oil to Biodiesel

    Science.gov (United States)

    Kabo, K. S.; Yacob, A. R.; Bakar, W. A. W. A.; Buang, N. A.; Bello, A. M.; Ruskam, A.

    2016-07-01

    Environmentally benign zinc oxide (ZnO) was modified with 0-15% (wt.) potassium through wet impregnation and used in transesterification of rice bran oil (RBO) to form biodiesel. The catalyst was characterized by X-Ray powder Diffraction (XRD), its basic sites determined by back titration and Response Surface Methodology (RSM) Box-Behnken Design (BBD) was used to optimize the modification process variables on the basic sites of the catalyst. The transesterification product, biodiesel was analyzed by Nuclear Magnetic Resonance (NMR) spectroscopy. The result reveals K-modified ZnO with highly increased basic sites. Quadratic model with high regression R2 = 0.9995 was obtained from the ANOVA of modification process, optimization at maximum basic sites criterion gave optimum modification conditions of K-loading = 8.5% (wt.), calcination temperature = 480 oC and time = 4 hours with response and basic sites = 8.14 mmol/g which is in close agreement with the experimental value of 7.64 mmol/g. The catalyst was used and a value of 95.53% biodiesel conversion was obtained and effect of potassium leaching was not significant in the process

  13. Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

    Science.gov (United States)

    Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

    2006-01-01

    Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice

  14. Neuronal process structure and growth proteins are targets of heavy PTM regulation during brain development

    DEFF Research Database (Denmark)

    Edwards, Alistair V G; Schwämmle, Veit; Larsen, Martin Røssel

    2014-01-01

    UNLABELLED: Brain development is a process requiring precise control of many different cell types. One method to achieve this is through specific and temporally regulated modification of proteins in order to alter structure and function. Post-translational modification (PTM) of proteins is known...... on protein-level events, this study also provides significant insight into detailed roles for individual modified proteins in the developing brain, helping to advance the understanding of the complex protein-driven processes that underlie development. Finally, the use of a novel bioinformatic analytical tool...... provides one of the most comprehensive sets of individual PTM site regulation data for mammalian brain tissue. This will provide a valuable resource for those wishing to perform comparisons or meta-analyses of large scale PTMomic data, as are becoming increasingly common. Furthermore, being focussed...

  15. RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

    Science.gov (United States)

    Pascali, Chiara; Teichmann, Martin

    2013-01-01

    RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

  16. Analysis of posttranslational modifications of proteins by tandem mass spectrometry

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Trelle, Morten B; Thingholm, Tine E

    2006-01-01

    -temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful...

  17. N-acetylation and phosphorylation of Sec complex subunits in the ER membrane

    Directory of Open Access Journals (Sweden)

    Soromani Christina

    2012-12-01

    Full Text Available Abstract Background Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER. It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p. Little is known about the biogenesis and regulation of individual Sec complex subunits. Results We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. Conclusions We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5

  18. Biochemical and Mass Spectrometry-Based Approaches to Profile SUMOylation in Human Cells

    DEFF Research Database (Denmark)

    Kessler, Benedikt M; Bursomanno, Sara; McGouran, Joanna F

    2017-01-01

    Posttranslational modification of proteins with the small ubiquitin-like modifier (SUMO) regulates protein function in the context of cell cycle and DNA repair. The occurrence of SUMOylation is less frequent as compared to protein modification with ubiquitin, and appears to be controlled by a sma...

  19. Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability

    NARCIS (Netherlands)

    Willems, A.P.; Gundogdu, M.; Kempers, M.J.E.; Giltay, J.C.; Pfundt, R.P.; Elferink, M.; Loza, B.F.; Fuijkschot, J.; Ferenbach, A.T.; Gassen, K.L. van; Aalten, D.M.F. van; Lefeber, D.J.

    2017-01-01

    N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and

  20. Push back to respond better: regulatory inhibition of the DNA double-strand break response.

    Science.gov (United States)

    Panier, Stephanie; Durocher, Daniel

    2013-10-01

    Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

  1. The SocioEconomic Analysis of Repository Siting (SEARS): Supplement to the SEARS technical documentation for the SEARS modifications

    International Nuclear Information System (INIS)

    Parpia, B.; Fannin, D.; Murdock, S.; Hamm, R.; Ransom-Nelson, W.; Leistritz, F.L.

    1985-09-01

    This supplement follows a format similar to that used in the SEARS Technical Documentation. Chapter 1 consists of a brief description of each of the modifications and then describes the alterations and additions to the relevant model components, subroutines and/or data files. Chapter 2 provides a partial model run showing the user- alterable procedures involved in the implementation of each of these modifications, and chapter 3 contains the schematic flowcharts and supplemental listings of the APL computer code altered as a result of these modifications. This report is part of the overall model documentation. 5 figs

  2. Covalent Modifications of Histone H3K9 Promote Binding of CHD3

    Directory of Open Access Journals (Sweden)

    Adam H. Tencer

    2017-10-01

    Full Text Available Chromatin remodeling is required for genome function and is facilitated by ATP-dependent complexes, such as nucleosome remodeling and deacetylase (NuRD. Among its core components is the chromodomain helicase DNA binding protein 3 (CHD3 whose functional significance is not well established. Here, we show that CHD3 co-localizes with the other NuRD subunits, including HDAC1, near the H3K9ac-enriched promoters of the NuRD target genes. The tandem PHD fingers of CHD3 bind histone H3 tails and posttranslational modifications that increase hydrophobicity of H3K9—methylation or acetylation (H3K9me3 or H3K9ac—enhance this interaction. Binding of CHD3 PHDs promotes H3K9Cme3-nucleosome unwrapping in vitro and perturbs the pericentric heterochromatin structure in vivo. Methylation or acetylation of H3K9 uniquely alleviates the intra-nucleosomal interaction of histone H3 tails, increasing H3K9 accessibility. Collectively, our data suggest that the targeting of covalently modified H3K9 by CHD3 might be essential in diverse functions of NuRD.

  3. Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

    Directory of Open Access Journals (Sweden)

    Suresh Marada

    2015-08-01

    Full Text Available The G protein-coupled receptor (GPCR Smoothened (Smo is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

  4. Deliberated opinion of the Environment Authority on the request of modification of the exploitation of the basic nuclear installation (INB) nr 93 'Georges Besse' by EURODIF-Production of the Tricastin site (Drome and Vaucluse)

    International Nuclear Information System (INIS)

    2011-01-01

    This report proposes an environmental review of the content of a request for the modification of the decree authorizing the creation of the 'Georges Besse' INB 93 (basic nuclear installation), a uranium enrichment plant exploited by EURODIF-production on the Tricastin site. This modification notably deals with the PRISME operation. The report describes and discusses the objectives of this operation, its context and the concerned operation program. It indicated the concerned procedures. It discusses the style and content of the impact study from a general point of view, and outlines weaknesses and methodological problems. It more particularly discusses the analysis of the present site condition, the project presentation and justification, the analysis of impacts on the environment and on health, of impacts related to waste management, of non-radiological impacts on water, of impacts on air and on biodiversity, and of social and economic impacts. It also addresses the analysis of impacts of construction works. It finally discusses the analysis of the risk management study

  5. Redox regulation of the Calvin-Benson cycle: something old, something new

    Directory of Open Access Journals (Sweden)

    Laure eMichelet

    2013-11-01

    Full Text Available Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin-Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin-Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin-Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses.

  6. Pattern of occurrence and occupancy of carbonylation sites in proteins

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max

    2011-01-01

    sites. Comparison of metal-catalyzed oxidation of two closely related proteins indicates that this type of carbonylation might not be very specific in proteins. Interestingly, carbonylated sites show a very strong tendency to cluster together in the protein primary sequence hinting at some sort......Proteins are targets for modification by reactive oxygen species, and carbonylation is an important irreversible modification that increases during oxidative stress. While information on protein carbonylation is accumulating, its pattern is not yet understood. We have made a meta...

  7. Investigating the role of (2S,4R)-4-hydroxyproline in elastin model peptides

    DEFF Research Database (Denmark)

    Bochicchio, Brigida; Laurita, Alessandro; Heinz, Andrea

    2013-01-01

    Post-translational modifications play a key role in defining the biological functions of proteins. Among them, the hydroxylation of proline producing the (2S,4R)-4-hydroxyproline (Hyp) is one of the most frequent modifications observed in vertebrates, being particularly abundant in the proteins o...

  8. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    Science.gov (United States)

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  9. Palmitoylation as a Functional Regulator of Neurotransmitter Receptors

    Directory of Open Access Journals (Sweden)

    Vladimir S. Naumenko

    2018-01-01

    Full Text Available The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs and ligand-gated ion channels (LICs. From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior.

  10. Notch-mediated post-translational control of Ngn3 protein stability regulates pancreatic patterning and cell fate commitment

    DEFF Research Database (Denmark)

    Qu, Xiaoling; Afelik, Solomon; Jensen, Jan Nygaard

    2013-01-01

    of ducts. On one hand, Ngn3 cell-intrinsically activates endocrine target genes; on the other, Ngn3 cell-extrinsically promotes lateral signaling via the Dll1>Notch>Hes1 pathway which substantially limits its ability to sustain endocrine formation. Prior to endocrine commitment, the Ngn3-mediated...... involves transcriptional repression as previously shown, but also incorporates a novel post-translational mechanism. In addition to its ability to promote endocrine fate, we provide evidence of a competing ability of Ngn3 in the patterning of multipotent progenitor cells in turn controlling the formation...

  11. A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics

    DEFF Research Database (Denmark)

    Eberl, H.C.; Mann, M.; Spruijt, C.G.

    2013-01-01

    Posttranslational modifications on core histones can serve as binding scaffolds for chromatin-associated proteins. Proteins that specifically bind to or "read" these modifications were previously identified in mass spectrometry-based proteomics screens based on stable isotope-labeling in cell lines...... the chromatin interaction landscape in mouse tissues, our workflow can be used for peptides with different modifications and cell types of any organism....

  12. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity

    Directory of Open Access Journals (Sweden)

    Nicolas Huguenin-Dezot

    2016-07-01

    Full Text Available Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

  13. Multiple layers of posttranslational regulation refine circadian clock activity in Arabidopsis.

    Science.gov (United States)

    Seo, Pil Joon; Mas, Paloma

    2014-01-01

    The circadian clock is a cellular time-keeper mechanism that regulates biological rhythms with a period of ~24 h. The circadian rhythms in metabolism, physiology, and development are synchronized by environmental cues such as light and temperature. In plants, proper matching of the internal circadian time with the external environment confers fitness advantages on plant survival and propagation. Accordingly, plants have evolved elaborated regulatory mechanisms that precisely control the circadian oscillations. Transcriptional feedback regulation of several clock components has been well characterized over the past years. However, the importance of additional regulatory mechanisms such as chromatin remodeling, protein complexes, protein phosphorylation, and stability is only starting to emerge. The multiple layers of circadian regulation enable plants to properly synchronize with the environmental cycles and to fine-tune the circadian oscillations. This review focuses on the diverse posttranslational events that regulate circadian clock function. We discuss the mechanistic insights explaining how plants articulate a high degree of complexity in their regulatory networks to maintain circadian homeostasis and to generate highly precise waveforms of circadian expression and activity.

  14. ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro

    International Nuclear Information System (INIS)

    Woehle, D.L.; Lueddecke, B.A.; Ludden, P.W.

    1990-01-01

    Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [α- 32 P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [α- 32 P]ATP- and [α- 32 P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [α- 32 P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes. A 32 P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H 3 32 PO 4 -grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification

  15. Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

    Science.gov (United States)

    Stoichevska, Violet; Peng, Yong Y; Vashi, Aditya V; Werkmeister, Jerome A; Dumsday, Geoff J; Ramshaw, John A M

    2017-03-01

    Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017. © 2016 Wiley Periodicals, Inc.

  16. Posttranslational protein S-palmitoylation and the compartmentalization of signaling molecules in neurons

    Directory of Open Access Journals (Sweden)

    SEAN I PATTERSON

    2002-01-01

    Full Text Available Protein domains play a fundamental role in the spatial and temporal organization of intracellular signaling systems. While protein phosphorylation has long been known to modify the interactions that underlie this organization, the dynamic cycling of lipids should now be included amongst the posttranslational processes determining specificity in signal transduction. The characteristics of this process are reminiscent of the properties of protein and lipid phosphorylation in determining compartmentalization through SH2 or PH domains. Recent studies have confirmed the functional importance of protein S-palmitoylation in the compartmentalization of signaling molecules that support normal physiological function in cell division and apoptosis, and synaptic transmission and neurite outgrowth. In neurons, S-palmitoylation and targeting of proteins to rafts are regulated differentially in development by a number of processes, including some related to synaptogenesis and synaptic plasticity. Alterations in the S-palmitoylation state of proteins substantially affect their cellular function, raising the possibility of new therapeutic targets in cancer and nervous system injury and disease.

  17. Post-translational processing and secretion of atrial natriuretic factor

    International Nuclear Information System (INIS)

    Shields, P.P.

    1988-01-01

    The post-translational processing and regulated secretion of atrial natriuretic factor (ANF) were studied in primary cultures of rat cardiac myocytes. Cultures were established from neonatal rat atria or ventricles, and were maintained for 7-14 days in complete serum free medium. The cultures contained high and constant levels of ANF-(1-126), the known storage form of the hormone in vivo. The cultures also secreted ANF-(1-126), instead of the known circulating form of the hormone, ANF-(99-126). However, the inclusion of the glucocorticoids dexamethasone or hydrocortisone in the culture medium increased the levels of ir-ANF contained and secreted by the cultures, and caused both atrial and ventricular cultures to secrete principally ANF-(99-126) instead of ANF-(1-126). The secreted peptide was shown to be authentic ANF-(99-126) by chromatographic, amino acid composition and radiosequence analysis, thus confirming that the cultures were accurately processing ANF to the in vivo circulating form in the presence of glucocorticoids. Glucocorticoids also caused an increase in size and clustering of atrial myocytes as determined by immunocytochemical analysis, but the morphological effects could be dissociated from the stimulation of ANF-(99-126) secretion by manipulating the timing of glucocorticoid exposure. The location of ANF-(99-126) formation was investigated using biosynthetically labeled 35 S-Cys-ANF-(1-126) in conjunction with actively processing cultures

  18. Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

    Science.gov (United States)

    Cao, Xueyan; Song, Dahe; Yang, Mei; Yang, Ning; Ye, Qing; Tao, Dongbing; Liu, Biao; Wu, Rina; Yue, Xiqing

    2017-11-29

    Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

  19. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-01-01

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

  20. Determination of total cyanide in Hanford Site high-level wastes

    Energy Technology Data Exchange (ETDEWEB)

    Winters, W.I. [Westinghouse Hanford Co., Richland, WA (United States); Pool, K.H. [Pacific Northwest Lab., Richland, WA (United States)

    1994-05-01

    Nickel ferrocyanide compounds (Na{sub 2-x}Cs{sub x}NiFe (CN){sub 6}) were produced in a scavenging process to remove {sup 137}Cs from Hanford Site single-shell tank waste supernates. Methods for determining total cyanide in Hanford Site high-level wastes are needed for the evaluation of potential exothermic reactions between cyanide and oxidizers such as nitrate and for safe storage, processing, and management of the wastes in compliance with regulatory requirements. Hanford Site laboratory experience in determining cyanide in high-level wastes is summarized. Modifications were made to standard cyanide methods to permit improved handling of high-level waste samples and to eliminate interferences found in Hanford Site waste matrices. Interferences and associated procedure modifications caused by high nitrates/nitrite concentrations, insoluble nickel ferrocyanides, and organic complexants are described.

  1. Determination of total cyanide in Hanford Site high-level wastes

    International Nuclear Information System (INIS)

    Winters, W.I.; Pool, K.H.

    1994-05-01

    Nickel ferrocyanide compounds (Na 2-x Cs x NiFe (CN) 6 ) were produced in a scavenging process to remove 137 Cs from Hanford Site single-shell tank waste supernates. Methods for determining total cyanide in Hanford Site high-level wastes are needed for the evaluation of potential exothermic reactions between cyanide and oxidizers such as nitrate and for safe storage, processing, and management of the wastes in compliance with regulatory requirements. Hanford Site laboratory experience in determining cyanide in high-level wastes is summarized. Modifications were made to standard cyanide methods to permit improved handling of high-level waste samples and to eliminate interferences found in Hanford Site waste matrices. Interferences and associated procedure modifications caused by high nitrates/nitrite concentrations, insoluble nickel ferrocyanides, and organic complexants are described

  2. The Deep Thioredoxome in Chlamydomonas reinhardtii: New Insights into Redox Regulation.

    Science.gov (United States)

    Pérez-Pérez, María Esther; Mauriès, Adeline; Maes, Alexandre; Tourasse, Nicolas J; Hamon, Marion; Lemaire, Stéphane D; Marchand, Christophe H

    2017-08-07

    Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  3. A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

    Science.gov (United States)

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2012-08-01

    1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

  4. Enzyme Technology of Peroxidases: Immobilization, Chemical and Genetic Modification

    Science.gov (United States)

    Longoria, Adriana; Tinoco, Raunel; Torres, Eduardo

    An overview of enzyme technology applied to peroxidases is made. Immobilization on organic, inorganic, and hybrid supports; chemical modification of amino acids and heme group; and genetic modification by site-directed and random mutagenesis are included. Different strategies that were carried out to improve peroxidase performance in terms of stability, selectivity, and catalytic activity are analyzed. Immobilization of peroxidases on inorganic and organic materials enhances the tolerance of peroxidases toward the conditions normally found in many industrial processes, such as the presence of an organic solvent and high temperature. In addition, it is shown that immobilization helps to increase the Total Turnover Number at levels high enough to justify the use of a peroxidase-based biocatalyst in a synthesis process. Chemical modification of peroxidases produces modified enzymes with higher thermostability and wider substrate variability. Finally, through mutagenesis approaches, it is possible to produce modified peroxidases capable of oxidizing nonnatural substrates with high catalytic activity and affinity.

  5. Savannah River Site reactor hardware design modification study

    International Nuclear Information System (INIS)

    Fisher, J.E.

    1990-03-01

    A study was undertaken to assess the merits of proposed design modifications to the SRS reactors. The evaluation was based on the responses calculated by the RELAP5 systems code to double-ended guillotine break loss-of-coolant-accidents (DEGB LOCAs). The three concepts evaluated were (a) elevated plenum inlet piping with a guard vessel and clamshell enclosures, (b) closure of both rotovalves in the affected loop, and (c) closure of the pump suction valve in the affected loop. Each concept included a fast reactor shutdown (to 65% power in 100 ms) and a 2-s ac pump trip. For the elevated piping design, system recovery was predicted for breaks in the plenum inlet or pump suction piping; response to the pump discharge break location did not show improvement compared to the present system configuration. The rotovalve closure design improved system response to plenum inlet or pump discharge breaks; recovery was not predicted for pump suction breaks. The pump suction valve closure design demonstrated system recovery for all break locations downstream of the valve. A combination of features is recommended to ensure liquid inventory recovery for all break locations. The elevated piping design performance during pump discharge breaks would be improved with addition of a dc pump trip in the affected loop. Valve closure design performance for a break location in the short section of piping between the reactor concrete shield and the pump suction valve would benefit from the clamshell enclosing that section of piping. 12 refs., 10 figs., 2 tabs

  6. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  7. PSNO: Predicting Cysteine S-Nitrosylation Sites by Incorporating Various Sequence-Derived Features into the General Form of Chou’s PseAAC

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2014-06-01

    Full Text Available S-nitrosylation (SNO is one of the most universal reversible post-translational modifications involved in many biological processes. Malfunction or dysregulation of SNO leads to a series of severe diseases, such as developmental abnormalities and various diseases. Therefore, the identification of SNO sites (SNOs provides insights into disease progression and drug development. In this paper, a new bioinformatics tool, named PSNO, is proposed to identify SNOs from protein sequences. Firstly, we explore various promising sequence-derived discriminative features, including the evolutionary profile, the predicted secondary structure and the physicochemical properties. Secondly, rather than simply combining the features, which may bring about information redundancy and unwanted noise, we use the relative entropy selection and incremental feature selection approach to select the optimal feature subsets. Thirdly, we train our model by the technique of the k-nearest neighbor algorithm. Using both informative features and an elaborate feature selection scheme, our method, PSNO, achieves good prediction performance with a mean Mathews correlation coefficient (MCC value of about 0.5119 on the training dataset using 10-fold cross-validation. These results indicate that PSNO can be used as a competitive predictor among the state-of-the-art SNOs prediction tools. A web-server, named PSNO, which implements the proposed method, is freely available at http://59.73.198.144:8088/PSNO/.

  8. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  9. Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth.

    Science.gov (United States)

    Carabetta, Valerie J; Greco, Todd M; Tanner, Andrew W; Cristea, Ileana M; Dubnau, David

    2016-05-01

    N ε -Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. The past decade highlighted N ε -lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis . To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell

  10. Mining the human tissue proteome for protein citrullination.

    Science.gov (United States)

    Lee, Chien-Yun; Wang, Dongxue; Wilhelm, Mathias; Zolg, Daniel Paul; Schmidt, Tobias; Schnatbaum, Karsten; Reimer, Ulf; Pontén, Fredrik; Uhlén, Mathias; Hahne, Hannes; Kuster, Bernhard

    2018-04-02

    Citrullination is a post-translational modification of arginine catalyzed by five peptidylarginine deiminases (PADs) in humans. The loss of a positive charge may cause structural or functional alterations and while the modification has been linked to several diseases including rheumatoid arthritis and cancer, its physiological or pathophysiological roles remain largely unclear. In part this is owing to limitations in available methodology able to robustly enrich, detect and localize the modification. As a result, only few citrullination sites have been identified on human proteins with high confidence. In this study, we mined data from mass spectrometry-based deep proteomic profiling of 30 human tissues to identify citrullination sites on endogenous proteins. Database searching of ~70 million tandem mass spectra yielded ~13,000 candidate spectra which were further triaged by spectrum quality metrics and the detection of the specific neutral loss of isocyanic acid from citrullinated peptides to reduce false positives. Because citrullination is easily confused with deamidation, we synthetized ~2,200 citrullinated and 1,300 deamidated peptides to build a library of reference spectra. This led to the validation of 375 citrullination sites on 209 human proteins. Further analysis showed that >80% of the identified modifications sites were new and for 56% of the proteins, citrullination was detected for the first time. Sequence motif analysis revealed a strong preference for Asp and Gly, residues around the citrullination site. Interestingly, while the modification was detected in 26 human tissues with the highest levels found in brain and lung, citrullination levels did not correlate well with protein expression of the PAD enzymes. Even though the current work represents the largest survey of protein citrullination to date, the modification was mostly detected on high abundant proteins arguing that the development of specific enrichment methods would be required in order

  11. Rates and factors associated with major modifications to first-line combination antiretroviral therapy: results from the Asia-Pacific region.

    Directory of Open Access Journals (Sweden)

    Stephen Wright

    Full Text Available In the Asia-Pacific region many countries have adopted the WHO's public health approach to HIV care and treatment. We performed exploratory analyses of the factors associated with first major modification to first-line combination antiretroviral therapy (ART in resource-rich and resource-limited countries in the region.We selected treatment naive HIV-positive adults from the Australian HIV Observational Database (AHOD and the TREAT Asia HIV Observational Database (TAHOD. We dichotomised each country's per capita income into high/upper-middle (T-H and lower-middle/low (T-L. Survival methods stratified by income were used to explore time to first major modification of first-line ART and associated factors. We defined a treatment modification as either initiation of a new class of antiretroviral (ARV or a substitution of two or more ARV agents from within the same ARV class.A total of 4250 patients had 961 major modifications to first-line ART in the first five years of therapy. The cumulative incidence (95% CI of treatment modification was 0.48 (0.44-0.52, 0.33 (0.30-0.36 and 0.21 (0.18-0.23 for AHOD, T-H and T-L respectively. We found no strong associations between typical patient characteristic factors and rates of treatment modification. In AHOD, relative to sites that monitor twice-yearly (both CD4 and HIV RNA-VL, quarterly monitoring corresponded with a doubling of the rate of treatment modifications. In T-H, relative to sites that monitor once-yearly (both CD4 and HIV RNA-VL, monitoring twice-yearly corresponded to a 1.8 factor increase in treatment modifications. In T-L, no sites on average monitored both CD4 & HIV RNA-VL concurrently once-yearly. We found no differences in rates of modifications for once- or twice-yearly CD4 count monitoring.Low-income countries tended to have lower rates of major modifications made to first-line ART compared to higher-income countries. In higher-income countries, an increased rate of RNA-VL monitoring was

  12. Arginine Methylation Regulates MEIS2 Nuclear Localization to Promote Neuronal Differentiation of Adult SVZ Progenitors.

    Science.gov (United States)

    Kolb, Jasmine; Anders-Maurer, Marie; Müller, Tanja; Hau, Ann-Christin; Grebbin, Britta Moyo; Kallenborn-Gerhardt, Wiebke; Behrends, Christian; Schulte, Dorothea

    2018-04-10

    Adult neurogenesis is regulated by stem cell niche-derived extrinsic factors and cell-intrinsic regulators, yet the mechanisms by which niche signals impinge on the activity of intrinsic neurogenic transcription factors remain poorly defined. Here, we report that MEIS2, an essential regulator of adult SVZ neurogenesis, is subject to posttranslational regulation in the SVZ olfactory bulb neurogenic system. Nuclear accumulation of MEIS2 in adult SVZ-derived progenitor cells follows downregulation of EGFR signaling and is modulated by methylation of MEIS2 on a conserved arginine, which lies in close proximity to nested binding sites for the nuclear export receptor CRM1 and the MEIS dimerization partner PBX1. Methylation impairs interaction with CRM1 without affecting PBX1 dimerization and thereby allows MEIS2 nuclear accumulation, a prerequisite for neuronal differentiation. Our results describe a form of posttranscriptional modulation of adult SVZ neurogenesis whereby an extrinsic signal fine-tunes neurogenesis through posttranslational modification of a transcriptional regulator of cell fate. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Rule-based modeling: a computational approach for studying biomolecular site dynamics in cell signaling systems

    Science.gov (United States)

    Chylek, Lily A.; Harris, Leonard A.; Tung, Chang-Shung; Faeder, James R.; Lopez, Carlos F.

    2013-01-01

    Rule-based modeling was developed to address the limitations of traditional approaches for modeling chemical kinetics in cell signaling systems. These systems consist of multiple interacting biomolecules (e.g., proteins), which themselves consist of multiple parts (e.g., domains, linear motifs, and sites of phosphorylation). Consequently, biomolecules that mediate information processing generally have the potential to interact in multiple ways, with the number of possible complexes and post-translational modification states tending to grow exponentially with the number of binary interactions considered. As a result, only large reaction networks capture all possible consequences of the molecular interactions that occur in a cell signaling system, which is problematic because traditional modeling approaches for chemical kinetics (e.g., ordinary differential equations) require explicit network specification. This problem is circumvented through representation of interactions in terms of local rules. With this approach, network specification is implicit and model specification is concise. Concise representation results in a coarse graining of chemical kinetics, which is introduced because all reactions implied by a rule inherit the rate law associated with that rule. Coarse graining can be appropriate if interactions are modular, and the coarseness of a model can be adjusted as needed. Rules can be specified using specialized model-specification languages, and recently developed tools designed for specification of rule-based models allow one to leverage powerful software engineering capabilities. A rule-based model comprises a set of rules, which can be processed by general-purpose simulation and analysis tools to achieve different objectives (e.g., to perform either a deterministic or stochastic simulation). PMID:24123887

  14. Rule-based modeling: a computational approach for studying biomolecular site dynamics in cell signaling systems.

    Science.gov (United States)

    Chylek, Lily A; Harris, Leonard A; Tung, Chang-Shung; Faeder, James R; Lopez, Carlos F; Hlavacek, William S

    2014-01-01

    Rule-based modeling was developed to address the limitations of traditional approaches for modeling chemical kinetics in cell signaling systems. These systems consist of multiple interacting biomolecules (e.g., proteins), which themselves consist of multiple parts (e.g., domains, linear motifs, and sites of phosphorylation). Consequently, biomolecules that mediate information processing generally have the potential to interact in multiple ways, with the number of possible complexes and posttranslational modification states tending to grow exponentially with the number of binary interactions considered. As a result, only large reaction networks capture all possible consequences of the molecular interactions that occur in a cell signaling system, which is problematic because traditional modeling approaches for chemical kinetics (e.g., ordinary differential equations) require explicit network specification. This problem is circumvented through representation of interactions in terms of local rules. With this approach, network specification is implicit and model specification is concise. Concise representation results in a coarse graining of chemical kinetics, which is introduced because all reactions implied by a rule inherit the rate law associated with that rule. Coarse graining can be appropriate if interactions are modular, and the coarseness of a model can be adjusted as needed. Rules can be specified using specialized model-specification languages, and recently developed tools designed for specification of rule-based models allow one to leverage powerful software engineering capabilities. A rule-based model comprises a set of rules, which can be processed by general-purpose simulation and analysis tools to achieve different objectives (e.g., to perform either a deterministic or stochastic simulation). © 2013 Wiley Periodicals, Inc.

  15. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

    DEFF Research Database (Denmark)

    Molina, Henrik; Horn, David M; Tang, Ning

    2007-01-01

    Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total...... of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 ( approximately 80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more...... fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single...

  16. Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses.

    Science.gov (United States)

    Zhao, Yi; Long, Marcus J C; Wang, Yiran; Zhang, Sheng; Aye, Yimon

    2018-02-28

    Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true k cat / K m conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.

  17. Runoff, sediment transport, and landform modifications near Sheffield, Illinois

    International Nuclear Information System (INIS)

    Gray, J.R.; deVries, M.P.

    1984-01-01

    Relations among precipitation, runoff, sediment transport, and landform modifications are being evaluated at an 8.1-hectare, low-level radioactive waste disposal site near Sheffield, IL. Rainfall, runoff, and sediment discharge are measured in three basins comprising two-thirds of the site area and in a 1.10-hectare basin in undisturbed terrain 0.5 kilometer south of the site. The effects of slope, land use, and the physical characteristics of surficial material on runoff and sediment transport are evaluated at four 0.001-hectare plots - two on site and two on the undisturbed watershed. Preliminary results indicate that 890 millimeters of precipitation from July 1, 1982, through June 30, 1983, produced 230 millimeters of runoff from the site, compared to 50 millimeters of runoff from the undisturbed basin. Storm-sediment yields from the site consistently exceed yields from the undisturbed area. Runoff and sediment yields from burial-trench covers are consistently lower than yields from the site. Over 110 collapse holes were documented at the site from December 1978 through December 1982. More than 70% of these collapses formed along the periphery of trenches

  18. Runoff, sediment transport, and landform modifications near Sheffield, Illinois

    International Nuclear Information System (INIS)

    Gray, J.R.

    1984-01-01

    Relations among runoff, sediment transport, landform modifications, and precipitation are being evaluated at a 20-acre, low-level radioactive-waste disposal site near Sheffield, Ill. Rainfall, runoff, and sediment discharge are measured in three basins comprising two-thirds of the site area and in a 3.5-acre basin in undisturbed terrain 0.3 mile south of the site. The effects of slope, land use, and the physical characteristics of surficial material on runoff and sediment transport are evaluated at four 110-square-foot plots - two on site and two on the undisturbed basin. Preliminary results indicate the mean annual precipitation of 35 in. from July 1, 1982, through June 30, 1984, produced a mean of 8 in. of runoff annually from the site, compared to less than 2 in. of runoff annually from the undisturbed basin. Storm-sediment yields from the site consistently exceed yields from the undisturbed basin. Runoff and sediment yields from burial-trench covers are consistently lower than yields from the site. Two hundred and forty-four collapse holes were documented at the site from November 7, 1978, through June 7, 1984. More than 70% of these collapses formed along the periphery of trenches

  19. A modification to the Gill-Thomas-Cosman (GTC) head frame for stereotactic radiosurgery of head and neck and cervical spine sites

    International Nuclear Information System (INIS)

    Qian, G.; Hasala, P.; Rashid, H.; Costantino, T.; Cangiane, L.; Lombardi, E.; Arbit, E; Lederman, G.

    1996-01-01

    PURPOSE/OBJECTIVE: Fractionated Stereotactic Radiosurgery (FSR) is used primarily to treat intracranial lesions. Many tumors which arise at or inferior to the base of skull and about the cervical spine area were not amenable to radiosurgery. Presented is a modification to the GTC head frame which permits stereotactic radiosurgery to be directed at head and neck tumors with accurate reproducible precision. MATERIALS AND METHODS: The original GTC head frame has two fixation points; an anterior individualized dental impression and a posterior occipital plate with a mold contoured to the occipital pertuberance. The height of the occipital plate is adjustable in its vertical plane. However, the position of the dental piece is fixed and, therefore, limits treatment to regions superior. Treatment of head and neck and cervical spine tumors has been accomplished with a modification of the dental apparatus. Presented is our new device; a bracket extension which attaches to the dental piece and allows the head frame to be lowered to multiple desired positions. The occipital plate is elevated accordingly to support the occipital pertuberance. The depth helmet is also modified for quality assurance. A Rando phantom was used to test the accuracy and reproducibility of the new equipment. Bracket extensions lowering the head frame by 3 cm and 8 cm, respectively, were tested for accuracy of relocation on the Rando Phantom. Each extension was tested 20 times and for each individual test 18 depth helmet positions were obtained. RESULTS: The standard deviation of the modified head frame for each depth helmet measurement ranged from 0.05 to 0.35mm (mean 0.20) as compared to the standard deviation for the original GTC head frame which was 0.13 to 0.34mm (mean 0.24). The new device maintains the accuracy and reproducibility of the original GTC head frame. CONCLUSION: The GTC relocatable head frame for FSR has been modified to treat tumors of the head and neck and cervical spine sites

  20. Preventing E-cadherin aberrant N-glycosylation at Asn-554 improves its critical function in gastric cancer

    Science.gov (United States)

    Carvalho, S; Catarino, TA; Dias, AM; Kato, M; Almeida, A; Hessling, B; Figueiredo, J; Gärtner, F; Sanches, JM; Ruppert, T; Miyoshi, E; Pierce, M; Carneiro, F; Kolarich, D; Seruca, R; Yamaguchi, Y; Taniguchi, N; Reis, CA; Pinho, SS

    2016-01-01

    E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. PMID:26189796