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Sample records for distinct dynamic post-translational

  1. Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation.

    PubMed

    Vitrac, Heidi; MacLean, David M; Karlstaedt, Anja; Taegtmeyer, Heinrich; Jayaraman, Vasanthi; Bogdanov, Mikhail; Dowhan, William

    2017-02-03

    Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

  2. Post-translational modification: nature’s escape from genetic imprisonment and the basis for dynamic information encoding

    PubMed Central

    Prabakaran, Sudhakaran; Lippens, Guy; Steen, Hanno; Gunawardena, Jeremy

    2012-01-01

    We discuss protein post-translational modification (PTM) from an information processing perspective. PTM at multiple sites on a protein creates a combinatorial explosion in the number of potential “mod-forms”, or global patterns of modification. Distinct mod-forms can elicit distinct downstream responses, so that the overall response depends partly on the effectiveness of a particular mod-form to elicit a response and partly on the stoichiometry of that mod-form in the molecular population. We introduce the “mod-form distribution”—the relative stoichiometries of each mod-form—as the most informative measure of a protein’s state. Distinct mod-form distributions may summarise information about distinct cellular and physiological conditions and allow downstream processes to interpret this information accordingly. Such information “encoding” by PTMs may facilitate evolution by weakening the need to directly link upstream conditions to downstream responses. Mod-form distributions provide a quantitative framework in which to interpret ideas of “PTM codes” that are emerging in several areas of biology, as we show by reviewing examples of ion channels, GPCRs, microtubules and transcriptional co-regulators. We focus particularly on examples other than the well known “histone code”, to emphasise the pervasive use of information encoding in molecular biology. Finally, we touch briefly on new methods for measuring mod-form distributions. PMID:22899623

  3. Post-translational modifications of the intrinsically disordered terminal domains of histone H1: effects on secondary structure and chromatin dynamics.

    PubMed

    Roque, A; Ponte, I; Suau, P

    2016-04-21

    H1 linker histones are involved both in the maintenance of chromatin higher-order structure and in gene regulation. H1 binds to linker DNA regions on the surface of the nucleosome. In higher eukaryotes, H1 contains three distinct domains: a short N-terminal domain (NTD), a central globular domain, and a long C-terminal domain (CTD). Terminal domains determine subtype specificity and to a large extent the linker DNA binding and chromatin condensing properties of histone H1. This review is focused on the recent numerous studies that have provided insights in the role of H1 terminal domains in chromatin dynamics. The N- and C-terminal domains behave as intrinsically disordered proteins with coupled binding and folding. We examine the potential kinetic advantages of intrinsic disorder in the recognition of the specific H1 binding sites in chromatin. As typical intrinsically disordered regions, H1 terminal domains are post-translationally modified. Post-translational modifications in the NTD determine the interaction of histone H1 with other proteins involved in heterochromatin formation and transcriptional regulation, while phosphorylation by cyclin-dependent kinases modulates the secondary structure of the CTD and chromatin condensation. We review the arguments in favor of the involvement of H1 hyperphosphorylation in metaphase chromatin condensation and of partial phosphorylation in interphase chromatin relaxation. In addition, the interplay of histone H1 and other chromatin architectural proteins, such as proteins of the high-mobility group, protamines, and MeCP2, is associated with changes in chromatin structure.

  4. Post-translational regulation of inflammasomes

    PubMed Central

    Yang, Jie; Liu, Zhonghua; Xiao, Tsan Sam

    2017-01-01

    Inflammasomes play essential roles in immune protection against microbial infections. However, excessive inflammation is implicated in various human diseases, including autoinflammatory syndromes, diabetes, multiple sclerosis, cardiovascular disorders and neurodegenerative diseases. Therefore, precise regulation of inflammasome activities is critical for adequate immune protection while limiting collateral tissue damage. In this review, we focus on the emerging roles of post-translational modifications (PTMs) that regulate activation of the NLRP3, NLRP1, NLRC4, AIM2 and IFI16 inflammasomes. We anticipate that these types of PTMs will be identified in other types of and less well-characterized inflammasomes. Because these highly diverse and versatile PTMs shape distinct inflammatory responses in response to infections and tissue damage, targeting the enzymes involved in these PTMs will undoubtedly offer opportunities for precise modulation of inflammasome activities under various pathophysiological conditions. PMID:27345727

  5. Post-translational regulation of endothelial nitric oxide synthase in vascular endothelium

    PubMed Central

    Qian, Jin; Fulton, David

    2013-01-01

    Nitric oxide (NO) is a short-lived gaseous signaling molecule. In blood vessels, it is synthesized in a dynamic fashion by endothelial nitric oxide synthase (eNOS) and influences vascular function via two distinct mechanisms, the activation of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)-dependent signaling and the S-nitrosylation of proteins with reactive thiols (S-nitrosylation). The regulation of eNOS activity and NO bioavailability is critical to maintain blood vessel function. The activity of eNOS and ability to generate NO is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Post-translational modifications acutely impact eNOS activity and dysregulation of these mechanisms compromise eNOS activity and foster the development of cardiovascular diseases (CVDs). This review will intergrate past and current literature on the post-translational modifications of eNOS in both health and disease. PMID:24379783

  6. Post-translational regulation enables robust p53 regulation

    PubMed Central

    2013-01-01

    Background 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. Results 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. Conclusions Our analyses suggest that the p53-Mdm2 feedback favors regulatory mechanisms that provide the optimal trade-offs for dynamic control. PMID:23992617

  7. Dynamic Changes of Post-Translationally Modified Forms of CXCL10 and Soluble DPP4 in HCV Subjects Receiving Interferon-Free Therapy

    PubMed Central

    Casrouge, Armanda; Masur, Henry; Kottilil, Shyam; Albert, Matthew L.; Duffy, Darragh

    2015-01-01

    Serum levels of the interferon (IFN)-stimulated chemokine CXCL10 are increased during chronic HCV infection and associate with outcome of IFN-based therapy. Elevated levels of NH2-terminal truncated CXCL10 (3-77aa), produced by DPP4 cleavage, negatively associate with spontaneous clearance of acute HCV infection and sustained virological response (SVR) with IFN-based therapy for chronic infection. The association of different CXCL10 forms and DPP4 with outcome during IFN-free HCV therapy has not been examined. Using novel Simoa assays, plasma was analyzed from HCV genotype-1 (GT1) subjects who relapsed (n = 11) or achieved SVR (n = 10) after sofosbuvir and ribavirin (SOF/RBV) treatment, and from SOF/RBV relapsers who achieved SVR with a subsequent SOF/ledipasvir regimen (n = 9). While the NH2-truncated form of CXCL10 was elevated in HCV infection relative to healthy controls, pre-treatment plasma concentrations of CXCL10 forms failed to stratify subjects based on treatment outcome to IFN-free regimens. However, a trend (statistically non-significant) towards elevated higher levels of total and long CXCL10 was observed pre-treatment in subjects who relapsed. All forms of CXCL10 decreased rapidly following treatment initiation and were again elevated in subjects who experienced HCV relapse, indicating that CXCL10 production may be associated with active viral replication. While soluble DPP4 (sDPP4) and NH2-truncated CXCL10 concentrations were highly correlated, on-treatment sDPP4 levels and activity declined more slowly than CXCL10, suggesting differential regulation. Conclusion These data suggest post-translationally modified forms of CXCL10 will not support the prediction of treatment outcome in HCV GT1 subjects treated with SOF/RBV. PMID:26181438

  8. MARQUIS: A Multiplex Method for Absolute Quantification of Peptides and Post-Translational Modifications

    PubMed Central

    Curran, Timothy G; Zhang, Yi; Ma, Daniel J.; Sarkaria, Jann N.; White, Forest M

    2014-01-01

    Absolute quantification of protein expression and post-translational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS — Multiplex Absolute Regressed Quantification with Internal Standards — a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labeled standard peptides to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms. PMID:25581283

  9. Metabolic regulation of histone post-translational modifications

    PubMed Central

    Fan, Jing; Krautkramer, Kimberly A.; Feldman, Jessica L.; Denu, John M.

    2015-01-01

    Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as co-substrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance. PMID:25562692

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

    PubMed

    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.

  11. Global Post-Translational Modification Discovery.

    PubMed

    Li, Qiyao; Shortreed, Michael R; Wenger, Craig D; Frey, Brian L; Schaffer, Leah V; Scalf, Mark; Smith, Lloyd M

    2017-03-01

    A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community.

  12. Post-Translational Modifications in Circadian Rhythms

    PubMed Central

    Mehra, Arun; Baker, Christopher L.; Loros, Jennifer J.; Dunlap, Jay C.

    2009-01-01

    The pace has quickened in circadian biology research. In particular, an abundance of results focused on post-translational modifications (PTMs) is sharpening our view of circadian molecular clockworks. PTMs affect nearly all aspects of clock biology; in some cases they are essential for clock function and in others, they provide layers of regulatory fine-tuning. Our goal is to review recent advances in clock PTMs, help make sense of emerging themes, and spotlight intriguing and perhaps controversial new findings. We focus on PTMs affecting the core functions of eukaryotic clocks, in particular the functionally related oscillators in Neurospora crassa, Drosophila melanogaster, and mammalian cells. PMID:19740663

  13. Co- and post-translational modifications in Rubisco: unanswered questions.

    PubMed

    Houtz, Robert L; Magnani, Roberta; Nayak, Nihar R; Dirk, Lynnette M A

    2008-01-01

    Both the large (LS) and small (SS) subunits of Rubisco are subject to a plethora of co- and post-translational modifications. With the exceptions of LS carbamylation and SS transit sequence processing, the remaining modifications, including deformylation, acetylation, methylation, and N-terminal proteolytic processing of the LS, are still biochemically and/or functionally undefined although they are found in nearly all forms of Rubisco from vascular plants. A collection of relatively unique enzymes catalyse these modifications, and several have been characterized in other organisms. Some of the observed modifications in the LS and SS clearly suggest novel changes in enzyme specificity and/or activity, and others have common features with other co- and post-translationally modifying enzymes. With the possible exception of Lys14 methylation in the LS, processing of both the LS and SS of Rubisco is by default an ordered process sequentially leading up to the final forms observed in the holoenzyme. An overview of the nature of structural modifications in the LS and SS of Rubisco is presented, and, where possible, the nature of the enzymes catalysing these modifications (either through similarity with other known enzymes or through direct enzymological characterization) is described. Overall, there are a distinct lack of functional and mechanistic observations for modifications in Rubisco and thus represent many potentially productive avenues for research.

  14. Transcriptional and post-translational regulation of pannexins.

    PubMed

    Boyce, Andrew K J; Epp, Anna L; Nagarajan, Archana; Swayne, Leigh Anne

    2017-03-07

    Pannexins are a 3-membered family of proteins that form large pore ion and metabolite channels in vertebrates. The impact of pannexins on vertebrate biology is intricately tied to where and when they are expressed, and how they are modified, once produced. The purpose of this review is therefore to outline our current understanding of transcriptional and post-translational regulation of pannexins. First, we briefly summarize their discovery and characteristics. Next, we describe several aspects of transcriptional regulation, including cell and tissue-specific expression, dynamic expression over development and disease, as well as new insights into the underlying molecular machinery involved. Following this, we delve into the role of post-translational modifications in the regulation of trafficking and channel properties, highlighting important work on glycosylation, phosphorylation, S-nitrosylation and proteolytic cleavage. Embedded throughout, we also highlight important knowledge gaps and avenues of future research. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

  15. Global Post-Translational Modification Discovery

    PubMed Central

    2016-01-01

    A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultrawide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community. PMID:28248113

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

    PubMed Central

    Osamor, Victor Chukwudi; Chinedu, Shalom N; Azuh, Dominic E; Iweala, Emeka Joshua; Ogunlana, Olubanke Olujoke

    2016-01-01

    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. PMID:27013864

  17. Mapping post-translational modifications of mammalian testicular specific histone variant TH2B in tetraploid and haploid germ cells and their implications on the dynamics of nucleosome structure.

    PubMed

    Pentakota, Satya Krishna; Sandhya, Sankaran; P Sikarwar, Arun; Chandra, Nagasuma; Satyanarayana Rao, Manchanahalli R

    2014-12-05

    Histones regulate a variety of chromatin templated events by their post-translational modifications (PTMs). Although there are extensive reports on the PTMs of canonical histones, the information on the histone variants remains very scanty. Here, we report the identification of different PTMs, such as acetylation, methylation, and phosphorylation of a major mammalian histone variant TH2B. Our mass spectrometric analysis has led to the identification of both conserved and unique modifications across tetraploid spermatocytes and haploid spermatids. We have also computationally derived the 3-dimensional model of a TH2B containing nucleosome in order to study the spatial orientation of the PTMs identified and their effect on nucleosome stability and DNA binding potential. From our nucleosome model, it is evident that substitution of specific amino acid residues in TH2B results in both differential histone-DNA and histone-histone contacts. Furthermore, we have also observed that acetylation on the N-terminal tail of TH2B weakens the interactions with the DNA. These results provide direct evidence that, similar to somatic H2B, the testis specific histone TH2B also undergoes multiple PTMs, suggesting the possibility of chromatin regulation by such covalent modifications in mammalian male germ cells.

  18. Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

    PubMed Central

    Arnison, Paul G.; Bibb, Mervyn J.; Bierbaum, Gabriele; Bowers, Albert A.; Bugni, Tim S.; Bulaj, Grzegorz; Camarero, Julio A.; Campopiano, Dominic J.; Challis, Gregory L.; Clardy, Jon; Cotter, Paul D.; Craik, David J.; Dawson, Michael; Dittmann, Elke; Donadio, Stefano; Dorrestein, Pieter C.; Entian, Karl-Dieter; Fischbach, Michael A.; Garavelli, John S.; Göransson, Ulf; Gruber, Christian W.; Haft, Daniel H.; Hemscheidt, Thomas K.; Hertweck, Christian; Hill, Colin; Horswill, Alexander R.; Jaspars, Marcel; Kelly, Wendy L.; Klinman, Judith P.; Kuipers, Oscar P.; Link, A. James; Liu, Wen; Marahiel, Mohamed A.; Mitchell, Douglas A.; Moll, Gert N.; Moore, Bradley S.; Müller, Rolf; Nair, Satish K.; Nes, Ingolf F.; Norris, Gillian E.; Olivera, Baldomero M.; Onaka, Hiroyasu; Patchett, Mark L.; Piel, Joern; Reaney, Martin J. T.; Rebuffat, Sylvie; Ross, R. Paul; Sahl, Hans-Georg; Schmidt, Eric W.; Selsted, Michael E.; Severinov, Konstantin; Shen, Ben; Sivonen, Kaarina; Smith, Leif; Stein, Torsten; Süssmuth, Roderich D.; Tagg, John R.; Tang, Gong-Li; Truman, Andrew W.; Vederas, John C.; Walsh, Christopher T.; Walton, Jonathan D.; Wenzel, Silke C.; Willey, Joanne M.; van der Donk, Wilfred A.

    2014-01-01

    This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed. PMID:23165928

  19. Post-translational modifications mediated by reactive nitrogen species

    PubMed Central

    del Río, Luis A; Barroso, Juan B

    2008-01-01

    In animal cells, nitric oxide and NO-derived molecules have been shown to mediate post-translational modifications such as S-nitrosylation and protein tyrosine nitration which are associated with cell signalling and pathological processes, respectively. In plant cells, knowledge of the function of these post-translational modifications under physiological and stress conditions is still very rudimentary. In this addendum, we briefly examine how reactive nitrogen species (RNS) can exert important effects on proteins that could mediate signalling processes in plants. PMID:19841652

  20. Mitochondrial post-translational modifications and metabolic control: sirtuins and beyond.

    PubMed

    Kulkarni, Sameer S; Cantó, Carles

    2016-02-17

    In order to maintain metabolic homeostasis, organisms adjust the capacity and efficiency of ATP generation to changes in energetic demand and supply. While the transcriptional control of mitochondrial biogenesis allows to fine tune mitochondrial respiratory capacity with long-term requirements for differential energy demand (e.g.: exercise training), bioenergetic adaptation also needs to take place within shorter time frames in order to properly fine-tune nutrient availability, energy production and demand, either in a circadian fashion or after a meal. These quick metabolic responses can be achieved through exquisite modulation of diverse post-translational modifications, which influence a variety of mitochondrial processes, including mitochondrial dynamics, fatty acid oxidation, lipogenesis and bioenergetic efficiency. All organisms are equipped with numerous enzymes that allow creating a virtually unlimited palette of post-translational modification landscapes. In this review, we will specially focus on the role of mitochondrial sirtuin enzymes as modulators of mitochondrial ac(et)ylation and the possible interactions with other post-translational modification events.

  1. Alteration and modulation of protein activity by varying post-translational modification

    DOEpatents

    Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

    2015-03-03

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

  2. Alteration and modulation of protein activity by varying post-translational modification

    DOEpatents

    Thompson, David N.; Reed, David W.; Thompson, Vicki S.; Lacey, Jeffrey A.; Apel, William A.

    2016-07-12

    Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

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

    SciTech Connect

    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.

  4. Post-Translational Modifications of Histones in Vertebrate Neurogenesis

    PubMed Central

    Mitrousis, Nikolaos; Tropepe, Vincent; Hermanson, Ola

    2015-01-01

    The process of neurogenesis, through which the entire nervous system of an organism is formed, has attracted immense scientific attention for decades. How can a single neural stem cell give rise to astrocytes, oligodendrocytes, and neurons? Furthermore, how is a neuron led to choose between the hundreds of different neuronal subtypes that the vertebrate CNS contains? Traditionally, niche signals and transcription factors have been on the spotlight. Recent research is increasingly demonstrating that the answer may partially lie in epigenetic regulation of gene expression. In this article, we comprehensively review the role of post-translational histone modifications in neurogenesis in both the embryonic and adult CNS. PMID:26733796

  5. Vienna-PTM web server: a toolkit for MD simulations of protein post-translational modifications

    PubMed Central

    Margreitter, Christian; Petrov, Drazen; Zagrovic, Bojan

    2013-01-01

    Post-translational modifications (PTMs) play a key role in numerous cellular processes by directly affecting structure, dynamics and interaction networks of target proteins. Despite their importance, our understanding of protein PTMs at the atomistic level is still largely incomplete. Molecular dynamics (MD) simulations, which provide high-resolution insight into biomolecular function and underlying mechanisms, are in principle ideally suited to tackle this problem. However, because of the challenges associated with the development of novel MD parameters and a general lack of suitable computational tools for incorporating PTMs in target protein structures, MD simulations of post-translationally modified proteins have historically lagged significantly behind the studies of unmodified proteins. Here, we present Vienna-PTM web server (http://vienna-ptm.univie.ac.at), a platform for automated introduction of PTMs of choice to protein 3D structures (PDB files) in a user-friendly visual environment. With 256 different enzymatic and non-enzymatic PTMs available, the server performs geometrically realistic introduction of modifications at sites of interests, as well as subsequent energy minimization. Finally, the server makes available force field parameters and input files needed to run MD simulations of modified proteins within the framework of the widely used GROMOS 54A7 and 45A3 force fields and GROMACS simulation package. PMID:23703210

  6. Linking Post-Translational Modifications and Variation of Phenotypic Traits*

    PubMed Central

    Albertin, Warren; Marullo, Philippe; Bely, Marina; Aigle, Michel; Bourgais, Aurélie; Langella, Olivier; Balliau, Thierry; Chevret, Didier; Valot, Benoît; da Silva, Telma; Dillmann, Christine; de Vienne, Dominique; Sicard, Delphine

    2013-01-01

    Enzymes can be post-translationally modified, leading to isoforms with different properties. The phenotypic consequences of the quantitative variability of isoforms have never been studied. We used quantitative proteomics to dissect the relationships between the abundances of the enzymes and isoforms of alcoholic fermentation, metabolic traits, and growth-related traits in Saccharomyces cerevisiae. Although the enzymatic pool allocated to the fermentation proteome was constant over the culture media and the strains considered, there was variation in abundance of individual enzymes and sometimes much more of their isoforms, which suggests the existence of selective constraints on total protein abundance and trade-offs between isoforms. Variations in abundance of some isoforms were significantly associated to metabolic traits and growth-related traits. In particular, cell size and maximum population size were highly correlated to the degree of N-terminal acetylation of the alcohol dehydrogenase. The fermentation proteome was found to be shaped by human selection, through the differential targeting of a few isoforms for each food-processing origin of strains. These results highlight the importance of post-translational modifications in the diversity of metabolic and life-history traits. PMID:23271801

  7. Dual Coordination of Post Translational Modifications in Human Protein Networks

    PubMed Central

    Woodsmith, Jonathan; Kamburov, Atanas; Stelzl, Ulrich

    2013-01-01

    Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling. PMID:23505349

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

    PubMed

    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.

  9. Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

    PubMed Central

    Appel, Mason J.; Bertozzi, Carolyn R.

    2015-01-01

    Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function. PMID:25514000

  10. Chaperone-assisted Post-translational Transport of Plastidic Type I Signal Peptidase 1*

    PubMed Central

    Endow, Joshua K.; Singhal, Rajneesh; Fernandez, Donna E.; Inoue, Kentaro

    2015-01-01

    Type I signal peptidase (SPase I) is an integral membrane Ser/Lys protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally, and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in post-translational transport of plastidic SPase I 1 (Plsp1) from Arabidopsis thaliana, which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP hydrolysis-dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1 and was strongly enhanced by exogenously added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kDa complex that co-migrated with the Cpn60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kDa complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1. PMID:26446787

  11. Post-translational modifications modulate ligand recognition by the third PDZ domain of the MAGUK protein PSD-95.

    PubMed

    Murciano-Calles, Javier; Corbi-Verge, Carles; Candel, Adela M; Luque, Irene; Martinez, Jose C

    2014-01-01

    The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2-β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

  12. Current strategies and findings in clinically relevant post-translational modification-specific proteomics

    PubMed Central

    Pagel, Oliver; Loroch, Stefan; Sickmann, Albert; Zahedi, René P

    2015-01-01

    Mass spectrometry-based proteomics has considerably extended our knowledge about the occurrence and dynamics of protein post-translational modifications (PTMs). So far, quantitative proteomics has been mainly used to study PTM regulation in cell culture models, providing new insights into the role of aberrant PTM patterns in human disease. However, continuous technological and methodical developments have paved the way for an increasing number of PTM-specific proteomic studies using clinical samples, often limited in sample amount. Thus, quantitative proteomics holds a great potential to discover, validate and accurately quantify biomarkers in body fluids and primary tissues. A major effort will be to improve the complete integration of robust but sensitive proteomics technology to clinical environments. Here, we discuss PTMs that are relevant for clinical research, with a focus on phosphorylation, glycosylation and proteolytic cleavage; furthermore, we give an overview on the current developments and novel findings in mass spectrometry-based PTM research. PMID:25955281

  13. Co- and/or post-translational modifications are critical for TCH4 XET activity

    NASA Technical Reports Server (NTRS)

    Campbell, P.; Braam, J.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.

  14. Post-translational modifications in PrP expand the conformational diversity of prions in vivo

    PubMed Central

    Aguilar-Calvo, Patricia; Xiao, Xiangzhu; Bett, Cyrus; Eraña, Hasier; Soldau, Katrin; Castilla, Joaquin; Nilsson, K. Peter R.; Surewicz, Witold K.; Sigurdson, Christina J.

    2017-01-01

    Misfolded prion protein aggregates (PrPSc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrPC, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPI-anchorless PrPSc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of β-sheets that encompass residues 139 and 186 of PrPSc. These findings show how variation in post-translational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease. PMID:28272426

  15. Lysine carboxylation: unveiling a spontaneous post-translational modification

    SciTech Connect

    Jimenez-Morales, David; Adamian, Larisa; Shi, Dashuang; Liang, Jie

    2014-01-01

    A computational method for the prediction of lysine carboxylation (KCX) in protein structures is described. The method accurately identifies misreported KCXs and predicts previously unknown KCX sites. The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown. In this work, the signature microenvironment of lysine-carboxylation sites has been characterized. In addition, a computational method called Predictor of Lysine Carboxylation (PreLysCar) for the detection of lysine carboxylation in proteins with available three-dimensional structures has been developed. The likely prevalence of lysine carboxylation in the proteome was assessed through large-scale computations. The results suggest that about 1.3% of large proteins may contain a carboxylated lysine residue. This unexpected prevalence of lysine carboxylation implies an enrichment of reactions in which it may play functional roles. The results also suggest that by switching enzymes on and off under appropriate physicochemical conditions spontaneous PTMs may serve as an important and widely used efficient biological machinery for regulation.

  16. Proteomic analysis of post translational modifications in cyanobacteria.

    PubMed

    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.

  17. Post-Translational Modifications of the TAK1-TAB Complex

    PubMed Central

    Hirata, Yusuke; Takahashi, Miki; Morishita, Tohru; Noguchi, Takuya; Matsuzawa, Atsushi

    2017-01-01

    Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family that is activated by growth factors and cytokines such as TGF-β, IL-1β, and TNF-α, and mediates a wide range of biological processes through activation of the nuclear factor-κB (NF-κB) and the mitogen-activated protein (MAP) kinase signaling pathways. It is well established that activation status of TAK1 is tightly regulated by forming a complex with its binding partners, TAK1-binding proteins (TAB1, TAB2, and TAB3). Interestingly, recent evidence indicates the importance of post-translational modifications (PTMs) of TAK1 and TABs in the regulation of TAK1 activation. To date, a number of PTMs of TAK1 and TABs have been revealed, and these PTMs appear to fine-tune and coordinate TAK1 activities depending on the cellular context. This review therefore focuses on recent advances in the understanding of the PTMs of the TAK1-TAB complex. PMID:28106845

  18. Contribution of Post-translational Phosphorylation to Sarcomere-Linked Cardiomyopathy Phenotypes

    PubMed Central

    Westfall, Margaret V.

    2016-01-01

    Secondary shifts develop in post-translational phosphorylation of sarcomeric proteins in multiple 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. PMID:27683560

  19. Forcefield_PTM: Ab Initio Charge and AMBER Forcefield Parameters for Frequently Occurring Post-Translational Modifications.

    PubMed

    Khoury, George A; Thompson, Jeff P; Smadbeck, James; Kieslich, Chris A; Floudas, Christodoulos A

    2013-12-10

    In this work, we introduce Forcefield_PTM, a set of AMBER forcefield parameters consistent with ff03 for 32 common post-translational modifications. Partial charges were calculated through ab initio calculations and a two-stage RESP-fitting procedure in an ether-like implicit solvent environment. The charges were found to be generally consistent with others previously reported for phosphorylated amino acids, and trimethyllysine, using different parameterization methods. Pairs of modified and their corresponding unmodified structures were curated from the PDB for both single and multiple modifications. Background structural similarity was assessed in the context of secondary and tertiary structures from the global dataset. Next, the charges derived for Forcefield_PTM were tested on a macroscopic scale using unrestrained all-atom Langevin molecular dynamics simulations in AMBER for 34 (17 pairs of modified/unmodified) systems in implicit solvent. Assessment was performed in the context of secondary structure preservation, stability in energies, and correlations between the modified and unmodified structure trajectories on the aggregate. As an illustration of their utility, the parameters were used to compare the structural stability of the phosphorylated and dephosphorylated forms of OdhI. Microscopic comparisons between quantum and AMBER single point energies along key χ torsions on several PTMs were performed and corrections to improve their agreement in terms of mean squared errors and squared correlation coefficients were parameterized. This forcefield for post-translational modifications in condensed-phase simulations can be applied to a number of biologically relevant and timely applications including protein structure prediction, protein and peptide design, docking, and to study the effect of PTMs on folding and dynamics. We make the derived parameters and an associated interactive webtool capable of performing post-translational modifications on proteins

  20. The regulation of BK channel activity by pre- and post-translational modifications.

    PubMed

    Kyle, Barry D; Braun, Andrew P

    2014-01-01

    Large conductance, Ca(2+)-activated K(+) (BK) channels represent an important pathway for the outward flux of K(+) ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca(2+)]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner.

  1. The regulation of BK channel activity by pre- and post-translational modifications

    PubMed Central

    Kyle, Barry D.; Braun, Andrew P.

    2014-01-01

    Large conductance, Ca2+-activated K+ (BK) channels represent an important pathway for the outward flux of K+ ions from the intracellular compartment in response to membrane depolarization, and/or an elevation in cytosolic free [Ca2+]. They are functionally expressed in a range of mammalian tissues (e.g., nerve and smooth muscles), where they can either enhance or dampen membrane excitability. The diversity of BK channel activity results from the considerable alternative mRNA splicing and post-translational modification (e.g., phosphorylation) of key domains within the pore-forming α subunit of the channel complex. Most of these modifications are regulated by distinct upstream cell signaling pathways that influence the structure and/or gating properties of the holo-channel and ultimately, cellular function. The channel complex may also contain auxiliary subunits that further affect channel gating and behavior, often in a tissue-specific manner. Recent studies in human and animal models have provided strong evidence that abnormal BK channel expression/function contributes to a range of pathologies in nerve and smooth muscle. By targeting the upstream regulatory events modulating BK channel behavior, it may be possible to therapeutically intervene and alter BK channel expression/function in a beneficial manner. PMID:25202279

  2. Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle.

    PubMed

    Lewis, Kevin M; Munske, Gerhard R; Byrd, Samuel S; Kang, Jeehoon; Cho, Hyun-Jai; Ríos, Eduardo; Kang, ChulHee

    2016-09-13

    Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc₂Man₁), while cardiac calsequestrin had five additional mannoses (GlcNAc₂Man₆). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca(2+)-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic "guiderail"-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca(2+) concentrations, and proper glycosylation, in turn, guarantees both effective Ca(2+) storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.

  3. Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle

    PubMed Central

    Lewis, Kevin M.; Munske, Gerhard R.; Byrd, Samuel S.; Kang, Jeehoon; Cho, Hyun-Jai; Ríos, Eduardo; Kang, ChulHee

    2016-01-01

    Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca2+-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca2+ concentrations, and proper glycosylation, in turn, guarantees both effective Ca2+ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site. PMID:27649144

  4. Protein post-translational modification analyses using on-chip immunoprobed isoelectric focusing.

    PubMed

    Tia, Samuel Q; Brown, Katharine; Chen, Danica; Herr, Amy E

    2013-03-05

    Post-translational modifications play a critical role in regulating protein function. Increasingly, determination of protein identity, estimation of abundance, and characterization of post-translational modifications are required for analysis of protein-mediated cell signaling networks. As such, we report an integrated and rapid multispectral immunoprobed isoelectric focusing technique for identifying specific proteins bearing post-translational modifications. Immunoprobed isoelectric focusing is composed of isoelectric focusing in a large pore-size polyacrylamide gel to determine protein pI followed by immobilization of pI-resolved proteins. Proteins are immobilized via covalent attachment to a channel-filling benzophenone-functionalized polyacrylamide gel via brief UV exposure (photoblot), followed by multispectral antibody-based detection. The assay correlates observed post-translational modifications to pI shifts relative to the unmodified protein of interest. During the electrokinetically driven antibody probing stage, we observed nonuniform electrophoretic probe mobility along the channel axis. The spatially varying mobility is attributed to nonuniform charge arising from covalent attachment of ampholytes to the benzophenone-functionalized gel matrix during the photoblotting step. Using the multistep microfluidic assay, phosphorylated and acetylated forms of heat shock protein 27 and superoxide dismutase 2 were detected, respectively. The assay reported protein isoforms in immune-purified sample and raw cell lysate in 2 hours with sample volume requirements of 2 μL. This new assay is well-matched to systems biology frameworks for study of protein post-translational modifications.

  5. Histone deacetylases: salesmen and customers in the post-translational modification market.

    PubMed

    Brandl, André; Heinzel, Thorsten; Krämer, Oliver H

    2009-04-01

    HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N-epsilon-acetylated lysine residues in histones and non-histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post-translational modification. Such structural alterations can determine the stability, localization, activity and protein-protein interactions of HDACs. This subsequently affects the modification of their substrates and the co-ordination of cellular signalling networks. Intriguingly, physiologically relevant non-histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post-translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post-translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.

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

    PubMed Central

    Axelsen, Lene N.; Calloe, Kirstine; Holstein-Rathlou, Niels-Henrik; Nielsen, Morten S.

    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 expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open 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, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720

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

    PubMed Central

    Laedermann, Cedric J.; Abriel, Hugues; Decosterd, Isabelle

    2015-01-01

    In the peripheral sensory nervous system the neuronal expression of voltage-gated sodium channels (Navs) is very important for the transmission of nociceptive information since they give rise to the upstroke of the action potential (AP). Navs are composed of nine 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 AP and consequently modify nociceptive 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 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 tools to alleviate pain. PMID

  8. Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury

    PubMed Central

    Osna, Natalia A; Carter, Wayne G; Ganesan, Murali; Kirpich, Irina A; McClain, Craig J; Petersen, Dennis R; Shearn, Colin T; Tomasi, Maria L; Kharbanda, Kusum K

    2016-01-01

    It is likely that the majority of proteins will undergo post-translational modification, be it enzymatic or non-enzymatic. These modified protein(s) regulate activity, localization and interaction with other cellular molecules thereby maintaining cellular hemostasis. Alcohol exposure significantly alters several of these post-translational modifications leading to impairments of many essential physiological processes. Here, we present new insights into novel modifications following ethanol exposure and their role in the initiation and progression of liver injury. This critical review condenses the proceedings of a symposium at the European Society for the Biomedical Research on Alcoholism Meeting held September 12-15, 2015, in Valencia, Spain. PMID:27468209

  9. Post-translational control of genetic circuits using Potyvirus proteases

    PubMed Central

    Fernandez-Rodriguez, Jesus; Voigt, Christopher A.

    2016-01-01

    Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. PMID:27298256

  10. Cyclic mechanical strain of myocytes modifies CapZβ1 post translationally via PKCε.

    PubMed

    Lin, Ying-Hsi; Swanson, Erik R; Li, Jieli; Mkrtschjan, Michael A; Russell, Brenda

    2015-10-01

    The heart is exquisitely sensitive to mechanical stimuli and adapts to increased demands for work by enlarging the cardiomyocytes. In order to determine links between mechano-transduction mechanisms and hypertrophy, neonatal rat ventricular myocytes (NRVM) were subjected to physiologic strain for analysis of the dynamics of the actin capping protein, CapZ, and its post-translational modifications (PTM). CapZ binding rates were assessed after strain by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) expressed by a GFP-CapZβ1 adenovirus. To assess the role of the protein kinase C epsilon isoform (PKCε), rest or cyclic strain were combined with specific PKCε activation by constitutively active PKCε, or by inhibition with dominant negative PKCε (dnPKCε) expression. Significant increases of CapZ FRAP kinetics with strain were blunted by dnPKCε, suggesting that PKCε is involved in mechano-transduction signaling. Similar combinations of strain and PKC regulation in NRVMs were studied by PTM profiles of CapZβ1 using quantitative two-dimensional gel electrophoresis. The significantly increased charge on CapZ seen with mechanical strain was reversed by the addition of dnPKCε. Potential clinical relevance was confirmed in vivo by PTMs of CapZ in the failing heart of one-year old transgenic mice over-expressing PKCε. Furthermore, with strain there was significant PKCε translocation to the Z-disc and co-localization with CapZβ1 or α-actinin, which was quantified on confocal images. A hypothetical model is presented proposing that one destination of the mechanotransduction signaling pathways might be for PTMs of CapZ thereby regulating actin capping and filament assembly.

  11. Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3.

    PubMed

    Toni, Lee S; Padilla, Pamela A

    2016-02-01

    Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.

  12. In vivo post-translational modifications of recombinant mussel adhesive protein in insect cells.

    PubMed

    Lim, Seonghye; Kim, Kyoung Ro; Choi, Yoo Seong; Kim, Dae-Kyum; Hwang, Daehee; Cha, Hyung Joon

    2011-01-01

    Mussel adhesive proteins (MAPs) have been suggested as promising bioadhesives for diverse application fields, including medical uses. Previously, we successfully constructed and produced a new type of functional recombinant MAP, fp-151, in a prokaryotic Escherichia coli expression system. Even though the E. coli-derived MAP showed several excellent features, such as high production yield and efficient purification, in vitro enzymatic modification is required to convert tyrosine residues to l-3,4-dihydroxyphenyl alanine (dopa) molecules for its adhesive ability, due to the intrinsic inability of E. coli to undergo post-translational modification. In this work, we produced a soluble recombinant MAP in insect Sf9 cells, which are widely used as an effective and convenient eukaryotic expression system for eukaryotic foreign proteins. Importantly, we found that insect-derived MAP contained converted dopa residues by in vivo post-translational modification. In addition, insect-derived MAP also had other post-translational modifications including phosphorylation of serine and hydroxylation of proline that originally occurred in some natural MAPs. To our knowledge, this is the first report on in vivo post-translational modifications of MAP containing dopa and other modified amino acid residues.

  13. Smoothing T cell roads to the tumor: Chemokine post-translational regulation.

    PubMed

    Molon, Barbara; Viola, Antonella; Bronte, Vincenzo

    2012-05-01

    We described a novel tumor-associated immunosuppressive mechanism based on post-translational modifications of chemokines by reactive nitrogen species (RNS). To overcome tumor immunosuppressive hindrances, we designed and developed a new drug, AT38, that inhibits RNS generation at the tumor site. Combinatorial approaches with AT38 boost the effectiveness of cancer immunotherapy protocols.

  14. New roles for old modifications: emerging roles of N-terminal post-translational modifications in development and disease.

    PubMed

    Tooley, John G; Schaner Tooley, Christine E

    2014-12-01

    The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with N-terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange.

  15. New roles for old modifications: Emerging roles of N-terminal post-translational modifications in development and disease

    PubMed Central

    Tooley, John G; Schaner Tooley, Christine E

    2014-01-01

    The importance of internal post-translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N-terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N-terminal PTMs are now beginning to be elucidated. New findings show that N-terminal methylation, along with N-terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N-terminal ubiquitylation and N-terminal propionylation. Here, will discuss the recent advances in the functional studies of N-terminal PTMs, recount the new N-terminal PTMs being identified, and briefly examine the possibility of dynamic N-terminal PTM exchange. PMID:25209108

  16. Distinct eye movement patterns enhance dynamic visual acuity

    PubMed Central

    Palidis, Dimitrios J.; Wyder-Hodge, Pearson A.; Fooken, Jolande; Spering, Miriam

    2017-01-01

    Dynamic visual acuity (DVA) is the ability to resolve fine spatial detail in dynamic objects during head fixation, or in static objects during head or body rotation. This ability is important for many activities such as ball sports, and a close relation has been shown between DVA and sports expertise. DVA tasks involve eye movements, yet, it is unclear which aspects of eye movements contribute to successful performance. Here we examined the relation between DVA and the kinematics of smooth pursuit and saccadic eye movements in a cohort of 23 varsity baseball players. In a computerized dynamic-object DVA test, observers reported the location of the gap in a small Landolt-C ring moving at various speeds while eye movements were recorded. Smooth pursuit kinematics—eye latency, acceleration, velocity gain, position error—and the direction and amplitude of saccadic eye movements were linked to perceptual performance. Results reveal that distinct eye movement patterns—minimizing eye position error, tracking smoothly, and inhibiting reverse saccades—were related to dynamic visual acuity. The close link between eye movement quality and DVA performance has important implications for the development of perceptual training programs to improve DVA. PMID:28187157

  17. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    DOE PAGES

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less

  18. Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

    SciTech Connect

    Wu, Meiye; Singh, Anup K.

    2014-07-15

    In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.

  19. Methane Seep Carbonates Host Distinct, Diverse, and Dynamic Microbial Assemblages

    PubMed Central

    Pasulka, Alexis L.; Marlow, Jeffrey J.; Grupe, Benjamin M.; Levin, Lisa A.

    2015-01-01

    ABSTRACT Marine methane seeps are globally distributed geologic features in which reduced fluids, including methane, are advected upward from the subsurface. As a result of alkalinity generation during sulfate-coupled methane oxidation, authigenic carbonates form slabs, nodules, and extensive pavements. These carbonates shape the landscape within methane seeps, persist long after methane flux is diminished, and in some cases are incorporated into the geologic record. In this study, microbial assemblages from 134 native and experimental samples across 5,500 km, representing a range of habitat substrates (carbonate nodules and slabs, sediment, bottom water, and wood) and seepage conditions (active and low activity), were analyzed to address two fundamental questions of seep microbial ecology: (i) whether carbonates host distinct microbial assemblages and (ii) how sensitive microbial assemblages are to habitat substrate type and temporal shifts in methane seepage flux. Through massively parallel 16S rRNA gene sequencing and statistical analysis, native carbonates are shown to be reservoirs of distinct and highly diverse seep microbial assemblages. Unique coupled transplantation and colonization experiments on the seafloor demonstrated that carbonate-associated microbial assemblages are resilient to seep quiescence and reactive to seep activation over 13 months. Various rates of response to simulated seep quiescence and activation are observed among similar phylogenies (e.g., Chloroflexi operational taxonomic units) and similar metabolisms (e.g., putative S oxidizers), demonstrating the wide range of microbial sensitivity to changes in seepage flux. These results imply that carbonates do not passively record a time-integrated history of seep microorganisms but rather host distinct, diverse, and dynamic microbial assemblages. PMID:26695630

  20. Post-translational modifications of tubulin and microtubule stability in adult rat ventricular myocytes and immortalized HL-1 cardiomyocytes.

    PubMed

    Belmadani, Souad; Poüs, Christian; Fischmeister, Rodolphe; Méry, Pierre-François

    2004-03-01

    Little is known about the subcellular distribution and the dynamics of tubulins in adult cardiac myocytes although both are modified during cardiac hypertrophy and heart failure. Using confocal microscopy, we examined post-translational modifications of tubulin in fully differentiated ventricular myocytes isolated from adult rat hearts, as well as in immortalized and dividing HL-1 cardiomyocytes. Detyrosinated Glu-alpha-tubulin was the most abundant post-translationally modified tubulin found in ventricular myocytes, while acetylated- and delta2-alpha-tubulins were found in lower amounts or absent. In contrast, dividing HL-1 cardiomyocytes exhibited high levels of tyrosinated or acetylated alpha-tubulins. A mild nocodazole treatment (0.1 microM, 1 h) disrupted microtubules in HL-1 myocytes, but not in adult ventricular myocytes. A stronger treatment (10 microM, 2 h) was required to disassemble tubulins in adult myocytes. Glu-alpha-tubulin containing microtubules were more resistant to nocodazole treatment in HL-1 cardiomyocytes than in ventricular myocytes. Endogenous activation of the cAMP pathway with the forskolin analog L858051 (20 microM) or the beta-adrenergic agonist isoprenaline (10 microM) disrupted the most labile microtubules in HL-1 cardiomyocytes. In contrast, isoprenaline (10 microM), cholera toxin (200 ng/ml, a G(S)-protein activator), L858051 (20 microM) or forskolin (10 microM) had no effect on the microtubule network in ventricular myocytes. In addition, intracellular Ca2+ accumulation induced either by thapsigargin (2 microM) or caffeine (10 mM) did not modify microtubule stability in ventricular myocytes. Our data demonstrate the unique stability of the microtubule network in adult cardiac myocytes. We speculate that microtubule stability is required to support cellular integrity during cardiac contraction.

  1. Conditional Analysis of Dynamically Distinct Regions in Stratified Turbulence

    NASA Astrophysics Data System (ADS)

    Portwood, Gavin; de Bruyn Kops, Stephen; Taylor, John; Salehipour, Hasem; Caulfield, Colm-Cille

    2016-11-01

    Stratified flows have been shown to exhibit broadly intermittent flow dynamics at large scales. In DNS of forced homogeneous stratified turbulence, we employ a conditional averaging technique to distinguish compositional flow regions which define the entire flow domain. Here, we condition on the vertical density gradient at inertial and buoyancy length scales to subdivide homogeneous stratified turbulence into three distinct regions that may be characterised by Gn ≡ ɛ / νN2 . We show that flows across the Fr-Re parameter space exhibit regions of (a) moderately 'quiescent' flow with few three-dimensional overturnings, (b) 'layered' turbulent regions which have constrained vertical length scales, and (c) three dimensional 'patches' of turbulence and that these regions may be characterised by Gn O (1) , Gn O (10) , and Gn O (100) , respectively. We conjecture that treating stratified turbulence as an instantaneous assemblage of these different regions in varying proportions may explain some of the apparently highly scattered flow dynamics and statistics previously reported in the literature. U.S. Office of Naval Research via Grant N00014-15-1-2248; U.K. Engineering and Physical Sciences Research Council Grant EP/K034529/1; U.S. DoD HPCMP Frontier Project FP-CFD-FY14-007.

  2. Fluorescence-activated sorting of fixed nuclei: a general method for studying nuclei from specific cell populations that preserves post-translational modifications.

    PubMed

    Marion-Poll, Lucile; Montalban, Enrica; Munier, Annie; Hervé, Denis; Girault, Jean-Antoine

    2014-04-01

    Long-lasting brain alterations that underlie learning and memory are triggered by synaptic activity. How activity can exert long-lasting effects on neurons is a major question in neuroscience. Signalling pathways from cytoplasm to nucleus and the resulting changes in transcription and epigenetic modifications are particularly relevant in this context. However, a major difficulty in their study comes from the cellular heterogeneity of brain tissue. A promising approach is to directly purify identified nuclei. Using mouse striatum we have developed a rapid and efficient method for isolating cell type-specific nuclei from fixed adult brain (fluorescence-activated sorting of fixed nuclei; FAST-FIN). Animals are quickly perfused with a formaldehyde fixative that stops enzymatic reactions and maintains the tissue in the state it was at the time of death, including nuclear localisation of soluble proteins such as GFP and differences in nuclear size between cell types. Tissue is subsequently dissociated with a Dounce homogeniser and nuclei prepared by centrifugation in an iodixanol density gradient. The purified fixed nuclei can then be immunostained with specific antibodies and analysed or sorted by flow cytometry. Simple criteria allow distinction of neurons and non-neuronal cells. Immunolabelling and transgenic mice that express fluorescent proteins can be used to identify specific cell populations, and the nuclei from these populations can be efficiently isolated, even rare cell types such as parvalbumin-expressing interneurons. FAST-FIN allows the preservation and study of dynamic and labile post-translational protein modifications. It should be applicable to other tissues and species, and allow study of DNA and its modifications.

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

    PubMed

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J; Rogowska-Wrzesinska, Adelina; Jensen, Ole N

    2015-12-01

    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. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human 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 represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

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

    PubMed Central

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J.; Rogowska-Wrzesinska, Adelina; Jensen, Ole N.

    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. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human 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 represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3. PMID:26424599

  5. An update on transcriptional and post-translational regulation of brain voltage-gated sodium channels.

    PubMed

    Onwuli, Donatus O; Beltran-Alvarez, Pedro

    2016-03-01

    Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

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

    PubMed Central

    Baud, Véronique; Collares, Davi

    2016-01-01

    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. PMID:27153093

  7. Post-translation modification in Archaea: Lessons from Haloferax volcanii and other haloarchaea

    PubMed Central

    Eichler, Jerry; Maupin-Furlow, Julie

    2012-01-01

    As an ever-growing number of genome sequences appear, it is becoming increasingly clear that factors other than genome sequence impart complexity to the proteome. Of the various sources of proteomic variability, post-translational modifications most greatly serve to expand the variety of proteins found in the cell. Likewise, modulating the rates at which different proteins are degraded also results in a constantly changing cellular protein profile. While both strategies for generating proteomic diversity are adopted by organisms across evolution, the responsible pathways and enzymes in Archaea are often less well described than are their eukaryotic and bacterial counterparts. Studies on halophilic archaea, in particular Haloferax volcanii, originally isolated from the Dead Sea, are helping to fill the void. In this review, recent developments concerning post-translational modifications and protein degradation in the haloarchaea are discussed. PMID:23167813

  8. In vivo regulation of chemokine activity by post-translational modification.

    PubMed

    Moelants, Eva A V; Mortier, Anneleen; Van Damme, Jo; Proost, Paul

    2013-07-01

    Cytokines and chemokines represent two important groups of proteins that control the immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation leading to various diseases, including rheumatoid arthritis, characterized by chronic inflammation and bone erosion. Chemokine activity is regulated at multiple levels, such as post-translational modification (PTM) of chemokines and their receptors by specific enzymes including proteases and peptidylarginine deiminases. Many in vitro experiments underscore the importance of post-translational processing of human chemokines. PTMs may enhance or reduce chemokine activity or may alter the receptor specificity of chemokine ligands. However, identification of chemokine isoforms in physiological in vivo settings forms the ultimate proof that PTM of chemokines is relevant in regulating the biological activity of these molecules. This review summarizes current knowledge on the in vivo role for PTMs in the regulation of chemokine activity.

  9. Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

    PubMed Central

    Su, Xiaodan; Ren, Chen; Freitas, Michael A

    2008-01-01

    Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. PMID:17425457

  10. Post-translational modifications of linker histone H1 variants in mammals

    NASA Astrophysics Data System (ADS)

    Starkova, T. Yu; Polyanichko, A. M.; Artamonova, T. O.; Khodorkovskii, M. A.; Kostyleva, E. I.; Chikhirzhina, E. V.; Tomilin, A. N.

    2017-02-01

    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2–H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

  11. Lysine post-translational modification of glyceraldehyde-3-phosphate dehydrogenase regulates hepatic and systemic metabolism.

    PubMed

    Bond, Simon T; Howlett, Kirsten F; Kowalski, Greg M; Mason, Shaun; Connor, Timothy; Cooper, Adrian; Streltsov, Victor; Bruce, Clinton R; Walder, Ken R; McGee, Sean L

    2017-03-03

    Reciprocal regulation of hepatic glycolysis and gluconeogenesis contributes to systemic metabolic homeostasis. Recent evidence from lower order organisms has found that reversible post-translational modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), particularly acetylation, contributes to the reciprocal regulation of glycolysis/gluconeogenesis. However, whether this occurs in mammalian hepatocytes in vitro or in vivo is unknown. Several proteomics studies have identified 4 lysine residues in critical regions of mammalian GAPDH that are altered by multiple post-translational modifications. In FAO hepatoma cells, mutation of all 4 lysine residues (4K-R GAPDH) to mimic their unmodified state reduced GAPDH glycolytic activity and glycolytic flux and increased gluconeogenic GAPDH activity and glucose production. Hepatic expression of 4K-R GAPDH in mice increased GAPDH gluconeogenic activity and the contribution of gluconeogenesis to endogenous glucose production in the unfed state. Consistent with the increased reliance on the energy-consuming gluconeogenic pathway, plasma free fatty acids and ketones were elevated in mice expressing 4K-R GAPDH, suggesting enhanced lipolysis and hepatic fatty acid oxidation. In normal mice, food withholding and refeeding, as well as hormonal regulators of reciprocal glycolysis/gluconeogenesis, such as insulin, glucagon, and norepinephrine, had no effect on global GAPDH acetylation. However, GAPDH acetylation was reduced in obese and type 2 diabetic db/db mice. These findings show that post-translational modification of GAPDH lysine residues regulates hepatic and systemic metabolism, revealing an unappreciated role for hepatic GAPDH in substrate selection and utilization.-Bond, S. T., Howlett, K. F., Kowalski, G. M., Mason, S., Connor, T., Cooper, A., Streltsov, V., Bruce, C. R., Walder, K. R., McGee, S. L. Lysine post-translational modification of glyceraldehyde-3-phosphate dehydrogenase regulates hepatic and systemic

  12. Post-translational modifications of linker histone H1 variants in mammals.

    PubMed

    Starkova, T Yu; Polyanichko, A M; Artamonova, T O; Khodorkovskii, M A; Kostyleva, E I; Chikhirzhina, E V; Tomilin, A N

    2017-02-16

    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

  13. Global Identification of Protein Post-translational Modifications in a Single-Pass Database Search.

    PubMed

    Shortreed, Michael R; Wenger, Craig D; Frey, Brian L; Sheynkman, Gloria M; Scalf, Mark; Keller, Mark P; Attie, Alan D; Smith, Lloyd M

    2015-11-06

    Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify post-translational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified over 2200 unique, high-confidence modified peptides comprising 26 different PTM types in a single-pass database search.

  14. Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

    PubMed

    Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

    2012-08-01

    Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

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

    PubMed Central

    2011-01-01

    Background Complete and accurate genome annotation is crucial for comprehensive and systematic studies of biological systems. 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. Results We experimentally annotated the bacterial pathogen Salmonella Typhimurium 14028, using "shotgun" proteomics to accurately uncover the translational landscape and post-translational features. The data provide protein-level experimental validation for approximately half of the predicted protein-coding genes in Salmonella and suggest revisions to several genes that appear to have incorrectly assigned translational start sites, including a potential novel alternate start codon. Additionally, we uncovered 12 non-annotated genes missed by gene prediction programs, as well as evidence suggesting a role for one of these novel ORFs in Salmonella pathogenesis. We also characterized post-translational features in the Salmonella genome, including chemical modifications and proteolytic cleavages. We find that bacteria have a much larger and more complex repertoire of chemical modifications than previously thought including several novel modifications. Our in vivo proteolysis data identified more than 130 signal peptide and N-terminal methionine cleavage events critical for protein function. Conclusion This work highlights several ways in which application of proteomics data can improve the quality of genome annotations to facilitate novel biological insights and provides a comprehensive proteome map of Salmonella as a resource for systems analysis. PMID:21867535

  16. Thiazolyl Peptide Antibiotic Biosynthesis: A Cascade of Post-translational Modifications on Ribosomal Nascent Proteins*

    PubMed Central

    Walsh, Christopher T.; Acker, Michael G.; Bowers, Albert A.

    2010-01-01

    Antibiotics of the thiocillin, GE2270A, and thiostrepton class, which block steps in bacterial protein synthesis, contain a trithiazolyl (tetrahydro)pyridine core that provides the architectural constraints for high affinity binding to either the 50 S ribosomal subunit or elongation factor Tu. These mature antibiotic scaffolds arise from a cascade of post-translational modifications on 50–60-residue prepeptide precursors that trim away the N-terminal leader sequences (∼40 residues) while the C-terminal 14–18 residues are converted into the mature scaffold. In the producing microbes, the genes encoding the prepeptide open reading frames are flanked in biosynthetic clusters by genes encoding post-translational modification enzymes that carry out lantibiotic-type dehydrations of Ser and Thr residues to dehydroamino acid side chains, cyclodehydration and oxidation of cysteines to thiazoles, and condensation of two dehydroalanine residues en route to the (tetrahydro)pyridine core. The trithiazolyl pyridine framework thus arises from post-translational modification of the peptide backbone of three Cys and two Ser residues of the prepeptide. PMID:20522549

  17. Reproducible Analysis of Post-Translational Modifications in Proteomes—Application to Human Mutations

    PubMed Central

    Holehouse, Alex S.; Naegle, Kristen M.

    2015-01-01

    Background Protein post-translational modifications (PTMs) are an important aspect of protein regulation. The number of PTMs discovered within the human proteome, and other proteomes, has been rapidly expanding in recent years. As a consequence of the rate in which new PTMs are identified, analysis done in one year may result in different conclusions when repeated in subsequent years. Among the various functional questions pertaining to PTMs, one important relationship to address is the interplay between modifications and mutations. Specifically, because the linear sequence surrounding a modification site often determines molecular recognition, it is hypothesized that mutations near sites of PTMs may be more likely to result in a detrimental effect on protein function, resulting in the development of disease. Methods and Results We wrote an application programming interface (API) to make analysis of ProteomeScout, a comprehensive database of PTMs and protein information, easy and reproducible. We used this API to analyze the relationship between PTMs and human mutations associated with disease (based on the ‘Clinical Significance’ annotation from dbSNP). Proteins containing pathogenic mutations demonstrated a significant study bias which was controlled for by analyzing only well-studied proteins, based on their having at least one pathogenic mutation. We found that pathogenic mutations are significantly more likely to lie within eight amino acids of a phosphoserine, phosphotyrosine or ubiquitination site when compared to mutations in general, based on a Fisher’s Exact test. Despite the skew of pathogenic mutations occurring on positively charged arginines, we could not account for this relationship based only on residue type. Finally, we hypothesize a potential mechanism for a pathogenic mutation on RAF1, based on its proximity to a phosphorylation site, which represents a subtle regulation difference that may explain why its biochemical effect has failed to

  18. Cancer serum biomarkers based on aberrant post-translational modifications of glycoproteins: Clinical value and discovery strategies.

    PubMed

    Silva, M Luísa S

    2015-12-01

    Due to the increase in life expectancy in the last decades, as well as changes in lifestyle, cancer has become one of the most common diseases both in developed and developing countries. Early detection remains the most promising approach to improve long-term survival of cancer patients and this may be achieved by efficient screening of biomarkers in biological fluids. Great efforts have been made to identify specific alterations during oncogenesis. Changes at the cellular glycosylation profiles are among such alterations. The "glycosylation machinery" of cells is affected by malignant transformation due to the altered expression of glycogens, leading to changes in glycan biosynthesis and diversity. Alterations in the post-translational modifications of proteins that occur in cancer result in the expression of antigenically distinct glycoproteins. Therefore, these aberrant and cancer-specific glycoproteins and the autoantibodies that are produced in response to their presence constitute targets for cancer biomarkers' search. Different strategies have been implemented for the discovery of cancer glycobiomarkers and are herein reviewed, along with their potentialities and limitations. Practical issues related with serum analysis are also addressed, as well as the challenges that this area faces in the near future.

  19. In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana.

    PubMed

    Chen, Jiajia; Gao, Jun; Peng, Maolin; Wang, Yi; Yu, Yanyan; Yang, Pengyuan; Jin, Hong

    2015-07-30

    Post-translational modifications (PTMs) on histone are highly correlated with genetic and epigenetic regulation of gene expression from chromatin. Mass spectrometry (MS) has developed to be an optimal tool for the identification and quantification of histone PTMs. Derivatization of histones with chemicals such as propionic anhydride, N-hydroxysuccinimide ester (NHS-propionate) has been widely used in histone PTMs analysis in bottom-up MS strategy, which requires high purity for histone samples. However, biological samples are not always prepared with high purity, containing detergents or other interferences in most cases. As an alternative approach, an adaptation of in gel derivatization method, termed In-gel NHS, is utilized for a broader application in histone PTMs analysis and it is shown to be a more time-saving preparation method. The proposed method was optimized for a better derivatization efficiency and displayed high reproducibility, indicating quantification of histone PTMs based on In-gel NHS was achievable. Without any traditional fussy histone purification procedures, we succeeded to quantitatively profile the histone PTMs from Arabidopsis with selective knock down of CLF (clf-29) and the original parental (col) with In-gel NHS method in a rapid way, which indicated the high specificity of CLF on H3K27me3 in Arabidopsis. In-gel NHS quantification results also suggest distinctive histone modification patterns in plants, which is invaluable foundation for future studies on histone modifications in plants.

  20. Coordinated Post-translational Responses of Aquaporins to Abiotic and Nutritional Stimuli in Arabidopsis Roots*

    PubMed Central

    di Pietro, Magali; Vialaret, Jérôme; Li, Guo-Wei; Hem, Sonia; Prado, Karine; Rossignol, Michel; Maurel, Christophe; Santoni, Véronique

    2013-01-01

    In plants, aquaporins play a crucial role in regulating root water transport in response to environmental and physiological cues. Controls achieved at the post-translational level are thought to be of critical importance for regulating aquaporin function. To investigate the general molecular mechanisms involved, we performed, using the model species Arabidopsis, a comprehensive proteomic analysis of root aquaporins in a large set of physiological contexts. We identified nine physiological treatments that modulate root hydraulics in time frames of minutes (NO and H2O2 treatments), hours (mannitol and NaCl treatments, exposure to darkness and reversal with sucrose, phosphate supply to phosphate-starved roots), or days (phosphate or nitrogen starvation). All treatments induced inhibition of root water transport except for sucrose supply to dark-grown plants and phosphate resupply to phosphate-starved plants, which had opposing effects. Using a robust label-free quantitative proteomic methodology, we identified 12 of 13 plasma membrane intrinsic protein (PIP) aquaporin isoforms, 4 of the 10 tonoplast intrinsic protein isoforms, and a diversity of post-translational modifications including phosphorylation, methylation, deamidation, and acetylation. A total of 55 aquaporin peptides displayed significant changes after treatments and enabled the identification of specific and as yet unknown patterns of response to stimuli. The data show that the regulation of PIP and tonoplast intrinsic protein abundance was involved in response to a few treatments (i.e. NaCl, NO, and nitrate starvation), whereas changes in the phosphorylation status of PIP aquaporins were positively correlated to changes in root hydraulic conductivity in the whole set of treatments. The identification of in vivo deamidated forms of aquaporins and their stimulus-induced changes in abundance may reflect a new mechanism of aquaporin regulation. The overall work provides deep insights into the in vivo post-translational

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

    PubMed Central

    Zaffagnini, Mirko; Fermani, Simona; Costa, Alex; Lemaire, Stéphane D.; Trost, Paolo

    2013-01-01

    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. PMID:24282406

  2. Regulation of voltage gated calcium channels by GPCRs and post-translational modification.

    PubMed

    Huang, Junting; Zamponi, Gerald W

    2016-10-18

    Calcium entry via voltage gated calcium channels mediates a wide range of physiological functions, whereas calcium channel dysregulation has been associated with numerous pathophysiological conditions. There are myriad cell signaling pathways that act on voltage gated calcium channels to fine tune their activities and to regulate their cell surface expression. These regulatory mechanisms include the activation of G protein-coupled receptors and downstream phosphorylation events, and their control over calcium channel trafficking through direct physical interactions. Calcium channels also undergo post-translational modifications that alter both function and density of the channels in the plasma membrane. Here we focus on select aspects of these regulatory mechanisms and highlight recent developments.

  3. Post-translational modifications in Pseudomonas aeruginosa revolutionized by proteomic analysis.

    PubMed

    Ouidir, Tassadit; Jouenne, Thierry; Hardouin, Julie

    2016-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable individuals. It is known that post-translational modifications (PTMs) play a key role in bacterial physiology. Their characterization is still challenging and the recent advances in proteomics allow large-scale and high-throughput analyses of PTMs. Here, we provide an overview of proteomic data about the modified proteins in P. aeruginosa. We emphasize the significant contribution of proteomics in knowledge enhancement of PTMs (phosphorylation, N-acetylation and glycosylation) and we discuss their importance in P. aeruginosa physiology.

  4. Clostridiolysin S, a Post-translationally Modified Biotoxin from Clostridium botulinum*

    PubMed Central

    Gonzalez, David J.; Lee, Shaun W.; Hensler, Mary E.; Markley, Andrew L.; Dahesh, Samira; Mitchell, Douglas A.; Bandeira, Nuno; Nizet, Victor; Dixon, Jack E.; Dorrestein, Pieter C.

    2010-01-01

    Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark β-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S. PMID:20581111

  5. Methods for Identifying and Examining HTLV-1 HBZ Post-translational Modifications.

    PubMed

    Al-Saleem, Jacob; Kvaratskhelia, Mamuka; Green, Patrick L

    2017-01-01

    Post-translational modifications (PTMs) are chemical alterations to individual amino acids that alter a protein's conformation, stability, and/or function. Several pathogenic viruses have been shown to encode proteins with PTMs, including human T-cell leukemia virus type 1 (HTLV-1) Tax and Rex regulatory proteins. HTLV-1 basic leucine zipper protein (HBZ) was hypothesized to feature PTMs due to its functional activities and interactions with cellular transcription factors and acetyltransferases. Here, we describe the approach used to identify, via mass spectrometry, the PTMs of HBZ. In addition, we describe methods to determine the functional relevance of the identified PTMs.

  6. Global histone post-translational modifications and cancer: Biomarkers for diagnosis, prognosis and treatment?

    PubMed Central

    Khan, Shafqat Ali; Reddy, Divya; Gupta, Sanjay

    2015-01-01

    Global alterations in epigenetic landscape are now recognized as a hallmark of cancer. Epigenetic mechanisms such as DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs are proven to have strong association with cancer. In particular, covalent post-translational modifications of histone proteins are known to play an important role in chromatin remodeling and thereby in regulation of gene expression. Further, histone modifications have also been associated with different aspects of carcinogenesis and have been studied for their role in the better management of cancer patients. In this review, we will explore and discuss how histone modifications are involved in cancer diagnosis, prognosis and treatment. PMID:26629316

  7. Dysbiosis May Trigger Autoimmune Diseases via Inappropriate Post-Translational Modification of Host Proteins

    PubMed Central

    Lerner, Aaron; Aminov, Rustam; Matthias, Torsten

    2016-01-01

    The gut ecosystem with myriads of microorganisms and the high concentration of immune system cells can be considered as a separate organ on its own. The balanced interaction between the host and microbial cells has been shaped during the long co-evolutionary process. In dysbiotic conditions, however, this balance is compromised and results in abnormal interaction between the host and microbiota. It is hypothesize here that the changed spectrum of microbial enzymes involved in post-translational modification of proteins (PTMP) may contribute to the aberrant modification of host proteins thus generating autoimmune responses by the host, resulting in autoimmune diseases. PMID:26903965

  8. Integrated proteomic analysis of post-translational modifications by serial enrichment

    PubMed Central

    Mertins, Philipp; Qiao, Jana W; Patel, Jinal; Udeshi, Namrata D; Clauser, Karl R; Mani, D R; Burgess, Michael W; Gillette, Michael A; Jaffe, Jacob D; Carr, Steven A

    2014-01-01

    We report a mass spectrometry–based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. this technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells. PMID:23749302

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

    PubMed

    Schwämmle, Veit; Aspalter, Claudia-Maria; Sidoli, Simone; Jensen, Ole N

    2014-07-01

    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 marks, the order and hierarchy of their deposition, and their distinct biological functions. We developed the database CrossTalkDB to organize observed and reported co-existing histone marks as revealed by MS experiments of histone proteins and their derived peptides. Statistical assessment revealed 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 or exist across species, thereby revealing general rules for cross-talk between histone marks. The observed features are in accordance with previously reported examples of cross-talk. We observed novel types of interplay among acetylated residues, revealing positive cross-talk between nearby acetylated 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.

  10. Archaeal S-layer glycoproteins: post-translational modification in the face of extremes

    PubMed Central

    Kandiba, Lina; Eichler, Jerry

    2014-01-01

    Corresponding to the sole or basic component of the surface (S)-layer surrounding the archaeal cell in most known cases, S-layer glycoproteins are in direct contact with the harsh environments that characterize niches where Archaea can thrive. Accordingly, early work examining archaeal S-layer glycoproteins focused on identifying those properties that allow members of this group of proteins to maintain their structural integrity in the face of extremes of temperature, pH, and salinity, as well as other physical challenges. However, with expansion of the list of archaeal strains serving as model systems, as well as growth in the number of molecular tools available for the manipulation of these strains, studies on archaeal S-layer glycoproteins are currently more likely to consider the various post-translational modifications these polypeptides undergo. For instance, archaeal S-layer glycoproteins can undergo proteolytic cleavage, both N- and O-glycosylation, lipid-modification and oligomerization. In this mini-review, recent findings related to the post-translational modification of archaeal S-layer glycoproteins are considered. PMID:25505464

  11. Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

    PubMed

    Houtz, R L; Stults, J T; Mulligan, R M; Tolbert, N E

    1989-03-01

    Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

  12. Whole proteome analysis of post-translational modifications: applications of mass-spectrometry for proteogenomic annotation

    SciTech Connect

    Gupta, Nitin; Tanner, Stephen; Jaitly, Navdeep; Adkins, Joshua N.; Lipton, Mary S.; Edwards, Robert; Romine, Margaret F.; Osterman, Andrei; Bafna, Vineet; Smith, Richard D.; Pevzner, Pavel A.

    2007-09-04

    While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.

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

    PubMed

    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.

  14. A cytoplasmic male sterility-associated mitochondrial peptide in common bean is post-translationally regulated.

    PubMed Central

    Sarria, R; Lyznik, A; Vallejos, C E; Mackenzie, S A

    1998-01-01

    Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally. PMID:9668139

  15. Review: Post-translational cross-talk between brassinosteroid and sucrose signaling.

    PubMed

    Kühn, Christina

    2016-07-01

    A direct link has been elucidated between brassinosteroid function and perception, and sucrose partitioning and transport. Sucrose regulation and brassinosteroid signaling cross-talk at various levels, including the well-described regulation of transcriptional gene expression: BZR-like transcription factors link the signaling pathways. Since brassinosteroid responses depend on light quality and quantity, a light-dependent alternative pathway was postulated. Here, the focus is on post-translational events. Recent identification of sucrose transporter-interacting partners raises the question whether brassinosteroid and sugars jointly affect plant innate immunity and plant symbiotic interactions. Membrane permeability and sensitivity depends on the number of cell surface receptors and transporters. More than one endocytic route has been assigned to specific components, including brassinosteroid-receptors. The number of such proteins at the plasma membrane relies on endocytic recycling, internalization and/or degradation. Therefore, vesicular membrane trafficking is gaining considerable attention with regard to plant immunity. The organization of pattern recognition receptors (PRRs), other receptors or transporters in membrane microdomains participate in endocytosis and the formation of specific intracellular compartments, potentially impacting biotic interactions. This minireview focuses on post-translational events affecting the subcellular compartmentation of membrane proteins involved in signaling, transport, and defense, and on the cross-talk between brassinosteroid signals and sugar availability.

  16. Global profiling of co- and post-translationally N-myristoylated proteomes in human cells

    PubMed Central

    Thinon, Emmanuelle; Serwa, Remigiusz A.; Broncel, Malgorzata; Brannigan, James A.; Brassat, Ute; Wright, Megan H.; Heal, William P.; Wilkinson, Anthony J.; Mann, David J.; Tate, Edward W.

    2014-01-01

    Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. PMID:25255805

  17. Differential protein levels and post-translational modifications in spinal cord injury of the rat.

    PubMed

    Afjehi-Sadat, Leila; Brejnikow, Mika; Kang, Sung Ung; Vishwanath, Vinay; Walder, Nadja; Herkner, Kurt; Redl, Heinz; Lubec, Gert

    2010-03-05

    Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

  18. Post-translational Claisen Condensation and Decarboxylation en Route to the Bicyclic Core of Pantocin A

    PubMed Central

    Ghodge, Swapnil V.; Biernat, Kristen A.; Bassett, Sarah Jane; Redinbo, Matthew R.; Bowers, Albert A.

    2016-01-01

    Pantocin A (PA) is a member of the growing family of ribosomally encoded and post-translationally modified peptide natural products (RiPPs). PA is much smaller than most known RiPPs, a tripeptide with a tight bicyclic core that appears to be cleaved from the middle of a larger 30-residue precursor peptide. We show here that the enzyme PaaA catalyzes the double dehydration and decarboxylation of two glutamic acid residues in the 30-residue precursor PaaP. Further truncates of PaaP leader and follower peptide sequences demonstrate the different impacts of these two regions on PaaA-mediated tailoring and delineate an essential role for the follower sequence in the decarboxylation step. The crystal structure of apo PaaA is reported, allowing identification of structural features that set PaaA apart from other homologous enzymes that typically do not catalyze such extended post-translational chemistry. Together, these data reveal how additional chemistry can be extracted from a ubiquitous enzyme family toward ribosomally derived peptide natural product biosynthesis and suggest that more examples of such enzymes likely exist in untapped genomic space. PMID:27088303

  19. Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

    SciTech Connect

    Kim, Jeongkwon; Zhang, Rui; Strittmatter, Eric F.; Smith, Richard D.; Zand, Robert

    2008-07-11

    Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the posttranslational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial.

  20. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry.

    PubMed

    Barondeau, David P; Kassmann, Carey J; Tainer, John A; Getzoff, Elizabeth D

    2005-02-15

    The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylidene-imidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.

  1. Effector-triggered post-translational modifications and their role in suppression of plant immunity

    PubMed Central

    Howden, Andrew J. M.; Huitema, Edgar

    2012-01-01

    Plant–pathogen interactions feature complex signaling exchanges between host and microbes that ultimately determine association outcomes. Plants deploy pattern recognition receptors to perceive pathogen-associated molecular patterns, mount pattern-triggered immunity (PTI), and fend off potential pathogens. In recent years an increasing number of defense-signaling components have been identified along with a mechanistic understanding of their regulation during immune responses. Post-translational modifications (PTMs) are now thought to play a crucial role in regulating defense signaling. In a bid to suppress PTI and infect their host, pathogens have evolved large repertoires of effectors that trigger susceptibility and allow colonization of host tissues. While great progress has been made in elucidating defense-signaling networks in plants and the activities of effectors in immune suppression, a critical gap exists in our understanding of effector mechanism-of-action. Given the importance of PTMs in the regulation of defense signaling, we will explore the question: how do effectors modify the post-translational status of host proteins and thus interfere with host processes required for immunity? We will consider how emerging proteomics-based experimental strategies may help us answer this important question and ultimately open the pathogens’ effector black box. PMID:22811685

  2. STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post-Translational Modifications

    PubMed Central

    Spencer, Jean L.; Bhatia, Vivek N.; Whelan, Stephen A.; Costello, Catherine E.

    2014-01-01

    The identification of protein post-translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post-Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi-sample comparison by collating, scoring, and ranking PTMs and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting. PMID:25422678

  3. Post-translational derepression of invertase activity in source leaves via down-regulation of invertase inhibitor expression is part of the plant defense response.

    PubMed

    Bonfig, Katharina B; Gabler, Andrea; Simon, Uwe K; Luschin-Ebengreuth, Nora; Hatz, Martina; Berger, Susanne; Muhammad, Naseem; Zeier, Jürgen; Sinha, Alok K; Roitsch, Thomas

    2010-11-01

    There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports

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

    PubMed

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

    2015-01-01

    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.

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

    SciTech Connect

    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.

  6. Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

    PubMed Central

    Hu, Hao; Gu, Xi; Xue, Liang-Jiao; Swamy, Prashant S.; Harding, Scott A.; Tsai, Chung-Jui

    2016-01-01

    Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem where high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes. PMID:27790223

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

    PubMed Central

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

    2015-01-01

    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. PMID:26635632

  8. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

    DOE PAGES

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; ...

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans . Our top-down analysis provided the confident identification of 55 proteins in the periplasm andmore » characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.« less

  9. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

    PubMed Central

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; Meng, Da; Zhao, Rui; Tolić, Nikola; Cao, Li; Shukla, Anil; Monroe, Matthew E.; Moore, Ronald J.; Lipton, Mary S.; Paša-Tolić, Ljiljana

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm. PMID:23555055

  10. Regulation of FXR transcriptional activity in health and disease: Emerging roles of FXR cofactors and post-translational modifications.

    PubMed

    Kemper, Jongsook Kim

    2011-08-01

    Abnormally elevated lipid and glucose levels due to the disruption of metabolic homeostasis play causative roles in the development of metabolic diseases. A cluster of metabolic conditions, including dyslipidemia, abdominal obesity, and insulin resistance, is referred to as metabolic syndrome, which has been increasing globally at an alarming rate. The primary nuclear bile acid receptor, Farnesoid X Receptor (FXR, NR1H4), plays important roles in controlling lipid and glucose levels by regulating expression of target genes in response to bile acid signaling in enterohepatic tissues. In this review, I discuss how signal-dependent FXR transcriptional activity is dynamically regulated under normal physiological conditions and how it is dysregulated in metabolic disease states. I focus on the emerging roles of post-translational modifications (PTMs) and transcriptional cofactors in modulating FXR transcriptional activity and pathways. Dysregulation of nuclear receptor transcriptional signaling due to aberrant PTMs and cofactor interactions are key determinants in the development of metabolic diseases. Therefore, targeting such abnormal PTMs and transcriptional cofactors of FXR in disease states may provide a new molecular strategy for development of pharmacological agents to treat metabolic syndrome. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

  11. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

    PubMed

    Martin, Dale D O; Vilas, Gonzalo L; Prescher, Jennifer A; Rajaiah, Gurram; Falck, John R; Bertozzi, Carolyn R; Berthiaume, Luc G

    2008-03-01

    Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.

  12. Post-translational modifications of chicken myelin basic protein charge components.

    PubMed

    Kim, Jeongkwon; Zhang, Rui; Strittmatter, Eric F; Smith, Richard D; Zand, Robert

    2009-02-01

    Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP's. Mammalian MBP's, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the post-translational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial. The C1 component lacks any phosphorylated sites, a finding in agreement with the analysis of other MBP species. It also had a single methylation at R105 as did the components C2 and C3. The C2 component contains ten phosphorylated sites (S7, S18, S33, S64, S73, T96, S113, S141, S164, and S168), and modified arginine to citrulline residues at R24, and R165. Component C3 contains eight phosphorylated sites (S7, S33, S64, T96, S113, S141, S164, and S168), and citrulline residues at Arginine 41, R24 and R165. Partial deamidation of glutamine residues Q71, Q101 and Q146 were present in addition to asparagine N90 that was found in all three charge components. The glutamine at residue 3 is partially deamidated in isomers C1 and C2, whereas glutamine 74 and asparagine 83 were found not to be deamidated. Comparison of the PTM's of MBP's isolated

  13. Live interaction distinctively shapes social gaze dynamics in rhesus macaques.

    PubMed

    Dal Monte, Olga; Piva, Matthew; Morris, Jason A; Chang, Steve W C

    2016-10-01

    The dynamic interaction of gaze between individuals is a hallmark of social cognition. However, very few studies have examined social gaze dynamics after mutual eye contact during real-time interactions. We used a highly quantifiable paradigm to assess social gaze dynamics between pairs of monkeys and modeled these dynamics using an exponential decay function to investigate sustained attention after mutual eye contact. When monkeys were interacting with real partners compared with static images and movies of the same monkeys, we found a significant increase in the proportion of fixations to the eyes and a smaller dispersion of fixations around the eyes, indicating enhanced focal attention to the eye region. Notably, dominance and familiarity between the interacting pairs induced separable components of gaze dynamics that were unique to live interactions. Gaze dynamics of dominant monkeys after mutual eye contact were associated with a greater number of fixations to the eyes, whereas those of familiar pairs were associated with a faster rate of decrease in this eye-directed attention. Our findings endorse the notion that certain key aspects of social cognition are only captured during interactive social contexts and dependent on the elapsed time relative to socially meaningful events.

  14. [Post-translational modification (PTM) bioinformatics in China: progresses and perspectives].

    PubMed

    Zexian, Liu; Yudong, Cai; Xuejiang, Guo; Ao, Li; Tingting, Li; Jianding, Qiu; Jian, Ren; Shaoping, Shi; Jiangning, Song; Minghui, Wang; Lu, Xie; Yu, Xue; Ziding, Zhang; Xingming, Zhao

    2015-07-01

    Post-translational modifications (PTMs) are essential for regulating conformational changes, activities and functions of proteins, and are involved in almost all cellular pathways and processes. Identification of protein PTMs is the basis for understanding cellular and molecular mechanisms. In contrast with labor-intensive and time-consuming experiments, the PTM prediction using various bioinformatics approaches can provide accurate, convenient, and efficient strategies and generate valuable information for further experimental consideration. In this review, we summarize the current progresses made by Chineses bioinformaticians in the field of PTM Bioinformatics, including the design and improvement of computational algorithms for predicting PTM substrates and sites, design and maintenance of online and offline tools, establishment of PTM-related databases and resources, and bioinformatics analysis of PTM proteomics data. Through comparing similar studies in China and other countries, we demonstrate both advantages and limitations of current PTM bioinformatics as well as perspectives for future studies in China.

  15. Cyclisation mechanisms in the biosynthesis of ribosomally synthesised and post-translationally modified peptides

    PubMed Central

    2016-01-01

    Summary Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a large class of natural products that are remarkably chemically diverse given an intrinsic requirement to be assembled from proteinogenic amino acids. The vast chemical space occupied by RiPPs means that they possess a wide variety of biological activities, and the class includes antibiotics, co-factors, signalling molecules, anticancer and anti-HIV compounds, and toxins. A considerable amount of RiPP chemical diversity is generated from cyclisation reactions, and the current mechanistic understanding of these reactions will be discussed here. These cyclisations involve a diverse array of chemical reactions, including 1,4-nucleophilic additions, [4 + 2] cycloadditions, ATP-dependent heterocyclisation to form thiazolines or oxazolines, and radical-mediated reactions between unactivated carbons. Future prospects for RiPP pathway discovery and characterisation will also be highlighted. PMID:27559376

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

    PubMed

    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.

  17. Post-translational Modifications in Heart Failure: Small Changes, Big Impact.

    PubMed

    Lee, Ahyoung; Oh, Jae Gyun; Gorski, Przemek A; Hajjar, Roger J; Kho, Changwon

    2016-04-01

    Heart failure is a complex disease process with various aetiologies and is a significant cause of morbidity and death world-wide. Post-translational modifications (PTMs) alter protein structure and provide functional diversity in terms of physiological functions of the heart. In addition, alterations in protein PTMs have been implicated in human disease pathogenesis. Small ubiquitin-like modifier mediated modification (SUMOylation) pathway was found to play essential roles in cardiac development and function. Abnormal SUMOylation has emerged as a new feature of heart failure pathology. In this review, we will highlight the importance of SUMOylation as a regulatory mechanism of SERCA2a function, and its therapeutic potential for the treatment of heart failure.

  18. Autophagy regulates the post-translational cleavage of BCL-2 and promotes neuronal survival.

    PubMed

    Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto

    2010-05-18

    B-cell lymphoma 2 protein (BCL-2) is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs) that are known to undergo massive programmed cell death (apoptosis) during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice.

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

    PubMed

    Stepper, Judith; Shastri, Shilpa; Loo, Trevor S; Preston, Joanne C; Novak, Petr; Man, Petr; Moore, Christopher H; Havlíček, Vladimír; Patchett, Mark L; Norris, Gillian E

    2011-02-18

    O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22.

  20. Post-Translational Modifications of Cardiac Mitochondrial Proteins in Cardiovascular Disease: Not Lost in Translation

    PubMed Central

    Marquez, Jubert; Lee, Sung Ryul; Kim, Nari

    2016-01-01

    Protein post-translational modifications (PTMs) are crucial in regulating cellular biology by playing key roles in processes such as the rapid on and off switching of signaling network and the regulation of enzymatic activities without affecting gene expressions. PTMs lead to conformational changes in the tertiary structure of protein and resultant regulation of protein function such as activation, inhibition, or signaling roles. PTMs such as phosphorylation, acetylation, and S-nitrosylation of specific sites in proteins have key roles in regulation of mitochondrial functions, thereby contributing to the progression to heart failure. Despite the extensive study of PTMs in mitochondrial proteins much remains unclear. Further research is yet to be undertaken to elucidate how changes in the proteins may lead to cardiovascular and metabolic disease progression in particular. We aimed to summarize the various types of PTMs that occur in mitochondrial proteins, which might be associated with heart failure. This study will increase the understanding of cardiovascular diseases through PTM. PMID:26798379

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Rhodopsin: the Functional Significance of Asn-Linked Glycosylation and Other Post-translational Modifications

    PubMed Central

    Murray, Anne R.; Fliesler, Steven J.; Al-Ubaidi, Muayyad R.

    2010-01-01

    Rhodopsin, the G-protein coupled receptor in retinal rod photoreceptors, is a highly conserved protein that undergoes several types of post-translational modifications. These modifications are essential to maintain the protein’s structure as well as its proper function in the visual transduction cycle. Rhodopsin is N-glycosylated at Asn-2 and Asn-15 in its extracellular N-terminal domain. Mutations within the glycosylation consensus sequences of rhodopsin cause autosomal dominant retinitis pigmentosa, a disease that leads to blindness. Several groups have studied the role of rhodopsin’s N-linked glycan chains in protein structure and function using a variety of approaches. These include the generation of a transgenic mouse model, study of a naturally occurring mutant animal model, in vivo pharmacological inhibition of glycosylation, and in vitro analyses using transfected COS-1 cells. These studies have provided insights into the possible role of rhodopsin glycosylation, but have yielded conflicting results. PMID:19941415

  3. The emerging immunological role of post-translational modifications by reactive nitrogen species in cancer microenvironment.

    PubMed

    De Sanctis, Francesco; Sandri, Sara; Ferrarini, Giovanna; Pagliarello, Irene; Sartoris, Silvia; Ugel, Stefano; Marigo, Ilaria; Molon, Barbara; Bronte, Vincenzo

    2014-01-01

    Under many inflammatory contexts, such as tumor progression, systemic and peripheral immune response is tailored by reactive nitrogen species (RNS)-dependent post-translational modifications, suggesting a biological function for these chemical alterations. RNS modify both soluble factors and receptors essential to induce and maintain a tumor-specific immune response, creating a "chemical barrier" that impairs effector T cell infiltration and functionality in tumor microenvironment and supports the escape phase of cancer. RNS generation during tumor growth mainly depends on nitric oxide production by both tumor cells and tumor-infiltrating myeloid cells that constitutively activate essential metabolic pathways of l-arginine catabolism. This review provides an overview of the potential immunological and biological role of RNS-induced modifications and addresses new approaches targeting RNS either in search of novel biomarkers or to improve anti-cancer treatment.

  4. MBSJ MCC Young Scientist Award 2010. Recent progress in research on small post-translationally modified peptide signals in plants.

    PubMed

    Matsubayashi, Yoshikatsu

    2012-01-01

    Peptide signaling plays a major role in various aspects of plant growth and development, as has been shown in recent biochemical, genetic and bioinformatic studies. There are over a dozen secreted peptides recognized in plants known to regulate cellular functions. To become functional, these secreted peptide signals often undergo post-translational modifications, such as tyrosine sulfation, proline hydroxylation, and hydroxyproline arabinosylation, and successive proteolytic processing. These types of ‘small post-translationally modified peptide signals’ are one of the major groups of peptide signals found in plants. In parallel with the discovery of peptide signals, specific receptors for such peptide signals were identified as being membrane-localized leucine-rich repeat receptor kinases. This short review highlights the recent progress in research on small post-translationally modified peptide signals, including our own research.

  5. Post-translational modifications of Desulfovibrio vulgaris Hildenborough sulfate reduction pathway proteins.

    PubMed

    Gaucher, Sara P; Redding, Alyssa M; Mukhopadhyay, Aindrila; Keasling, Jay D; Singh, Anup K

    2008-06-01

    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 aspect of 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

  6. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

    PubMed Central

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

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

    SciTech Connect

    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

  8. NCOA3-mediated upregulation of mucin expression via transcriptional and post-translational changes during the development of pancreatic cancer

    PubMed Central

    Kumar, S; Das, S; Rachagani, S; Kaur, S; Joshi, S; Johansson, SL; Ponnusamy, MP; Jain, M; Batra, SK

    2015-01-01

    Pancreatic cancer (PC) is characterized by aberrant overexpression of mucins that contribute to its pathogenesis. Although the inflammatory cytokines contribute to mucin overexpression, the mucin profile of PC is markedly distinct from that of normal or inflamed pancreas. We postulated that de novo expression of various mucins in PC involves chromatin modifications. Analysis of chromatin modifying enzymes by PCR array identified differential expression of NCOA3 in MUC4-expressing PC cell lines. Immunohistochemistry analysis in tumor tissues from patients and spontaneous mouse models, and microarray analysis following the knockdown of NCOA3 were performed to elucidate its role in mucin regulation and overall impact on PC. Silencing of NCOA3 in PC cell lines resulted in significant downregulation of two most differentially expressed mucins in PC, MUC4 and MUC1 (P<0.01). Immunohistochemistry analysis in PC tissues and metastatic lesions established an association between NCOA3 and mucin (MUC1 and MUC4) expression. Spontaneous mouse model of PC (K-rasG12D; Pdx-1cre) showed early expression of Ncoa3 during preneoplastic lesions. Mechanistically, NCOA3 knockdown abrogated retinoic acid-mediated MUC4 upregulation by restricting MUC4 promoter accessibility as demonstrated by micrococcus nuclease digestion (P<0.05) and chromatin immuno-precipitation analysis. NCOA3 also created pro-inflammatory conditions by upregulating chemokines like CXCL1, 2, 5 and CCL20 (P<0.001). AKT, ubiquitin C, ERK1/2 and NF-κB occupied dominant nodes in the networks significantly modulated after NCOA3 silencing. In addition, NCOA3 stabilized mucins post translationally through fucosylation by FUT8, as the knockdown of FUT8 resulted in the downregulation of MUC4 and MUC1 at protein levels. PMID:25531332

  9. Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies

    PubMed Central

    2012-01-01

    Introduction Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. Methods We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. Results We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Conclusions Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified. PMID:22300536

  10. In vivo and in vitro post-translational polymorphism of chicken natural auto-antibodies.

    PubMed

    Bergstra, Tamara J; Smeets, Kim; Nieuwland, Mike G B; Parmentier, Henk K

    2010-08-01

    Natural antibodies (NAb) perform many important functions in various immune responses and are often polyreactive of nature with low binding affinity. Natural auto-antibodies (N(A)Ab) are NAb binding at least one auto-antigen. Polyreactivity of N(A)Ab has been proposed to rest on post-translational polymorphism of the immunoglobulin F(ab)(2) fragment caused by various locally present oxidizing agents, salts and lower or higher pH. Challenge with pathogen-associated molecular patterns (PAMP), such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA), respectively, may underlie N(A)Ab polymorphism by the activation of inflammatory cells whose products affect the three-dimensional structure of N(A)Ab F(ab)(2) fragments. We evaluated by Western blotting the effects of subcutaneous administered LPS and LTA, respectively, on binding characteristics of chicken N(A)Ab towards the 'auto-antigen' chicken-liver-cell-lysate (CCL) in situ prior to (day 0) and 3 days after subcutaneous challenge, as well as the effect of different in vitro maltreatments in the form of oxidizing agents: 5mM hydrogen peroxide, 10mM hydrogen peroxide, pH 2.6, and pH 2.0, aqua dest, and phosphate buffered saline (PBS) as control, respectively, on chicken N(A)Ab polymorphism. On both days 0 and 3 after challenge, N(A)Ab in plasma from all chickens bound to CCL. No significant differences of in vivo or in vitro maltreatments were found on the number of CCL fragments bound by the N(A)Ab. However, significant differences in the staining patterns of individual CCL molecular weight-identified fragments (MWIF) were found. The sum (Sigma) of newly stained fragments and disappeared fragments (SigmaMWIF) that were bound by plasma samples was significantly different between in vivo LPS or in vivo LTA challenged birds. Also, significant differences in the percentages of extinction intensity of these SigmaMWIF were found. In addition, the plasma samples obtained at day 0 and day 3 from both LTA and LPS

  11. Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides.

    PubMed

    Chew, Su Chuen; Kundukad, Binu; Seviour, Thomas; van der Maarel, Johan R C; Yang, Liang; Rice, Scott A; Doyle, Patrick; Kjelleberg, Staffan

    2014-08-05

    Biofilms are densely populated communities of microbial cells protected and held together by a matrix of extracellular polymeric substances. The structure and rheological properties of the matrix at the microscale influence the retention and transport of molecules and cells in the biofilm, thereby dictating population and community behavior. Despite its importance, quantitative descriptions of the matrix microstructure and microrheology are limited. Here, particle-tracking microrheology in combination with genetic approaches was used to spatially and temporally study the rheological contributions of the major exopolysaccharides Pel and Psl in Pseudomonas aeruginosa biofilms. Psl increased the elasticity and effective cross-linking within the matrix, which strengthened its scaffold and appeared to facilitate the formation of microcolonies. Conversely, Pel reduced effective cross-linking within the matrix. Without Psl, the matrix becomes more viscous, which facilitates biofilm spreading. The wild-type biofilm decreased in effective cross-linking over time, which would be advantageous for the spreading and colonization of new surfaces. This suggests that there are regulatory mechanisms to control production of the exopolysaccharides that serve to remodel the matrix of developing biofilms. The exopolysaccharides were also found to have profound effects on the spatial organization and integration of P. aeruginosa in a mixed-species biofilm model of P. aeruginosa-Staphylococcus aureus. Pel was required for close association of the two species in mixed-species microcolonies. In contrast, Psl was important for P. aeruginosa to form single-species biofilms on top of S. aureus biofilms. Our results demonstrate that Pel and Psl have distinct physical properties and functional roles during biofilm formation. Importance: Most bacteria grow as biofilms in the environment or in association with eukaryotic hosts. Removal of biofilms that form on surfaces is a challenge in clinical

  12. Reversible post-translational modification of proteins by nitrated fatty acids in vivo.

    PubMed

    Batthyany, Carlos; Schopfer, Francisco J; Baker, Paul R S; Durán, Rosario; Baker, Laura M S; Huang, Yingying; Cerveñansky, Carlos; Branchaud, Bruce P; Freeman, Bruce A

    2006-07-21

    Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The

  13. Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders.

    PubMed

    dos Santos-Pinto, José Roberto Aparecido; Lamprecht, Günther; Chen, Wei-Qiang; Heo, Seok; Hardy, John George; Priewalder, Helga; Scheibel, Thomas Rainer; Palma, Mario Sergio; Lubec, Gert

    2014-06-13

    Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk

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

    PubMed Central

    Ghahremani, Mina; Stigter, Kyla A.; Plaxton, William

    2016-01-01

    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. PMID:28248235

  15. Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.

    PubMed

    Bandiera, Silvio; Weidlich, Simone; Harth, Volker; Broede, Peter; Ko, Yun; Friedberg, Thomas

    2005-02-01

    Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues.

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

    PubMed Central

    Kurotani, Atsushi; Sakurai, Tetsuya

    2015-01-01

    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. PMID:26307970

  17. Software Analysis of Uncorrelated MS1 Peaks for Discovery of Post-Translational Modifications.

    PubMed

    Pascal, Bruce D; West, Graham M; Scharager-Tapia, Catherina; Flefil, Ricardo; Moroni, Tina; Martinez-Acedo, Pablo; Griffin, Patrick R; Carvalloza, Anthony C

    2015-12-01

    The goal in proteomics to identify all peptides in a complex mixture has been largely addressed using various LC MS/MS approaches, such as data dependent acquisition, SRM/MRM, and data independent acquisition instrumentation. Despite these developments, many peptides remain unsequenced, often due to low abundance, poor fragmentation patterns, or data analysis difficulties. Many of the unidentified peptides exhibit strong evidence in high resolution MS(1) data and are frequently post-translationally modified, playing a significant role in biological processes. Proteomics Workbench (PWB) software was developed to automate the detection and visualization of all possible peptides in MS(1) data, reveal candidate peptides not initially identified, and build inclusion lists for subsequent MS(2) analysis to uncover new identifications. We used this software on existing data on the autophagy regulating kinase Ulk1 as a proof of concept for this method, as we had already manually identified a number of phosphorylation sites Dorsey, F. C. et al (J. Proteome. Res. 8(11), 5253-5263 (2009)). PWB found all previously identified sites of phosphorylation. The software has been made freely available at http://www.proteomicsworkbench.com . Graphical Abstract ᅟ.

  18. Polygalacturonases from Moniliophthora perniciosa are regulated by fermentable carbon sources and possible post-translational modifications.

    PubMed

    Argôlo Santos Carvalho, Heliana; de Andrade Silva, Edson Mario; Carvalho Santos, Stenio; Micheli, Fabienne

    2013-11-01

    We report the first molecular and in silico analysis of Monilophthora perniciosa polygalacturonases (PGs). Three MpPG genes (MpPG1, MpPG2 and MpPG3) were identified and analyzed at transcriptional level, by RT-qPCR, in dikaryotic M. perniciosa mycelium grown on solid-bran based medium and on liquid medium supplemented with different fermentable and non-fermentable carbon sources. The MpPG genes presented different expression patterns suggesting different individual regulation. However, all are mainly regulated by fermentable carbon sources (galactose and mannose). The integrated analysis of PG gene expression and systems biology (using MpG1 and MpG2 orthologs in Neurospora crassa, named NCU06961 and NCU02369, respectively) allowed identifying some possible mechanism of protein regulation during the necrotrophic fungal phase. MpPG1-NCU06961 and MpPG2-NCU02369 directly or indirectly interacted with central and highly connected proteins involved in protein synthesis and protein regulation associated to post-translational modifications, in cell wall metabolism, and in cellular metabolism related to energy production. This analysis also allowed the identification of key proteins for further studies of M. perniciosa development and/or for disease management, such as MpPG2, a pectin methylesterase, an acetolactate synthase and the small ubiquitin-like modifier SMT3-like.

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

    PubMed Central

    Hashiguchi, Akiko; Komatsu, Setsuko

    2016-01-01

    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. PMID:28248251

  20. The Flavone Luteolin Suppresses SREBP-2 Expression and Post-Translational Activation in Hepatic Cells

    PubMed Central

    Wong, Tsz Yan; Lin, Shu-mei; Leung, Lai K.

    2015-01-01

    High blood cholesterol has been associated with cardiovascular diseases. The enzyme HMG CoA reductase (HMGCR) is responsible for cholesterol synthesis, and inhibitors of this enzyme (statins) have been used clinically to control blood cholesterol. Sterol regulatory element binding protein (SREBP) -2 is a key transcription factor in cholesterol metabolism, and HMGCR is a target gene of SREBP-2. Attenuating SREBP-2 activity could potentially minimize the expression of HMGCR. Luteolin is a flavone that is commonly detected in plant foods. In the present study, Luteolin suppressed the expression of SREBP-2 at concentrations as low as 1 μM in the hepatic cell lines WRL and HepG2. This flavone also prevented the nuclear translocation of SREBP-2. Post-translational processing of SREBP-2 protein was required for nuclear translocation. Luteolin partially blocked this activation route through increased AMP kinase (AMPK) activation. At the transcriptional level, the mRNA and protein expression of SREBP-2 were reduced through luteolin. A reporter gene assay also verified that the transcription of SREBF2 was weakened in response to this flavone. The reduced expression and protein processing of SREBP-2 resulted in decreased nuclear translocation. Thus, the transcription of HMGCR was also decreased after luteolin treatment. In summary, the results of the present study showed that luteolin modulates HMGCR transcription by decreasing the expression and nuclear translocation of SREBP-2. PMID:26302339

  1. De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins

    SciTech Connect

    Shen, Yufeng; Tolic, Nikola; Hixson, Kim K.; Purvine, Samuel O.; Anderson, Gordon A.; Smith, Richard D.

    2008-10-15

    De novo sequencing has a promise to discover the protein post-translation modifications; however, such approach is still in their infancy and not widely applied for proteomics practices due to its limited reliability. In this work, we describe a de novo sequencing approach for discovery of protein modifications through identification of the UStags (Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry for peptides and polypeptides in a yeast lysate, and the de novo sequences obtained were filtered to define a more limited set of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags’ prefix and suffix sequences and the UStags themselves) were used to infer the possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances of yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. Random matching of the de novo sequences to the predicted sequences were examined with use of two random (false) databases, and ~3% false discovery rates were estimated for the de novo-UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity are described. The de novo-UStag complements the UStag method previously reported by enabling discovery of new protein modifications.

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

    PubMed

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

    PubMed Central

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

    2016-01-01

    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. PMID:27174170

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

    PubMed Central

    Mendes Maia, Teresa; Gogendeau, Delphine; Pennetier, Carole; Janke, Carsten; Basto, Renata

    2014-01-01

    Summary 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. PMID:24414207

  5. Post-translational Modifications and Protein Quality Control in Motor Neuron and Polyglutamine Diseases

    PubMed Central

    Sambataro, Fabio; Pennuto, Maria

    2017-01-01

    Neurodegenerative diseases, including motor neuron and polyglutamine (polyQ) diseases, are a broad class of neurological disorders. These diseases are characterized by neuronal dysfunction and death, and by the accumulation of toxic aggregation-prone proteins in the forms of inclusions and micro-aggregates. Protein quality control is a cellular mechanism to reduce the burden of accumulation of misfolded proteins, a function that results from the coordinated actions of chaperones and degradation systems, such as the ubiquitin-proteasome system (UPS) and autophagy-lysosomal degradation system. The rate of turnover, aggregation and degradation of the disease-causing proteins is modulated by post-translational modifications (PTMs), such as phosphorylation, arginine methylation, palmitoylation, acetylation, SUMOylation, ubiquitination, and proteolytic cleavage. Here, we describe how PTMs of proteins linked to motor neuron and polyQ diseases can either enhance or suppress protein quality control check and protein aggregation and degradation. The identification of molecular strategies targeting these modifications may offer novel avenues for the treatment of these yet incurable diseases.

  6. Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin.

    PubMed

    Loebel, D; Scaloni, A; Paolini, S; Fini, C; Ferrara, L; Breer, H; Pelosi, P

    2000-09-01

    Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichia coli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

  7. Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes.

    PubMed

    Ishikawa, Koji; Makanae, Koji; Iwasaki, Shintaro; Ingolia, Nicholas T; Moriya, Hisao

    2017-01-01

    Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of subunits of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems.

  8. Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs)

    PubMed Central

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Valderrama, Raquel; Mata-Pérez, Capilla; Padilla, María N.; Corpas, Francisco J.; Barroso, Juan B.

    2016-01-01

    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. PMID:26909095

  9. Characterization of Proteoforms with Unknown Post-translational Modifications Using the MIScore

    SciTech Connect

    Kou, Qiang; Zhu, Binhai; Wu, Si; Ansong, Charles; Tolić, Nikola; Paša-Tolić, Ljiljana; Liu, Xiaowen

    2016-08-05

    Various proteoforms may be generated from a single gene due to primary structure alterations (PSAs) such as genetic variations, alternative splicing, and post-translational modifications (PTMs). Top-down mass spectrometry is capable of analyzing intact proteins and identifying patterns of multiple PSAs, making it the method of choice for studying complex proteoforms. In top-down proteomics, proteoform identification is often performed by searching tandem mass spectra against a protein sequence database that contains only one reference protein sequence for each gene or transcript variant in a proteome. Because of the incompleteness of the protein database, an identified proteoform may contain unknown PSAs compared with the reference sequence. Proteoform characterization is to identify and localize PSAs in a proteoform. Although many software tools have been proposed for proteoform identification by top-down mass spectrometry, the characterization of proteoforms in identified proteoform-spectrum matches still relies mainly on manual annotation. We propose to use the Modification Identification Score (MIScore), which is based on Bayesian models, to automatically identify and localize PTMs in proteoforms. Experiments showed that the MIScore is accurate in identifying and localizing one or two modifications.

  10. Enhanced top-down characterization of histone post-translational modifications

    SciTech Connect

    Tian, Zhixin; Tolić, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Paša-Tolić, Ljiljana

    2012-01-01

    Background: Multiple post-translational modifications (PTMs) on core histones often work synergistically to fine tune chromatin structure and functions, generating a “histone code” that can be interpreted by a variety of chromatin interacting proteins. Although previous bottom-up and middle-down proteomic approaches have been developed for limited characterization of PTMs on histone N-terminal tails, high-throughput methods for comprehensive identification of PTMs distributed along the entire primary amino acid sequence are yet to be implemented. Results: Here we report a novel online two-dimensional liquid chromatography - tandem mass spectrometry (2D LC–MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The metal-free LC system with reverse phase separation followed by weak cation exchange – hydrophilic interaction chromatography (WCX-HILIC) and online Orbitrap Velos tandem mass spectrometry allowed for unambiguous identification of over 700 histone isoforms from a single 2D LC–MS/MS analysis of 7.5 µg of purified core histones. In comparison with previous offline top-down analysis of H4, this online study identified 100 additional isoforms from 100-fold less sample. This platform enabled comprehensive characterization of histone modifications, including those beyond tail regions, with dramatically improved throughput and sensitivity compared to more traditional platforms. Isoforms identified included those with combinatorial PTMs extending well beyond the N-terminal tail regions as well as a large number of phosphorylated isoforms.

  11. Post-Translational Dosage Compensation Buffers Genetic Perturbations to Stoichiometry of Protein Complexes

    PubMed Central

    Makanae, Koji; Iwasaki, Shintaro; Moriya, Hisao

    2017-01-01

    Understanding buffering mechanisms for various perturbations is essential for understanding robustness in cellular systems. Protein-level dosage compensation, which arises when changes in gene copy number do not translate linearly into protein level, is one mechanism for buffering against genetic perturbations. Here, we present an approach to identify genes with dosage compensation by increasing the copy number of individual genes using the genetic tug-of-war technique. Our screen of chromosome I suggests that dosage-compensated genes constitute approximately 10% of the genome and consist predominantly of subunits of multi-protein complexes. Importantly, because subunit levels are regulated in a stoichiometry-dependent manner, dosage compensation plays a crucial role in maintaining subunit stoichiometries. Indeed, we observed changes in the levels of a complex when its subunit stoichiometries were perturbed. We further analyzed compensation mechanisms using a proteasome-defective mutant as well as ribosome profiling, which provided strong evidence for compensation by ubiquitin-dependent degradation but not reduced translational efficiency. Thus, our study provides a systematic understanding of dosage compensation and highlights that this post-translational regulation is a critical aspect of robustness in cellular systems. PMID:28121980

  12. Seed storage proteins of the globulin family are cleaved post-translationally in wheat embryos

    PubMed Central

    2012-01-01

    Background The 7S globulins are plant seed storage proteins that have been associated with the development of a number of human diseases, including peanut allergy. Immune reactivity to the wheat seed storage protein globulin-3 (Glo-3) has been associated with the development of the autoimmune disease type 1 diabetes in diabetes-prone rats and mice, as well as in a subset of human patients. Findings The present study characterized native wheat Glo-3 in salt-soluble wheat seed protein extracts. Glo-3-like peptides were observed primarily in the wheat embryo. Glo-3-like proteins varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting with anti-Glo-3A antibodies. Five major polypeptide spots were identified by mass spectrometry and N-terminal sequencing as belonging to the Glo-3 family. Conclusions These results in combination with our previous findings have allowed for the development of a hypothetical model of the post-translational events contributing to the wheat 7S globulin profile in mature wheat kernels. PMID:22838494

  13. Translational and post-translational regulation of mouse cation transport regulator homolog 1

    PubMed Central

    Nomura, Yuki; Hirata, Yoko; Kiuchi, Kazutoshi; Oh-hashi, Kentaro

    2016-01-01

    Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5′ untranslated region (5′UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5′UTR. The proteasome pathway predominantly participated in the stability of the Chac1 protein; however, its expression was remarkably up-regulated by co-transfection with ubiquitin genes. Using an immunoprecipitation assay, we revealed that ubiquitin molecule was directly conjugated to Chac1, and that mutated Chac1 with all lysine residues replaced by arginine was also ubiquitinated. Finally, we showed that WT Chac1 but not ΔChac1 reduced the intracellular level of glutathione. Taken together, our results suggest that the Chac1 protein expression is regulated in translational and post-translational fashion due to the Kozak-like sequence in the 5′UTR and the ubiquitin-mediated pathways. The bidirectional roles of ubiquitination in regulating Chac1 stabilization might give us a new insight into understanding the homeostasis of glutathione under pathophysiological conditions. PMID:27302742

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

    PubMed

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

    2016-05-13

    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.

  15. Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin

    NASA Astrophysics Data System (ADS)

    Henze, Andrea; Homann, Thomas; Rohn, Isabelle; Aschner, Michael; Link, Christopher D.; Kleuser, Burkhard; Schweigert, Florian J.; Schwerdtle, Tanja; Bornhorst, Julia

    2016-11-01

    The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.

  16. Post-translational modifications in rheumatoid arthritis and atherosclerosis: Focus on citrullination and carbamylation.

    PubMed

    Spinelli, Francesca Romana; Pecani, Arbi; Conti, Fabrizio; Mancini, Riccardo; Alessandri, Cristiano; Valesini, Guido

    2016-09-01

    Coronary heart disease is the main cause of mortality in patients with rheumatoid arthritis (RA), a disease known to be associated with accelerated atherosclerosis. The role of inflammation and immunity in atherosclerotic process offers possible explanations for the increased cardiovascular risk in patients with RA. The immune response to citrullinated peptides has been extensively studied in RA; antibodies directed to citrullinated peptides are now a cornerstone for RA diagnosis. However, few studies have investigated the response to citrullinated peptides and the development of atherosclerotic plaque. Antibodies to carbamylated proteins can be detected before the clinical onset of RA, suggesting a potential predictive role for these antibodies; on the other hand, carbamylation of lipoproteins has been described in patients with cardiovascular disease. This review examines the role of citrullination and carbamylation, two post-translational protein modifications that appear to be involved in the pathogenesis of both RA and atherosclerosis, expanding the similarities between these two diseases. Further investigation on the role of the immune response to modified proteins may contribute to a better comprehension of cardiovascular disease in patients with RA.

  17. Importance of post-translational modifications on the function of key haemostatic proteins.

    PubMed

    Karlaftis, Vasiliki; Perera, Sachin; Monagle, Paul; Ignjatovic, Vera

    2016-01-01

    Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.

  18. Software Analysis of Uncorrelated MS1 Peaks for Discovery of Post-Translational Modifications

    NASA Astrophysics Data System (ADS)

    Pascal, Bruce D.; West, Graham M.; Scharager-Tapia, Catherina; Flefil, Ricardo; Moroni, Tina; Martinez-Acedo, Pablo; Griffin, Patrick R.; Carvalloza, Anthony C.

    2015-12-01

    The goal in proteomics to identify all peptides in a complex mixture has been largely addressed using various LC MS/MS approaches, such as data dependent acquisition, SRM/MRM, and data independent acquisition instrumentation. Despite these developments, many peptides remain unsequenced, often due to low abundance, poor fragmentation patterns, or data analysis difficulties. Many of the unidentified peptides exhibit strong evidence in high resolution MS1 data and are frequently post-translationally modified, playing a significant role in biological processes. Proteomics Workbench (PWB) software was developed to automate the detection and visualization of all possible peptides in MS1 data, reveal candidate peptides not initially identified, and build inclusion lists for subsequent MS2 analysis to uncover new identifications. We used this software on existing data on the autophagy regulating kinase Ulk1 as a proof of concept for this method, as we had already manually identified a number of phosphorylation sites Dorsey, F. C. et al (J. Proteome. Res. 8(11), 5253-5263 (2009)). PWB found all previously identified sites of phosphorylation. The software has been made freely available at http://www.proteomicsworkbench.com .

  19. PTMOracle: A Cytoscape App for Covisualizing and Coanalyzing Post-Translational Modifications in Protein Interaction Networks.

    PubMed

    Tay, Aidan P; Pang, Chi Nam Ignatius; Winter, Daniel L; Wilkins, Marc R

    2017-04-06

    Post-translational modifications of proteins (PTMs) act as key regulators of protein activity and of protein-protein interactions (PPIs). To date, it has been difficult to comprehensively explore functional links between PTMs and PPIs. To address this, we developed PTMOracle, a Cytoscape app for coanalyzing PTMs within PPI networks. PTMOracle also allows extensive data to be integrated and coanalyzed with PPI networks, allowing the role of domains, motifs, and disordered regions to be considered. For proteins of interest, or a whole proteome, PTMOracle can generate network visualizations to reveal complex PTM-associated relationships. This is assisted by OraclePainter for coloring proteins by modifications, OracleTools for network analytics, and OracleResults for exploring tabulated findings. To illustrate the use of PTMOracle, we investigate PTM-associated relationships and their role in PPIs in four case studies. In the yeast interactome and its rich set of PTMs, we construct and explore histone-associated and domain-domain interaction networks and show how integrative approaches can predict kinases involved in phosphodegrons. In the human interactome, a phosphotyrosine-associated network is analyzed but highlights the sparse nature of human PPI networks and lack of PTM-associated data. PTMOracle is open source and available at the Cytoscape app store: http://apps.cytoscape.org/apps/ptmoracle .

  20. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

    PubMed Central

    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David

    2015-01-01

    Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. PMID:25680960

  1. Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin

    PubMed Central

    Henze, Andrea; Homann, Thomas; Rohn, Isabelle; Aschner, Michael; Link, Christopher D.; Kleuser, Burkhard; Schweigert, Florian J.; Schwerdtle, Tanja; Bornhorst, Julia

    2016-01-01

    The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization – time of flight – mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling. PMID:27869126

  2. Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances

    PubMed Central

    Grabsztunowicz, Magda; Koskela, Minna M.; Mulo, Paula

    2017-01-01

    Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation. PMID:28280500

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26865024

  4. Post-translational biosynthesis of the protein-derived cofactor tryptophan tryptophylquinone

    PubMed Central

    Davidson, Victor L.; Wilmot, Carrie M.

    2014-01-01

    Methylamine dehydrogenase (MADH) catalyzes the oxidative deamination of methylamine to formaldehyde and ammonia. Tryptophan tryptophylquinone (TTQ) is the protein-derived cofactor of MADH that is required for these catalytic activities. TTQ is biosynthesized through the post-translational modification of two Trp residues within MADH, during which the indole rings of two Trp side chains are cross-linked and two oxygen atoms are inserted into one of the indole rings. MauG is a c-type diheme enzyme that catalyzes the final three reactions in TTQ formation. In total, this is a six-electron oxidation process requiring three cycles of MauG-dependent two-electron oxidation events using either H2O2 or O2. The MauG redox form that is responsible for the catalytic activity is an unprecedented bis-Fe(IV) species. The amino acids of MADH that are modified are ~ 40 Å from the site where MauG binds oxygen, and the reaction proceeds by a hole hopping electron transfer mechanism. This review will address these highly unusual aspects of the long range catalytic reaction that is mediated by MauG. PMID:23746262

  5. Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications*

    PubMed Central

    Halim, Adnan; Carlsson, Michael C.; Madsen, Caroline Benedicte; Brand, Stephanie; Møller, Svenning Rune; Olsen, Carl Erik; Vakhrushev, Sergey Y.; Brimnes, Jens; Wurtzen, Peter Adler; Ipsen, Henrik; Petersen, Bent L.; Wandall, Hans H.

    2015-01-01

    Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM1 characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines. PMID:25389185

  6. Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk*

    PubMed Central

    Huang, Yuanhua; Xu, Bosen; Zhou, Xueya; Li, Ying; Lu, Ming; Jiang, Rui; Li, Tingting

    2015-01-01

    Post-translational modification (PTM)1 plays an important role in regulating the functions of proteins. PTMs of multiple residues on one protein may work together to determine a functional outcome, which is known as PTM cross-talk. Identification of PTM cross-talks is an emerging theme in proteomics and has elicited great interest, but their properties remain to be systematically characterized. To this end, we collected 193 PTM cross-talk pairs in 77 human proteins from the literature and then tested location preference and co-evolution at the residue and modification levels. We found that cross-talk events preferentially occurred among nearby PTM sites, especially in disordered protein regions, and cross-talk pairs tended to co-evolve. Given the properties of PTM cross-talk pairs, a naïve Bayes classifier integrating different features was built to predict cross-talks for pairwise combination of PTM sites. By using a 10-fold cross-validation, the integrated prediction model showed an area under the receiver operating characteristic (ROC) curve of 0.833, superior to using any individual feature alone. The prediction performance was also demonstrated to be robust to the biases in the collected PTM cross-talk pairs. The integrated approach has the potential for large-scale prioritization of PTM cross-talk candidates for functional validation and was implemented as a web server available at http://bioinfo.bjmu.edu.cn/ptm-x/. PMID:25605461

  7. A novel mechanism of post-translational modulation of HMGA functions by the histone chaperone nucleophosmin

    PubMed Central

    Arnoldo, Laura; Sgarra, Riccardo; Chiefari, Eusebio; Iiritano, Stefania; Arcidiacono, Biagio; Pegoraro, Silvia; Pellarin, Ilenia; Brunetti, Antonio; Manfioletti, Guidalberto

    2015-01-01

    High Mobility Group A are non-histone nuclear proteins that regulate chromatin plasticity and accessibility, playing an important role both in physiology and pathology. Their activity is controlled by transcriptional, post-transcriptional, and post-translational mechanisms. In this study we provide evidence for a novel modulatory mechanism for HMGA functions. We show that HMGAs are complexed in vivo with the histone chaperone nucleophosmin (NPM1), that this interaction requires the histone-binding domain of NPM1, and that NPM1 modulates both DNA-binding affinity and specificity of HMGAs. By focusing on two human genes whose expression is directly regulated by HMGA1, the Insulin receptor (INSR) and the Insulin-like growth factor-binding protein 1 (IGFBP1) genes, we demonstrated that occupancy of their promoters by HMGA1 was NPM1-dependent, reflecting a mechanism in which the activity of these cis-regulatory elements is directly modulated by NPM1 leading to changes in gene expression. HMGAs need short stretches of AT-rich nucleosome-free regions to bind to DNA. Therefore, many putative HMGA binding sites are present within the genome. Our findings indicate that NPM1, by exerting a chaperoning activity towards HMGAs, may act as a master regulator in the control of DNA occupancy by these proteins and hence in HMGA-mediated gene expression. PMID:25711412

  8. FBXW7 modulates cellular stress response and metastatic potential via HSF1 post-translational modification

    PubMed Central

    Aranda-Orgilles, Beatriz; Lui, Kevin; Aydin, Iraz T.; Trimarchi, Thomas; Darvishian, Farbod; Salvaggio, Christine; Zhong, Judy; Bhatt, Kamala; Chen, Emily I.; Celebi, Julide T.; Lazaris, Charalampos; Tsirigos, Aristotelis; Osman, Iman; Hernando, Eva; Aifantis, Iannis

    2015-01-01

    Heat-shock factor 1 (HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has been evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase FBXW7 α interacts with HSF1 through a conserved motif phosphorylated by GSK3β and ERK1. FBXW7α ubiquitylates HSF1 and loss of FBXW7α results in impaired degradation of nuclear HSF1 and defective heat-shock response attenuation. FBXW7α is either mutated or transcriptionally downregulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. FBXW7α deficiency and subsequent HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the HSF1 transcriptional program both in the presence of exogenous stress and in cancer. PMID:25720964

  9. [Ocular diseases caused by accumulation of proteins with post-translational modifications].

    PubMed

    Kaji, Yuichi

    2009-03-01

    Long-term duration of lifestyle-related diseases including diabetes induces various ocular diseases. For this reason, the development of lifestyle-related ocular diseases is closely related to the aging process. In the present study, we tried to reveal the molecular mechanism of lifestyle-related ocular diseases, especially diabetic complications of the eyes, in relation to aging. To unify the molecular mechanisms of diabetic complications and aging changes of the eyes, we focused on two kinds of nonenzymatic post-translational modification products: advanced glycation end products (AGEs) and D-amino acids. We found that the accumulation of proteins rich in AGEs and D-amino acids plays a central role in the development of both diabetic complications and such changes of the eyes as diabetic retinopathy, diabetic keratopathy, pinguecula, spheroidal degeneration of the cornea, and drusen. In addition, decreased function in AGE-modified and D-amino acid-containing proteins is a factor in the development of diabetic complications and aging changes in eyes. In this way, posttranslational changes in molecules and amino acids are important contributing factors in the development of diabetic complications and aging changes in eyes. In conclusion, accumulation of AGE-modified and D-amino acid-containing proteins is the molecular mechanism of both diabetic complications and the aging changes in eyes.

  10. Lysine Glutarylation Is a Protein Post-Translational Modification Regulated by SIRT5

    PubMed Central

    Tan, Minjia; Peng, Chao; Anderson, Kristin A.; Chhoy, Peter; Xie, Zhongyu; Dai, Lunzhi; Park, Jeong Soon; Chen, Yue; Huang, He; Zhang, Yi; Ro, Jennifer; Wagner, Gregory R.; Green, Michelle F.; Madsen, Andreas S.; Schmiesing, Jessica; Peterson, Brett S.; Xu, Guofeng; Ilkayeva, Olga R.; Muehlbauer, Michael J.; Braulke, Thomas; Mühlhausen, Chris; Backos, Donald S.; Olsen, Christian A.; McGuire, Peter J.; Pletcher, Scott D.; Lombard, David B.; Hirschey, Matthew D.; Zhao, Yingming

    2014-01-01

    We report the identification and characterization of a five-carbon protein post-translational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric academia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5. PMID:24703693

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Aberrant post-translational modifications compromise human myosin motor function in old age.

    PubMed

    Li, Meishan; Ogilvie, Hannah; Ochala, Julien; Artemenko, Konstantin; Iwamoto, Hiroyuki; Yagi, Naoto; Bergquist, Jonas; Larsson, Lars

    2015-04-01

    Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

  13. Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible

    NASA Technical Reports Server (NTRS)

    Parat, M. O.; Fox, P. L.

    2001-01-01

    Caveolin-1 is a palmitoylated protein involved in assembly of signaling molecules in plasma membrane subdomains termed caveolae and in intracellular cholesterol transport. Three cysteine residues in the C terminus of caveolin-1 are subject to palmitoylation, which is not necessary for caveolar targeting of caveolin-1. Protein palmitoylation is a post-translational and reversible modification that may be regulated and that in turn may regulate conformation, membrane association, protein-protein interactions, and intracellular localization of the target protein. We have undertaken a detailed analysis of [(3)H]palmitate incorporation into caveolin-1 in aortic endothelial cells. The linkage of palmitate to caveolin-1 was hydroxylamine-sensitive and thus presumably a thioester bond. However, contrary to expectations, palmitate incorporation was blocked completely by the protein synthesis inhibitors cycloheximide and puromycin. In parallel experiments to show specificity, palmitoylation of aortic endothelial cell-specific nitric-oxide synthase was unaffected by these reagents. Inhibitors of protein trafficking, brefeldin A and monensin, blocked caveolin-1 palmitoylation, indicating that the modification was not cotranslational but rather required caveolin-1 transport from the endoplasmic reticulum and Golgi to the plasma membrane. In addition, immunophilin chaperones that form complexes with caveolin-1, i.e. FK506-binding protein 52, cyclophilin A, and cyclophilin 40, were not necessary for caveolin-1 palmitoylation because agents that bind immunophilins did not inhibit palmitoylation. Pulse-chase experiments showed that caveolin-1 palmitoylation is essentially irreversible because the release of [(3)H]palmitate was not significant even after 24 h. These results show that [(3)H]palmitate incorporation is limited to newly synthesized caveolin-1, not because incorporation only occurs during synthesis but because the continuous presence of palmitate on caveolin-1 prevents

  14. Post-translational oxidative modification and inactivation of mitochondrial complex I in epileptogenesis.

    PubMed

    Ryan, Kristen; Backos, Donald S; Reigan, Philip; Patel, Manisha

    2012-08-15

    Mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy development (epileptogenesis) is unknown. One consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (PTM). We hypothesize that complex I (CI), a protein complex of the mitochondrial electron transport chain, is a target for oxidant-induced PTMs, such as carbonylation, leading to impaired function during epileptogenesis. The goal of this study was to determine whether oxidative modifications occur and what impact they have on CI enzymatic activity in the rat hippocampus in response to kainate (KA)-induced epileptogenesis. Rats were injected with a single high dose of KA or vehicle and evidence for CI modifications was measured during the acute, latent, and chronic stages of epilepsy. Mitochondrial-specific carbonylation was increased acutely (48 h) and chronically (6 week), coincident with decreased CI activity. Mass spectrometry analysis of immunocaptured CI identified specific metal catalyzed carbonylation to Arg76 within the 75 kDa subunit concomitant with inhibition of CI activity during epileptogenesis. Computational-based molecular modeling studies revealed that Arg76 is in close proximity to the active site of CI and carbonylation of the residue is predicted to induce substantial structural alterations to the protein complex. These data provide evidence for the occurrence of a specific and irreversible oxidative modification of an important mitochondrial enzyme complex critical for cellular bioenergetics during the process of epileptogenesis.

  15. [Post-translational ligation and function of dual-vector transferred split CFTR gene].

    PubMed

    Zhu, Fu-Xiang; Liu, Ze-Long; Qu, Hui-Ge; Chi, Xiao-Yan

    2010-01-01

    The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

  16. Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

    PubMed Central

    Widschwendter, Martin; Sun, Dahui; Sieburg, Hans B.; Spruck, Charles

    2010-01-01

    Background Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens. Methodology/Principal Findings In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay. Conclusions/Significance This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research. PMID:20596523

  17. The three 'P's of mitophagy: PARKIN, PINK1, and post-translational modifications.

    PubMed

    Durcan, Thomas M; Fon, Edward A

    2015-05-15

    Two Parkinson's disease (PD)-associated proteins, the mitochondrial kinase PINK1 and the E3-ubiquitin (Ub) ligase PARKIN, are central to mitochondrial quality control. In this pathway, PINK1 accumulates on defective mitochondria, eliciting the translocation of PARKIN from the cytosol to mediate the clearance of damaged mitochondria via autophagy (mitophagy). Throughout the different stages of mitophagy, post-translational modifications (PTMs) are critical for the regulation of PINK1 and PARKIN activity and function. Indeed, activation and recruitment of PARKIN onto damaged mitochondria involves PINK1-mediated phosphorylation of both PARKIN and Ub. Through a stepwise cascade, PARKIN is converted from an autoinhibited enzyme into an active phospho-Ub-dependent E3 ligase. Upon activation, PARKIN ubiquitinates itself in concert with many different mitochondrial substrates. The Ub conjugates attached to these substrates can in turn be phosphorylated by PINK1, which triggers further cycles of PARKIN recruitment and activation. This feed-forward amplification loop regulates both PARKIN activity and mitophagy. However, the precise steps and sequence of PTMs in this cascade are only now being uncovered. For instance, the Ub conjugates assembled by PARKIN consist predominantly of noncanonical K6-linked Ub chains. Moreover, these modifications are reversible and can be disassembled by deubiquitinating enzymes (DUBs), including Ub-specific protease 8 (USP8), USP15, and USP30. However, PINK1-mediated phosphorylation of Ub can impede the activity of these DUBs, adding a new layer of complexity to the regulation of PARKIN-mediated mitophagy by PTMs. It is therefore evident that further insight into how PTMs regulate the PINK1-PARKIN pathway will be critical for our understanding of mitochondrial quality control.

  18. [Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function].

    PubMed

    Zhu, Fuxiang; Gong, Xiandi; Liu, Zelong; Yang, Shude; Qu, Huige; Chi, Xiaoyan

    2010-12-01

    Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.

  19. Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53*

    PubMed Central

    DeHart, Caroline J.; Chahal, Jasdave S.; Flint, S. J.; Perlman, David H.

    2014-01-01

    The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues—for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated. PMID:24056736

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

    PubMed

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-09-15

    G protein-coupled receptors are divided into three classes (A, B and C) based on homology of their seven transmembrane domains. Class C is the smallest class with 22 human receptor subtypes including eight metabotropic glutamate (mGlu1-8) receptors, two GABAB receptors (GABAB1 and GABAB2), three 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 review the existence of post-translational modifications in class C G protein-coupled receptors and their regulatory roles, with particular focus on glycosylation, phosphorylation, ubiquitination, SUMOylation, disulphide bonding and lipidation.

  1. Post-translational folding of the influenza C virus glycoprotein HEF: defective processing in cells expressing the cloned gene.

    PubMed

    Szepanski, S; Veit, M; Pleschka, S; Klenk, H D; Schmidt, M F; Herrler, G

    1994-05-01

    The post-translational processing of the influenza C virus glycoprotein HEF was analysed. In cells infected with influenza C virus, HEF protein is synthesized as a glycosylated 80K polypeptide. A post-translational conformational rearrangement involving the formation of intramolecular disulphide bonds results in a decrease in its electrophoretic mobility. Therefore, SDS-PAGE under non-reducing conditions suggests an Mr of about 100K, whereas under reducing conditions an 80K protein is observed which is in accordance with the sequence data. The 100K form was detected 10 min after synthesis of HEF, and transport to the cell surface took about 60 min. This result indicates that the conformational change presumably occurs in the endoplasmic reticulum. A difference in post-translational processing was observed when the HEF gene was expressed in the absence of other influenza C virus genes. In cells infected with recombinant simian virus 40, the 80K precursor was synthesized, but this protein was neither converted to the 100K form nor transported to the cell surface. Deletion of the short cytoplasmic tail of HEF (Arg-Thr-Lys) or replacement of the two basic amino acids by hydrophobic (Ile) or acidic residues (Glu) resulted in HEF protein which was partially converted to the 100K form. Influenza C virus glycoprotein obtained after transient expression of the HEF gene using the vaccinia virus system was completely converted to the 100K form. However, in neither expression system was HEF transported to the cell surface. The possibility is discussed that the interaction of HEF with another viral protein is required for the post-translational folding and transport of this glycoprotein. The M protein of influenza C virus is suggested as a candidate for the chaperone which might interact with the cytoplasmic tail of HEF.

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

    PubMed

    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.

  3. PTHGRN: unraveling post-translational hierarchical gene regulatory networks using PPI, ChIP-seq and gene expression data.

    PubMed

    Guan, Daogang; Shao, Jiaofang; Zhao, Zhongying; Wang, Panwen; Qin, Jing; Deng, Youping; Boheler, Kenneth R; Wang, Junwen; Yan, Bin

    2014-07-01

    Interactions among transcriptional factors (TFs), cofactors and other proteins or enzymes can affect transcriptional regulatory capabilities of eukaryotic organisms. Post-translational modifications (PTMs) cooperate with TFs and epigenetic alterations to constitute a hierarchical complexity in transcriptional gene regulation. While clearly implicated in biological processes, our understanding of these complex regulatory mechanisms is still limited and incomplete. Various online software have been proposed for uncovering transcriptional and epigenetic regulatory networks, however, there is a lack of effective web-based software capable of constructing underlying interactive organizations between post-translational and transcriptional regulatory components. Here, we present an open web server, post-translational hierarchical gene regulatory network (PTHGRN) to unravel relationships among PTMs, TFs, epigenetic modifications and gene expression. PTHGRN utilizes a graphical Gaussian model with partial least squares regression-based methodology, and is able to integrate protein-protein interactions, ChIP-seq and gene expression data and to capture essential regulation features behind high-throughput data. The server provides an integrative platform for users to analyze ready-to-use public high-throughput Omics resources or upload their own data for systems biology study. Users can choose various parameters in the method, build network topologies of interests and dissect their associations with biological functions. Application of the software to stem cell and breast cancer demonstrates that it is an effective tool for understanding regulatory mechanisms in biological complex systems. PTHGRN web server is publically available at web site http://www.byanbioinfo.org/pthgrn.

  4. Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification.

    PubMed

    Tsai, Kuang-Lei; Sun, Yuh-Ju; Huang, Cheng-Yang; Yang, Jer-Yen; Hung, Mien-Chie; Hsiao, Chwan-Deng

    2007-01-01

    FOXO3a is a transcription factor of the FOXO family. The FOXO proteins participate in multiple signaling pathways, and their transcriptional activity is regulated by several post-translational mechanisms, including phosphorylation, acetylation and ubiquitination. Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics. To understand how FOXO mediate transcriptional activity, we report here the 2.7 A crystal structure of the DNA-binding domain of FOXO3a (FOXO3a-DBD) bound to a 13-bp DNA duplex containing a FOXO consensus binding sequence (GTAAACA). Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins. In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.

  5. Post-translational Modification of LipL32 during Leptospira interrogans Infection

    PubMed Central

    Witchell, Timothy D.; Eshghi, Azad; Nally, Jarlath E.; Hof, Rebecca; Boulanger, Martin J.; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Cameron, Caroline E.

    2014-01-01

    Background Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. Methodology/Principal Findings Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. Conclusions/Significance The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although

  6. Profiles of Epigenetic Histone Post-translational Modifications at Type 1 Diabetes Susceptible Genes*

    PubMed Central

    Miao, Feng; Chen, Zhuo; Zhang, Lingxiao; Liu, Zheng; Wu, Xiwei; Yuan, Yate-Ching; Natarajan, Rama

    2012-01-01

    Both genetic and environmental factors are implicated in type 1 diabetes (T1D). Because environmental factors can trigger epigenetic changes, we hypothesized that variations in histone post-translational modifications (PTMs) at the promoter/enhancer regions of T1D susceptible genes may be associated with T1D. We therefore evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy nondiabetic controls, and explored their connections to T1D. We used the chromatin immunoprecipitation-linked to microarray approach to profile key histone PTMs, including H3-lysine 4 trimethylation (H3K4me3), H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac), and H4K16Ac at genes within the T1D susceptible loci in lymphocytes, and H3K4me3, H3K9me2, H3K9Ac, and H4K16Ac at the insulin-dependent diabetes mellitus 1 region in monocytes of T1D patients and healthy controls separately. We screened for potential variations in histone PTMs using computational methods to compare datasets from T1D and controls. Interestingly, we observed marked variations in H3K9Ac levels at the upstream regions of HLA-DRB1 and HLA-DQB1 within the insulin-dependent diabetes mellitus 1 locus in T1D monocytes relative to controls. Additional experiments with THP-1 monocytes demonstrated increased expression of HLA-DRB1 and HLA-DQB1 in response to interferon-γ and TNF-α treatment that were accompanied by changes in H3K9Ac at the same promoter regions as that seen in the patient monocytes. These results suggest that the H3K9Ac status of HLA-DRB1 and HLA-DQB1, two genes highly associated with T1D, may be relevant to their regulation and transcriptional response toward external stimuli. Thus, the promoter/enhancer architecture and chromatin status of key susceptible loci could be important determinants in their functional association to T1D susceptibility. PMID:22431725

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

    PubMed

    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

  8. The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation.

    PubMed

    Meijer, Inez M J; Kerperien, JoAnn; Sotoca, Ana M; van Zoelen, Everardus J J; van Leeuwen, Jeroen E M

    2013-04-01

    domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.

  9. Post-translational control of collagen fibrillogenesis in mineralizing cultures of chick osteoblasts

    NASA Technical Reports Server (NTRS)

    Gerstenfeld, L. C.; Riva, A.; Hodgens, K.; Eyre, D. R.; Landis, W. J.

    1993-01-01

    Cultured osteoblasts from chick embryo calvaria were used as a model system to investigate the post-translational extracellular mechanisms controlling the macroassembly of collagen fibrils. The results of these studies demonstrated that cultured osteoblasts secreted a collagenous extracellular matrix that assembled and mineralized in a defined temporal and spatial sequence. The assembly of collagen occurred in a polarized fashion, such that successive orthogonal arrays of fibrils formed between successive cell layers proceeding from the culture surface toward the media. Mineralization followed in the same manner, being observed first in the deepest and oldest fibril layers. Collagen fibrillogenesis, the kinetics of cross-link formation, and collagen stability in the extracellular matrix of the cultures were examined over a 30 day culture period. Between days 8 and 12 in culture, collagen fibril diameters increased from < 30 nm to an average of 30-45 nm. Thereafter, diameters ranged in size from 20 to 200 nm. Quantitation of the collagen cross-linking residues, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP), showed that these mature cross-links increased from undetectable levels to concentrations found in normal chick bone. Analysis of the kinetics of their formation by pulse-chase labeling the cultures with [3H]lysine showed a doubling time of approximately 5 days. The relationships between cross-link formation, fibrillogenesis, and collagen stability were examined in cultures treated with beta-aminopropionitrile (beta-APN), a potent inhibitor of lysyl oxidase and cross-link formation. In beta-APN-treated cultures, total collagen synthesis was increased twofold, with no change in mRNA levels for type I collagen, whereas the amount of collagen accumulated in the cell layer was decreased by 50% and mineral deposition was reduced. The rate of collagen retention in the matrix was assessed by pulse-chase analysis of [3H]proline over a 16 day period in

  10. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic acid post-translational modifications.

    PubMed

    Paulech, Jana; Liddy, Kiersten A; Engholm-Keller, Kasper; White, Melanie Y; Cordwell, Stuart J

    2015-03-01

    Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post-translational modification (pKa Cys-SO3H < 0) creates a unique charge distribution when localized on tryptic peptides at acidic pH that can be utilized for their purification. The method is based on electrostatic repulsion of Cys-SO2H/SO3H-containing peptides from cationic resins (i.e. "negative" selection) followed by "positive" selection using hydrophilic interaction liquid chromatography. Modification of strong cation exchange protocols decreased the complexity of initial flowthrough fractions by allowing for hydrophobic retention of neutral peptides. Coupling of strong cation exchange and hydrophilic interaction liquid chromatography allowed for increased enrichment of Cys-SO2H/SO3H (up to 80%) from other modified peptides. We identified 181 Cys-SO2H/SO3H sites from rat myocardial tissue subjected to physiologically relevant concentrations of H2O2 (<100 μm) or to ischemia/reperfusion (I/R) injury via Langendorff perfusion. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in energy utilization and contractility, as well as those involved in oxidative damage and repair.

  11. Post-translational Activation of Glutamate Cysteine Ligase with Dimercaprol: A Novel Mechanism of Inhibiting Neuroinflammation in vitro.

    PubMed

    McElroy, Pallavi B; Sri Hari, Ashwini; Day, Brian J; Patel, Manisha

    2017-02-15

    Neuroinflammation and oxidative stress are hallmarks of various neurological diseases. However, whether and how the redox processes control neuroinflammation is incompletely understood. We hypothesized that increasing cellular glutathione (GSH) levels would inhibit neuroinflammation. A series of thiol compounds were identified to elevate cellular GSH levels by a novel approach i.e. post-translational activation of glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH biosynthesis. These small thiolcontaining compounds were examined for their ability to increase intracellular GSH levels in a murine microglial cell line (BV2), of which dimercaprol [2,3-dimercapto-1-propanol (DMP)] was found to be the most effective compound. DMP increased GCL activity, decreased LPS-induced production of pro-inflammatory cytokines and iNOS induction in BV2 cells in a concentrationdependent manner. DMP's ability to elevate GSH levels and attenuate LPS-induced proinflammatory cytokine production was inhibited by buthionine sulfoximine, an inhibitor of GCL. DMP increased the expression of GCL holoenzyme without altering the expression of its subunits or Nrf2 target proteins (NQO1 and HO-1), suggesting a post-translational mechanism. DMP attenuated LPS-induced mitogen activated protein (MAP) kinase activation in BV2 cells suggesting the MAP kinase pathway as the signaling mechanism underlying DMP's effect. Finally, DMP's ability to increase GSH via GCL activation was observed in mixed cerebrocortical cultures and N27 dopaminergic cells. Together, the data demonstrate a novel mechanism of GSH elevation by posttranslational activation of GCL. Post-translational activation of GCL offers a novel targeted approach to control inflammation in chronic neuronal disorders associated with impaired adaptive responses.

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

    PubMed

    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

  13. Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications.

    PubMed

    Marionneau, Céline; Abriel, Hugues

    2015-05-01

    The cardiac voltage-gated Na(+) channel, Na(V)1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of Na(V)1.5 function, as caused by mutations in its gene SCN5A, may lead to many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of Na(V)1.5 that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of Na(V)1.5 protein expression and function tightly depends on genetic mechanisms, recent studies have demonstrated that Na(V)1.5 is the target of various post-translational modifications that are pivotal not only in physiological conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca(2+)/Calmodulin-dependent protein Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of Na(V)1.5. Modern and sensitive mass spectrometry approaches, applied directly to channel proteins that were purified from native cardiac tissues, have enabled the determination of the precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand the roles of these modifications on the expression and the function of Na(V)1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with Na(V)1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components.

  14. Post-Translational Incorporation of L-Phenylalanine into the C-Terminus of α-Tubulin as a Possible Cause of Neuronal Dysfunction

    PubMed Central

    Ditamo, Yanina; Dentesano, Yanela M.; Purro, Silvia A.; Arce, Carlos A.; Bisig, C. Gastón

    2016-01-01

    α-Tubulin C-terminus undergoes post-translational, cyclic tyrosination/detyrosination, and L-Phenylalanine (Phe) can be incorporated in place of tyrosine. Using cultured mouse brain-derived cells and an antibody specific to Phe-tubulin, we showed that: (i) Phe incorporation into tubulin is reversible; (ii) such incorporation is not due to de novo synthesis; (iii) the proportion of modified tubulin is significant; (iv) Phe incorporation reduces cell proliferation without affecting cell viability; (v) the rate of neurite retraction declines as level of C-terminal Phe incorporation increases; (vi) this inhibitory effect of Phe on neurite retraction is blocked by the co-presence of tyrosine; (vii) microtubule dynamics is reduced when Phe-tubulin level in cells is high as a result of exogenous Phe addition and returns to normal values when Phe is removed; moreover, microtubule dynamics is also reduced when Phe-tubulin is expressed (plasmid transfection). It is known that Phe levels are greatly elevated in blood of phenylketonuria (PKU) patients. The molecular mechanism underlying the brain dysfunction characteristic of PKU is unknown. Beyond the differences between human and mouse cells, it is conceivable the possibility that Phe incorporation into tubulin is the first event (or among the initial events) in the molecular pathways leading to brain dysfunctions that characterize PKU. PMID:27905536

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

    PubMed

    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.

  16. New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products.

    PubMed

    Ortega, Manuel A; van der Donk, Wilfred A

    2016-01-21

    Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of structurally diverse natural products. Their biological activities and unique biosynthetic pathways have sparked a growing interest in RiPPs. Furthermore, the relatively low genetic complexity associated with RiPP biosynthesis makes them excellent candidates for synthetic biology applications. This Review highlights recent developments in the understanding of the biosynthesis of several bacterial RiPP family members, the use of the RiPP biosynthetic machinery for generating novel macrocyclic peptides, and the implementation of tools designed to guide the discovery and characterization of novel RiPPs.

  17. Dynamic Changes in Amygdala Psychophysiological Connectivity Reveal Distinct Neural Networks for Facial Expressions of Basic Emotions

    PubMed Central

    Diano, Matteo; Tamietto, Marco; Celeghin, Alessia; Weiskrantz, Lawrence; Tatu, Mona-Karina; Bagnis, Arianna; Duca, Sergio; Geminiani, Giuliano; Cauda, Franco; Costa, Tommaso

    2017-01-01

    The quest to characterize the neural signature distinctive of different basic emotions has recently come under renewed scrutiny. Here we investigated whether facial expressions of different basic emotions modulate the functional connectivity of the amygdala with the rest of the brain. To this end, we presented seventeen healthy participants (8 females) with facial expressions of anger, disgust, fear, happiness, sadness and emotional neutrality and analyzed amygdala’s psychophysiological interaction (PPI). In fact, PPI can reveal how inter-regional amygdala communications change dynamically depending on perception of various emotional expressions to recruit different brain networks, compared to the functional interactions it entertains during perception of neutral expressions. We found that for each emotion the amygdala recruited a distinctive and spatially distributed set of structures to interact with. These changes in amygdala connectional patters characterize the dynamic signature prototypical of individual emotion processing, and seemingly represent a neural mechanism that serves to implement the distinctive influence that each emotion exerts on perceptual, cognitive, and motor responses. Besides these differences, all emotions enhanced amygdala functional integration with premotor cortices compared to neutral faces. The present findings thus concur to reconceptualise the structure-function relation between brain-emotion from the traditional one-to-one mapping toward a network-based and dynamic perspective. PMID:28345642

  18. Dynamic Changes in Amygdala Psychophysiological Connectivity Reveal Distinct Neural Networks for Facial Expressions of Basic Emotions.

    PubMed

    Diano, Matteo; Tamietto, Marco; Celeghin, Alessia; Weiskrantz, Lawrence; Tatu, Mona-Karina; Bagnis, Arianna; Duca, Sergio; Geminiani, Giuliano; Cauda, Franco; Costa, Tommaso

    2017-03-27

    The quest to characterize the neural signature distinctive of different basic emotions has recently come under renewed scrutiny. Here we investigated whether facial expressions of different basic emotions modulate the functional connectivity of the amygdala with the rest of the brain. To this end, we presented seventeen healthy participants (8 females) with facial expressions of anger, disgust, fear, happiness, sadness and emotional neutrality and analyzed amygdala's psychophysiological interaction (PPI). In fact, PPI can reveal how inter-regional amygdala communications change dynamically depending on perception of various emotional expressions to recruit different brain networks, compared to the functional interactions it entertains during perception of neutral expressions. We found that for each emotion the amygdala recruited a distinctive and spatially distributed set of structures to interact with. These changes in amygdala connectional patters characterize the dynamic signature prototypical of individual emotion processing, and seemingly represent a neural mechanism that serves to implement the distinctive influence that each emotion exerts on perceptual, cognitive, and motor responses. Besides these differences, all emotions enhanced amygdala functional integration with premotor cortices compared to neutral faces. The present findings thus concur to reconceptualise the structure-function relation between brain-emotion from the traditional one-to-one mapping toward a network-based and dynamic perspective.

  19. Abstract and Effector-Selective Decision Signals Exhibit Qualitatively Distinct Dynamics before Delayed Perceptual Reports

    PubMed Central

    Twomey, Deirdre M.; Kelly, Simon P.

    2016-01-01

    Electrophysiological research has isolated neural signatures of decision formation in a variety of brain regions. Studies in rodents and monkeys have focused primarily on effector-selective signals that translate the emerging decision into a specific motor plan, but, more recently, research on the human brain has identified an abstract signature of evidence accumulation that does not appear to play any direct role in action preparation. The functional dissociations between these distinct signal types have only begun to be characterized, and their dynamics during decisions with deferred actions with or without foreknowledge of stimulus-effector mapping, a commonly studied task scenario in single-unit and functional imaging investigations, have not been established. Here we traced the dynamics of distinct abstract and effector-selective decision signals in the form of the broad-band centro-parietal positivity (CPP) and limb-selective β-band (8–16 and 18–30 Hz) EEG activity, respectively, during delayed-reported motion direction decisions with and without foreknowledge of direction-response mapping. With foreknowledge, the CPP and β-band signals exhibited a similar gradual build-up following evidence onset, but whereas choice-predictive β-band activity persisted up until the delayed response, the CPP dropped toward baseline after peaking. Without foreknowledge, the CPP exhibited identical dynamics, whereas choice-selective β-band activity was eliminated. These findings highlight qualitative functional distinctions between effector-selective and abstract decision signals and are of relevance to the assumptions founding functional neuroimaging investigations of decision-making. SIGNIFICANCE STATEMENT Neural signatures of evidence accumulation have been isolated in numerous brain regions. Although animal neurophysiology has largely concentrated on effector-selective decision signals that translate the emerging decision into a specific motor plan, recent research

  20. Post-translational modifications of proliferating cell nuclear antigen: A key signal integrator for DNA damage response (Review).

    PubMed

    Zhu, Qiong; Chang, Yuxiao; Yang, Jin; Wei, Quanfang

    2014-05-01

    Previous studies have shown that the post-translational modifications of proliferating cell nuclear antigen (PCNA) may be crucial in influencing the cellular choice between different pathways, such as the cell cycle checkpoint, DNA repair or apoptosis pathways, in order to maintain genomic stability. DNA damage leads to replication stress and the subsequent induction of PCNA modification by small ubiquitin (Ub)-related modifiers and Ub, which has been identified to affect multiple biological processes of genomic DNA. Thus far, much has been learned concerning the behavior of modified PCNA as a key signal integrator in response to DNA damage. In humans and yeast, modified PCNA activates DNA damage bypass via an error-prone or error-free pathway to prevent the breakage of DNA replication forks, which may potentially induce double-strand breaks and subsequent chromosomal rearrangements. However, the exact mechanisms by which these pathways work and by what means the modified PCNA is involved in these processes remain elusive. Thus, the improved understanding of PCNA modification and its implications for DNA damage response may provide us with more insight into the mechanisms by which human cells regulate aberrant recombination events, and cancer initiation and development. The present review focuses on the post-translational modifications of PCNA and its important functions in mediating mammalian cellular response to different types of DNA damage.

  1. Multiple {gamma}-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

    SciTech Connect

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

    2010-03-26

    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 {gamma}-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 {gamma}-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.

  2. The K+ battery-regulating Arabidopsis K+ channel AKT2 is under the control of multiple post-translational steps

    PubMed Central

    Michard, Erwan; Rocha, Marcio; Gomez-Porras, Judith L; González, Wendy; Corrâa, Luiz Gustavo Guedes; Ramírez-Aguilar, Santiago J; Cuin, Tracey Ann

    2011-01-01

    Potassium (K+) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K+ in plant homeostasis was shown. It was demonstrated that K+ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K+ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K+ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K+ channel. PMID:21445013

  3. The role of post-translational modifications in fine-tuning BLM helicase function during DNA repair

    PubMed Central

    Böhm, Stefanie; Bernstein, Kara Anne

    2014-01-01

    RecQ-like helicases are a highly conserved family of proteins which are critical for preserving genome integrity. Genome instability is considered a hallmark of cancer and mutations within three of the five human RECQ genes cause hereditary syndromes that are associated with cancer predisposition. The human RecQ-like helicase BLM has a central role in DNA damage signaling, repair, replication, and telomere maintenance. BLM and its budding yeast orthologue Sgs1 unwind double-stranded DNA intermediates. Intriguingly, BLM functions in both a pro- and anti-recombinogenic manner upon replicative damage, acting on similar substrates. Thus, BLM activity must be intricately controlled to prevent illegitimate recombination events that could have detrimental effects on genome integrity. In recent years it has become evident that post-translational modifications (PTMs) of BLM allow a fine-tuning of its function. To date, BLM phosphorylation, ubiquitination, and SUMOylation have been identified, in turn regulating its subcellular localization, protein-protein interactions, and protein stability. In this review, we will discuss the cellular context of when and how these different modifications of BLM occur. We will reflect on the current model of how PTMs control BLM function during DNA damage repair and compare this to what is known about post-translational regulation of the budding yeast orthologue Sgs1. Finally, we will provide an outlook towards future research, in particular to dissect the cross-talk between the individual PTMs on BLM. PMID:25150915

  4. Update on phosphate and charged post-translationally modified amino acid parameters in the GROMOS force field.

    PubMed

    Margreitter, Christian; Reif, Maria M; Oostenbrink, Chris

    2017-04-15

    In this study, we propose newly derived parameters for phosphate ions in the context of the GROMOS force field parameter sets. The non-bonded parameters used up to now lead to a hydration free energy, which renders the dihydrogen phosphate ion too hydrophobic when compared to experimentally derived values, making a reparametrization of the phosphate moiety necessary. Phosphate species are of great importance in biomolecular simulations not only because of their crucial role in the backbone of nucleic acids but also as they represent one of the most important types of post-translational modifications to protein side-chains and are an integral part in many lipids. Our re-parametrization of the free dihydrogen phosphate (H 2PO 4-) and three derivatives (methyl phosphate, dimethyl phosphate, and phenyl phosphate) leads, in conjunction with the previously updated charged side-chains in the GROMOS parameter set 54A8, to new nucleic acid backbone parameters and a 54A8 version of the widely used GROMOS protein post-translational modification parameter set. © 2017 Wiley Periodicals, Inc.

  5. A hybrid two-component system of Tannerella forsythia affects autoaggregation and post-translational modification of surface proteins.

    PubMed

    Niwa, Daisuke; Nishikawa, Kiyoshi; Nakamura, Hiroshi

    2011-05-01

    Tannerella forsythia is a Gram-negative oral anaerobe closely associated with both periodontal and periapical diseases. The ORF TF0022 of strain ATCC 43037 encodes a hybrid two-component system consisting of an N-terminal histidine kinase and a C-terminal response regulator. Disruption of the TF0022 locus enhanced autoaggregation of the broth-cultured cells. Comparative proteome analyses revealed that two S-layer proteins in the TF0022 mutant exhibited decreased apparent masses by denaturing gel electrophoresis, suggesting a deficiency in post-translational modification. Furthermore, the mutant decreased the production of a glycosyltransferase encoded by TF1061 that is located in a putative glycosylation-related gene cluster. Quantitative real-time PCR revealed reduced transcription of TF1061 and the associated genes in the TF0022 mutant. These results indicate that TF0022 upregulates the expression of the glycosylation-related genes and suggest modulation of the autoaggregation of T. forsythia cells by a possible post-translational modification of cell-surface components.

  6. Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guerrero, Andres; Lerno, Larry; Barile, Daniela; Lebrilla, Carlito B.

    2015-03-01

    Bovine κ-caseinoglycomacropeptide (GMP) is a highly modified peptide from κ-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before.

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

    PubMed

    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.

  8. Post-translational modifications in regulation of pathogen surveillance and signaling in plants: The inside- (and perturbations from) outside story.

    PubMed

    Bhattacharjee, Saikat; Noor, Jewel Jameeta; Gohain, Bornali; Gulabani, Hitika; Dnyaneshwar, Ingole Kishor; Singla, Ankit

    2015-07-01

    In its lifetime a plant is exposed to pathogens of diverse types. Although methods of surveillance are broadly pathogen-individualized, immune signaling ultimately connect to common core networks maintained by key protein hubs. Defense elicitations modulate these hubs to re-allocate energy from central metabolic pathway into processes that execute immunity. Because unregulated defenses severely decrease growth and productivity of the host, signaling regulators within the networks function to achieve cellular equilibrium once the threat is minimized. Protein modifications by post-translational processes regulate the molecular switches and crosstalks between interconnected pathways spatially and temporally. Covalent modification of host targets connected to hubs are strategies used by most virulent effectors and result in re-routing signals to suppress host defenses. Resistance is a result of activation of specialized classes of receptors that short-circuit effector activities by co-localizing via post-translational modifications (PTMs) with effector targets. Despite advancement in proteome methodologies, our understanding of how PTMs regulate plant defenses remains elusive. This review presents protein-modifications as forefront regulators of plant innate immunity.

  9. SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana.

    PubMed

    Castro, Pedro Humberto; Verde, Nuno; Lourenço, Tiago; Magalhães, Alexandre Papadopoulos; Tavares, Rui Manuel; Bejarano, Eduardo Rodríguez; Azevedo, Herlânder

    2015-12-01

    Post-translational modification mechanisms function as switches that mediate the balance between optimum growth and the response to environmental stimuli, by regulating the activity of key proteins. SUMO (small ubiquitin-like modifier) attachment, or sumoylation, is a post-translational modification that is essential for the plant stress response, also modulating hormonal circuits to co-ordinate developmental processes. The Arabidopsis SUMO E3 ligase SAP and Miz 1 (SIZ1) is the major SUMO conjugation enhancer in response to stress, and is implicated in several aspects of plant development. Here we report that known SUMO targets are over-represented in multiple carbohydrate-related proteins, suggesting a functional link between sumoylation and sugar metabolism and signaling in plants. We subsequently observed that SUMO-conjugated proteins accumulate in response to high doses of sugar in a SIZ1-dependent manner, and that the null siz1 mutant displays increased expression of sucrose and starch catabolic genes and shows reduced starch levels. We demonstrated that SIZ1 controls germination time and post-germination growth via osmotic and sugar-dependent signaling, respectively. Glucose was specifically linked to SUMO-sugar interplay, with high levels inducing root growth inhibition and aberrant root hair morphology in siz1. The use of sugar analogs and sugar marker gene expression analysis allowed us to implicate SIZ1 in a signaling pathway dependent on glucose metabolism, probably involving modulation of SNF1-related kinase 1 (SnRK1) activity.

  10. Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

    PubMed Central

    Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

    2015-01-01

    Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

  11. Sinorhizobium meliloti Controls Nitric Oxide-Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein.

    PubMed

    Blanquet, Pauline; Silva, Liliana; Catrice, Olivier; Bruand, Claude; Carvalho, Helena; Meilhoc, Eliane

    2015-12-01

    Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

  12. The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-like modifiers.

    PubMed

    Estruch, Sara B; Graham, Sarah A; Deriziotis, Pelagia; Fisher, Simon E

    2016-02-12

    Mutations affecting the transcription factor FOXP2 cause a rare form of severe speech and language disorder. Although it is clear that sufficient FOXP2 expression is crucial for normal brain development, little is known about how this transcription factor is regulated. To investigate post-translational mechanisms for FOXP2 regulation, we searched for protein interaction partners of FOXP2, and identified members of the PIAS family as novel FOXP2 interactors. PIAS proteins mediate post-translational modification of a range of target proteins with small ubiquitin-like modifiers (SUMOs). We found that FOXP2 can be modified with all three human SUMO proteins and that PIAS1 promotes this process. An aetiological FOXP2 mutation found in a family with speech and language disorder markedly reduced FOXP2 SUMOylation. We demonstrate that FOXP2 is SUMOylated at a single major site, which is conserved in all FOXP2 vertebrate orthologues and in the paralogues FOXP1 and FOXP4. Abolishing this site did not lead to detectable changes in FOXP2 subcellular localization, stability, dimerization or transcriptional repression in cellular assays, but the conservation of this site suggests a potential role for SUMOylation in regulating FOXP2 activity in vivo.

  13. Post-translational environmental switch of RadA activity by extein–intein interactions in protein splicing

    PubMed Central

    Topilina, Natalya I.; Novikova, Olga; Stanger, Matthew; Banavali, Nilesh K.; Belfort, Marlene

    2015-01-01

    Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control. PMID:26101259

  14. Cadmium affects microtubule organization and post-translational modifications of tubulin in seedlings of soybean (Glycine max L.)

    PubMed Central

    Gzyl, Jarosław; Chmielowska-Bąk, Jagna; Przymusiński, Roman; Gwóźdź, Edward A.

    2015-01-01

    Cadmium (Cd) is a non-essential heavy metal, toxic to all living organisms. The microtubule (MT) cytoskeleton appears to be one of the main targets of Cd action. In this study we present, with the use of various immunological approaches, the effect of Cd at moderate (85 μM) and high (170 μM) concentrations on the structure and functioning of the MT cytoskeleton in the root cells of soybean seedlings. As the result of heavy metal action, root growth was significantly diminished and was accompanied by a reduction in mitotic activity and disturbance in the structure of the MT arrays, including randomization of the cortical MT arrangement, distorted mitotic arrays and complete depolymerization of the MTs. Biochemical analysis revealed decreased levels of various α- and β-tubulin isoforms with a parallel down-regulation of most examined α-tubulin genes. Simultaneously, Cd treatment led to differentiated changes in the level of tubulin post-translational modifications, including tyrosination, detyrosination, acetylation, and polyglutamylation. Decreased tyrosination and polyglutamylation of particular tubulin isoforms accompanied by increase in the level of specific detyrosinated and acetylated isoforms implies augmented stability and reduced turnover of the MTs during stress conditions. Taken together, the obtained results indicate the significant impact of Cd on gene expression levels and subsequent post-translational processing of tubulin, which may be related to the impairment of MT cytoskeleton functioning in root cells. PMID:26594217

  15. All post-translational modifications except propeptide cleavage are required for optimal secretion of coagulation factor VII.

    PubMed

    Bolt, Gert; Steenstrup, Thomas D; Kristensen, Claus

    2007-11-01

    Human coagulation factor VII (FVII) has two N-glycosylation sites (N145 and N322) and two O-glycosylation sites (S52 and S60). In transiently transfected COS-7 cells, all combinations of N- and O-glycosylation knock-out mutations reduced the release of FVII to the medium. Pulse-chase analysis of CHO-K1 cell lines expressing recombinant FVII demonstrated that virtually all wild-type FVII synthesized was secreted from the cells, whereas both N- and O-glycosylation knock-out mutations induced partial intracellular degradation of the synthesized FVII. Likewise, two thirds of the FVII synthesized in vitamin K-depleted and warfarin-treated CHO cells was degraded intracellularly, demonstrating the importance of gamma-carboxylation for the secretion of FVII. The furin inhibitor decanoyl-R-V-K-R-chloromethylketone inhibited propeptide cleavage, but FVII with propeptide appeared to be secreted equally well as FVII without propeptide. Propeptide cleavage was not inhibited by vitamin K depletion and warfarin treatment, suggesting that for FVII, correct gamma-carboxylation is not required for optimal processing of the propeptide. In conclusion, all post-translational modifications of FVII except propeptide cleavage were important for complete secretion of the synthesized FVII and to avoid intracellular degradation. Thus, the extensive post-translational modification of FVII seems critical for the intracellular stability of the protein and is required for keeping the protein in the secretory pathway.

  16. The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-like modifiers

    PubMed Central

    Estruch, Sara B.; Graham, Sarah A.; Deriziotis, Pelagia; Fisher, Simon E.

    2016-01-01

    Mutations affecting the transcription factor FOXP2 cause a rare form of severe speech and language disorder. Although it is clear that sufficient FOXP2 expression is crucial for normal brain development, little is known about how this transcription factor is regulated. To investigate post-translational mechanisms for FOXP2 regulation, we searched for protein interaction partners of FOXP2, and identified members of the PIAS family as novel FOXP2 interactors. PIAS proteins mediate post-translational modification of a range of target proteins with small ubiquitin-like modifiers (SUMOs). We found that FOXP2 can be modified with all three human SUMO proteins and that PIAS1 promotes this process. An aetiological FOXP2 mutation found in a family with speech and language disorder markedly reduced FOXP2 SUMOylation. We demonstrate that FOXP2 is SUMOylated at a single major site, which is conserved in all FOXP2 vertebrate orthologues and in the paralogues FOXP1 and FOXP4. Abolishing this site did not lead to detectable changes in FOXP2 subcellular localization, stability, dimerization or transcriptional repression in cellular assays, but the conservation of this site suggests a potential role for SUMOylation in regulating FOXP2 activity in vivo. PMID:26867680

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

    PubMed

    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.

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

    PubMed

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

    2011-11-01

    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.

  19. Fluctuation of similarity (FLUS) to detect transitions between distinct dynamical regimes in short time series

    PubMed Central

    Malik, Nishant; Marwan, Norbert; Zou, Yong; Mucha, Peter J.; Kurths, Jürgen

    2016-01-01

    A method to identify distinct dynamical regimes and transitions between those regimes in a short univariate time series was recently introduced [1], employing the computation of fluctuations in a measure of nonlinear similarity based on local recurrence properties. In the present work, we describe the details of the analytical relationships between this newly introduced measure and the well known concepts of attractor dimensions and Lyapunov exponents. We show that the new measure has linear dependence on the effective dimension of the attractor and it measures the variations in the sum of the Lyapunov spectrum. To illustrate the practical usefulness of the method, we identify various types of dynamical transitions in different nonlinear models. We present testbed examples for the new method’s robustness against noise and missing values in the time series. We also use this method to analyze time series of social dynamics, specifically an analysis of the U.S. crime record time series from 1975 to 1993. Using this method, we find that dynamical complexity in robberies was influenced by the unemployment rate until the late 1980’s. We have also observed a dynamical transition in homicide and robbery rates in the late 1980’s and early 1990’s, leading to increase in the dynamical complexity of these rates. PMID:25019852

  20. Fluctuation of similarity to detect transitions between distinct dynamical regimes in short time series

    NASA Astrophysics Data System (ADS)

    Malik, Nishant; Marwan, Norbert; Zou, Yong; Mucha, Peter J.; Kurths, Jürgen

    2014-06-01

    A method to identify distinct dynamical regimes and transitions between those regimes in a short univariate time series was recently introduced [N. Malik et al., Europhys. Lett. 97, 40009 (2012), 10.1209/0295-5075/97/40009], employing the computation of fluctuations in a measure of nonlinear similarity based on local recurrence properties. In this work, we describe the details of the analytical relationships between this newly introduced measure and the well-known concepts of attractor dimensions and Lyapunov exponents. We show that the new measure has linear dependence on the effective dimension of the attractor and it measures the variations in the sum of the Lyapunov spectrum. To illustrate the practical usefulness of the method, we identify various types of dynamical transitions in different nonlinear models. We present testbed examples for the new method's robustness against noise and missing values in the time series. We also use this method to analyze time series of social dynamics, specifically an analysis of the US crime record time series from 1975 to 1993. Using this method, we find that dynamical complexity in robberies was influenced by the unemployment rate until the late 1980s. We have also observed a dynamical transition in homicide and robbery rates in the late 1980s and early 1990s, leading to increase in the dynamical complexity of these rates.

  1. Fluctuation of similarity to detect transitions between distinct dynamical regimes in short time series.

    PubMed

    Malik, Nishant; Marwan, Norbert; Zou, Yong; Mucha, Peter J; Kurths, Jürgen

    2014-06-01

    A method to identify distinct dynamical regimes and transitions between those regimes in a short univariate time series was recently introduced [N. Malik et al., Europhys. Lett. 97, 40009 (2012)], employing the computation of fluctuations in a measure of nonlinear similarity based on local recurrence properties. In this work, we describe the details of the analytical relationships between this newly introduced measure and the well-known concepts of attractor dimensions and Lyapunov exponents. We show that the new measure has linear dependence on the effective dimension of the attractor and it measures the variations in the sum of the Lyapunov spectrum. To illustrate the practical usefulness of the method, we identify various types of dynamical transitions in different nonlinear models. We present testbed examples for the new method's robustness against noise and missing values in the time series. We also use this method to analyze time series of social dynamics, specifically an analysis of the US crime record time series from 1975 to 1993. Using this method, we find that dynamical complexity in robberies was influenced by the unemployment rate until the late 1980s. We have also observed a dynamical transition in homicide and robbery rates in the late 1980s and early 1990s, leading to increase in the dynamical complexity of these rates.

  2. Reassortment and distinct evolutionary dynamics of Rift Valley Fever virus genomic segments.

    PubMed

    Freire, Caio C M; Iamarino, Atila; Soumaré, Peinda O Ly; Faye, Ousmane; Sall, Amadou A; Zanotto, Paolo M A

    2015-06-23

    Rift Valley Fever virus (RVFV) is a member of Bunyaviridae family that causes a febrile disease affecting mainly ruminants and occasionally humans in Africa, with symptoms that range from mid to severe. RVFV has a tri-segmented ssRNA genome that permits reassortment and could generate more virulent strains. In this study, we reveal the importance of reassortment for RVFV evolution using viral gene genealogy inference and phylodynamics. We uncovered seven events of reassortment that originated RVFV lineages with discordant origins among segments. Moreover, we also found that despite similar selection regimens, the three segments have distinct evolutionary dynamics; the longer segment L evolves at a significant lower rate. Episodes of discordance between population size estimates per segment also coincided with reassortment dating. Our results show that RVFV segments are decoupled enough to have distinct demographic histories and to evolve under different molecular rates.

  3. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    PubMed Central

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-01-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics. PMID:27872481

  4. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    NASA Astrophysics Data System (ADS)

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-11-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  5. Protein Glycosylation in Archaea: A Post-Translational Modification to Enhance Extremophilic Protein Stability

    DTIC Science & Technology

    2010-01-15

    covalent linkage of oligosaccharides to select target asparagine residues. However, little had been known of this process in Archaea. As such, this...archaeal oligosaccharide transferase differs from its eukaryal counterpart. In further distinction from the eukaryal N-glycosylation process, in which the...lipid-linked oligosaccharide precursor is not chemically modified (27), transient and permanent modification of dolichol-linked glycans have been

  6. Reconstructing dynamic mental models of facial expressions in prosopagnosia reveals distinct representations for identity and expression.

    PubMed

    Richoz, Anne-Raphaëlle; Jack, Rachael E; Garrod, Oliver G B; Schyns, Philippe G; Caldara, Roberto

    2015-04-01

    The human face transmits a wealth of signals that readily provide crucial information for social interactions, such as facial identity and emotional expression. Yet, a fundamental question remains unresolved: does the face information for identity and emotional expression categorization tap into common or distinct representational systems? To address this question we tested PS, a pure case of acquired prosopagnosia with bilateral occipitotemporal lesions anatomically sparing the regions that are assumed to contribute to facial expression (de)coding (i.e., the amygdala, the insula and the posterior superior temporal sulcus--pSTS). We previously demonstrated that PS does not use information from the eye region to identify faces, but relies on the suboptimal mouth region. PS's abnormal information use for identity, coupled with her neural dissociation, provides a unique opportunity to probe the existence of a dichotomy in the face representational system. To reconstruct the mental models of the six basic facial expressions of emotion in PS and age-matched healthy observers, we used a novel reverse correlation technique tracking information use on dynamic faces. PS was comparable to controls, using all facial features to (de)code facial expressions with the exception of fear. PS's normal (de)coding of dynamic facial expressions suggests that the face system relies either on distinct representational systems for identity and expression, or dissociable cortical pathways to access them. Interestingly, PS showed a selective impairment for categorizing many static facial expressions, which could be accounted for by her lesion in the right inferior occipital gyrus. PS's advantage for dynamic facial expressions might instead relate to a functionally distinct and sufficient cortical pathway directly connecting the early visual cortex to the spared pSTS. Altogether, our data provide critical insights on the healthy and impaired face systems, question evidence of deficits

  7. Quasi-static and Dynamic Nanoindentation of Soft and Spatially Distinct Materials and Structures

    NASA Astrophysics Data System (ADS)

    Tian, Nannan

    Quasi-static nanoindentation has been used to assess the mechanical properties of soft and spatially distinct materials for several years. Most of the soft materials exhibit time-dependent (viscoelastic) behavior; thereby dynamic nanoindentation analysis increased the possibility of obtaining an accurate mechanical response from the materials. Normally, the heterogonous microstructure of specimens can result in experimental error when analyzing nanoindentation results. The accurate assessment of nanoindentation on soft materials with spatially distinct structures is not fully understood in previous studies. Some existing features in specimens, such as the residual stresses generated during polymer parts processing, also significantly influences nanoindentation data analysis. The objective of this study is to systematically consider some of the uncertainties when it comes to characterize soft materials by nanoindentation and thus develop several improved characterization methods, and provide guidance for future measurement. The study sought to clear out four main uncertainties within nanoindentation analysis for viscoelastic and heterogeneous materials: 1. Does the free edge close to the indents affect the dynamic nanoindentation results? How can we improve the analysis method? 2. How should indentation results be utilized to estimate the potential residual stresses? 3. Could we perform the statistical nanoindnentation to obtain the comparable results of volume fraction of individual phases in heterogeneous materials? 4. During nanoindentation, what is the appropriate combination of the loading rate, unloading rate and the holding time setup in terms of viscoelastic materials? In this dissertation, the correlation of quasi-static nanoindentation analysis methods with the structural compliance, residual stress, sampling volume and various relaxation processes will be covered in the following chapters. Dynamic nanoindentation will be used to access the time

  8. The application of targeted mass spectrometry-based strategies to the detection and localization of post-translational modifications.

    PubMed

    Chicooree, Navin; Unwin, Richard D; Griffiths, John R

    2015-01-01

    This review describes some of the more interesting and imaginative ways in which mass spectrometry has been utilized to study a number of important post-translational modifications over the past two decades; from circa 1990 to 2013. A diverse range of modifications is covered, including citrullination, sulfation, hydroxylation and sumoylation. A summary of the biological role of each modification described, along with some brief mechanistic detail, is also included. Emphasis has been placed on strategies specifically aimed at detecting target modifications, as opposed to more serendipitous modification discovery approaches, which rely upon straightforward product ion scanning methods. The authors have intentionally excluded from this review both phosphorylation and glycosylation since these major modifications have been extensively reviewed elsewhere.

  9. Post-Translational Regulation of miRNA Pathway Components, AGO1 and HYL1, in Plants

    PubMed Central

    Cho, Seok Keun; Ryu, Moon Young; Shah, Pratik; Poulsen, Christian Peter; Yang, Seong Wook

    2016-01-01

    Post-translational modifications (PTMs) of proteins are essential to increase the functional diversity of the proteome. By adding chemical groups to proteins, or degrading entire proteins by phosphorylation, glycosylation, ubiquitination, neddylation, acetylation, lipidation, and proteolysis, the complexity of the proteome increases, and this then influences most biological processes. Although small RNAs are crucial regulatory elements for gene expression in most eukaryotes, PTMs of small RNA microprocessor and RNA silencing components have not been extensively investigated in plants. To date, several studies have shown that the proteolytic regulation of AGOs is important for host-pathogen interactions. DRB4 is regulated by the ubiquitin-proteasome system, and the degradation of HYL1 is modulated by a de-etiolation repressor, COP1, and an unknown cytoplasmic protease. Here, we discuss current findings on the PTMs of microprocessor and RNA silencing components in plants. PMID:27440184

  10. Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase

    PubMed Central

    Webb, Michael E.; Yorke, Briony A.; Kershaw, Tom; Lovelock, Sarah; Lobley, Carina M. C.; Kilkenny, Mairi L.; Smith, Alison G.; Blundell, Tom L.; Pearson, Arwen R.; Abell, Chris

    2014-01-01

    Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation. PMID:24699660

  11. Threonine 57 is required for the post-translational activation of Escherichia coli aspartate α-decarboxylase.

    PubMed

    Webb, Michael E; Yorke, Briony A; Kershaw, Tom; Lovelock, Sarah; Lobley, Carina M C; Kilkenny, Mairi L; Smith, Alison G; Blundell, Tom L; Pearson, Arwen R; Abell, Chris

    2014-04-01

    Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formed via the intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

  12. Transcriptional and post-translational regulation of Bim controls apoptosis in melatonin-treated human renal cancer Caki cells.

    PubMed

    Park, Eun Jung; Woo, Seon Min; Min, Kyoung-Jin; Kwon, Taeg Kyu

    2014-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) has recently gained attention as an anticancer agent and for combined cancer therapy. In this study, we investigated the underlying molecular mechanisms of the effects of melatonin on cancer cell death. Treatment with melatonin induced apoptosis and upregulated the expression of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim) in renal cancer Caki cells. Furthermore, downregulation of Bim expression by siRNA markedly reduced melatonin-mediated apoptosis. Melatonin increased Bim mRNA expression through the induction of Sp1 and E2F1 expression and transcriptional activity. We found that melatonin also modulated Bim protein stability through the inhibition of proteasome activity. However, melatonin-induced Bim upregulation was independent of melatonin's antioxidant properties and the melatonin receptor. Taken together, our results suggest that melatonin induces apoptosis through the upregulation of Bim expression at the transcriptional level and at the post-translational level.

  13. Glutathionylation of the aquaporin-2 water channel: a novel post-translational modification modulated by the oxidative stress.

    PubMed

    Tamma, Grazia; Ranieri, Marianna; Di Mise, Annarita; Centrone, Mariangela; Svelto, Maria; Valenti, Giovanna

    2014-10-03

    Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.

  14. Transcriptional and post-translational modifications of B-Raf in quinol-thioether induced tuberous sclerosis renal cell carcinoma.

    PubMed

    Cohen, Jennifer D; Labenski, Matthew; Mastrandrea, Nicholas J; Canatsey, Ryan D; Monks, Terrence J; Lau, Serrine S

    2016-08-01

    Increased activity of B-Raf has been identified in approximately 7% of human cancers. Treatment of Eker rats (Tsc-2(EK/+) ), bearing a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) gene, with the nephrocarcinogen 2,3,5-tris-(glutathion-S-yl) hydroquinone (TGHQ) results in loss of the wild-type allele of Tsc-2 in renal preneoplastic lesions and tumors. These tumors have increased protein expression of B-Raf, C-Raf (Raf-1), and increased expression and activity of ERK kinase. Similar changes are observed in Raf kinases following TGHQ-mediated transformation of primary renal epithelial cells derived from Tsc-2(EK/+) rats (QTRRE cells), cells that are also null for tuberin. Herein, we utilized LC-MS/MS to identify constitutive phosphorylation of S345 and S483 in both 100- and 95-kDa forms of B-Raf in QTRRE cells. Using microRotofor liquid-phase isoelectric focusing, we identified four fractions of B-Raf that contain different post-translational modification profiles in QTRRE cells. Amplification of the kinase domain of B-Raf from QTRRE cells, outer-stripe of the outer medulla of 8-month TGHQ- or vehicle-treated Tsc-2(+/+) and Tsc-2(EK/+) rats, as well as tumors excised from 8-month TGHQ-treated Tsc-2(EK/+) rats revealed three splice variants of B-Raf within the kinase domain. These splice variants differed by approximately 340, 544, and 600 bp; confirmed by sequencing. No point mutations within the kinase domain of B-Raf were identified. In addition, B-Raf/Raf-1/14-3-3 complex formation in the QTRRE cells was decreased by sorafenib, with concomitant selective decreases in p-ERK levels. Transcriptional and post-translational characterization of critical kinases, such as B-Raf, may contribute to the progression of tuberous sclerosis RCC. (246/250) © 2015 Wiley Periodicals, Inc.

  15. Theoretical study of the pre- and post-translational effects of adenine and thymine tautomers and methyl derivatives.

    PubMed

    Gardner, Noel; Magers, David; Hill, Glake

    2013-09-01

    The study of pre-translational effects (ionization, tautomerization) and post-translational effects (methylation) of adenine and thymine has only recently been the focus of some studies. These effects can potentially help regulate gene expression as well as potentially disrupt normal gene function. Because of this wide array of roles, greater insight into these effects in deoxyribonucleic acids (DNA) are paramount. There has been considerable research of each phenomenon (tautomerization, methylation and ionization) individually. In this work, we attempt to shed light upon the pre-translational effects and post translational effects of adenine and thymine by investigating the electron affinities (EAs) and ionization potentials (IPs) of the major and minor tautomers and their methyl derivatives. We performed all calculations using the density functional theory (DFT) B3LYP functional accompanied with 6-311G(d,p), 6-311+G(d,p) and 6-311++G(df,pd) basis sets. Our results reveal that the thymine tautomer has a higher EA and IP than the adenine tautomers. The higher EA suggests that an electron that attaches to the AT base pair would predominately attach to the thymine instead of adenine. The higher IP would suggest that an electron that is removed from the AT base pair would be predominately removed from the adenine within the base pair. Understanding how tautomerization, ionization and methylation differences change effects, discourages, or promotes one another is lacking. In this work, we begin the steps of integrating these effects with one another, to gain a greater understanding of molecular changes in DNA bases.

  16. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness.

  17. Budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications.

    PubMed

    Szymanski, Eva Paige; Kerscher, Oliver

    2013-10-30

    Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and

  18. Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation.

    PubMed

    Van Roey, Kim; Davey, Norman E

    2015-12-01

    A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules.

  19. The mitochondrial elongation factors MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics

    SciTech Connect

    Liu, Tong; Yu, Rong; Jin, Shao-Bo; Han, Liwei; Lendahl, Urban; Zhao, Jian; Nistér, Monica

    2013-11-01

    Mitochondria are dynamic organelles whose morphology is regulated by a complex balance of fission and fusion processes, and we still know relatively little about how mitochondrial dynamics is regulated. MIEF1 (also called MiD51) has recently been characterized as a key regulator of mitochondrial dynamics and in this report we explore the functions of its paralog MIEF2 (also called MiD49), to learn to what extent MIEF2 is functionally distinct from MIEF1. We show that MIEF1 and MIEF2 have many functions in common. Both are anchored in the mitochondrial outer membrane, recruit Drp1 from the cytoplasm to the mitochondrial surface and cause mitochondrial fusion, and MIEF2, like MIEF1, can interact with Drp1 and hFis1. MIEF1 and MIEF2, however, also differ in certain aspects. MIEF1 and MIEF2 are differentially expressed in human tissues during development. When overexpressed, MIEF2 exerts a stronger fusion-promoting effect than MIEF1, and in line with this, hFis1 and Mff can only partially revert the MIEF2-induced fusion phenotype, whereas MIEF1-induced fusion is reverted to a larger extent by hFis1 and Mff. MIEF2 forms high molecular weight oligomers, while MIEF1 is largely present as a dimer. Furthermore, MIEF1 and MIEF2 use distinct domains for oligomerization: in MIEF1, the region from amino acid residues 109–154 is required, whereas oligomerization of MIEF2 depends on amino acid residues 1 to 49, i.e. the N-terminal end. We also show that oligomerization of MIEF1 is not required for its mitochondrial localization and interaction with Drp1. In conclusion, our data suggest that the mitochondrial regulators MIEF1 and MIEF2 exert partially distinct functions in mitochondrial dynamics. - Highlights: • MIEF1 and MIEF2 recruit Drp1 to mitochondria and cause mitochondrial fusion. • MIEF2, like MIEF1, can interact with Drp1 and hFis1. • MIEF1 and MIEF2 are differentially expressed in human tissues during development. • MIEF2 exerts a stronger fusion

  20. Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

    PubMed Central

    Myers, Kenneth A.; Applegate, Kathryn T.

    2011-01-01

    During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end–tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II–mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs. PMID:21263030

  1. Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages.

    PubMed

    Dietrich, Jens-Erik; Panavaite, Laura; Gunther, Stefan; Wennekamp, Sebastian; Groner, Anna C; Pigge, Anton; Salvenmoser, Stefanie; Trono, Didier; Hufnagel, Lars; Hiiragi, Takashi

    2015-08-01

    Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability.

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

    SciTech Connect

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

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Thrombin-cleaved fragments of milk-osteopontin effect hydroxyapatite formation differently. Black-Right-Pointing-Pointer N- and C-terminal fragments promoted hydroxyapatite formation and growth. Black-Right-Pointing-Pointer A central fragment inhibited hydroxyapatite formation and growth. Black-Right-Pointing-Pointer 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.

  3. Metrics for the Human Proteome Project 2016: Progress on Identifying and Characterizing the Human Proteome, Including Post-Translational Modifications.

    PubMed

    Omenn, Gilbert S; Lane, Lydie; Lundberg, Emma K; Beavis, Ronald C; Overall, Christopher M; Deutsch, Eric W

    2016-11-04

    The HUPO Human Proteome Project (HPP) has two overall goals: (1) stepwise completion of the protein parts list-the draft human proteome including confidently identifying and characterizing at least one protein product from each protein-coding gene, with increasing emphasis on sequence variants, post-translational modifications (PTMs), and splice isoforms of those proteins; and (2) making proteomics an integrated counterpart to genomics throughout the biomedical and life sciences community. PeptideAtlas and GPMDB reanalyze all major human mass spectrometry data sets available through ProteomeXchange with standardized protocols and stringent quality filters; neXtProt curates and integrates mass spectrometry and other findings to present the most up to date authorative compendium of the human proteome. The HPP Guidelines for Mass Spectrometry Data Interpretation version 2.1 were applied to manuscripts submitted for this 2016 C-HPP-led special issue [ www.thehpp.org/guidelines ]. The Human Proteome presented as neXtProt version 2016-02 has 16,518 confident protein identifications (Protein Existence [PE] Level 1), up from 13,664 at 2012-12, 15,646 at 2013-09, and 16,491 at 2014-10. There are 485 proteins that would have been PE1 under the Guidelines v1.0 from 2012 but now have insufficient evidence due to the agreed-upon more stringent Guidelines v2.0 to reduce false positives. neXtProt and PeptideAtlas now both require two non-nested, uniquely mapping (proteotypic) peptides of at least 9 aa in length. There are 2,949 missing proteins (PE2+3+4) as the baseline for submissions for this fourth annual C-HPP special issue of Journal of Proteome Research. PeptideAtlas has 14,629 canonical (plus 1187 uncertain and 1755 redundant) entries. GPMDB has 16,190 EC4 entries, and the Human Protein Atlas has 10,475 entries with supportive evidence. neXtProt, PeptideAtlas, and GPMDB are rich resources of information about post-translational modifications (PTMs), single amino acid

  4. Distinct modes of centromere protein dynamics during cell cycle progression in Drosophila S2R+ cells.

    PubMed

    Lidsky, Peter V; Sprenger, Frank; Lehner, Christian F

    2013-10-15

    Centromeres are specified epigenetically in animal cells. Therefore, faithful chromosome inheritance requires accurate maintenance of epigenetic centromere marks during progression through the cell cycle. Clarification of the mechanisms that control centromere protein behavior during the cell cycle should profit from the relatively simple protein composition of Drosophila centromeres. Thus we have analyzed the dynamics of the three key players Cid/Cenp-A, Cenp-C and Cal1 in S2R+ cells using quantitative microscopy and fluorescence recovery after photobleaching, in combination with novel fluorescent cell cycle markers. As revealed by the observed protein abundances and mobilities, centromeres proceed through at least five distinct states during the cell cycle, distinguished in part by unexpected Cid behavior. In addition to the predominant Cid loading onto centromeres during G1, a considerable but transient increase was detected during early mitosis. A low level of Cid loading was detected in late S and G2, starting at the reported time of centromere DNA replication. Our results reveal the complexities of Drosophila centromere protein dynamics and its intricate coordination with cell cycle progression.

  5. System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics

    PubMed Central

    Zuleger, Nikolaj; Kelly, David A.; Richardson, A. Christine; Kerr, Alastair R. W.; Goldberg, Martin W.; Goryachev, Andrew B.

    2011-01-01

    The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum–inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane. PMID:21444689

  6. Stat5 signaling specifies basal versus stress erythropoietic responses through distinct binary and graded dynamic modalities.

    PubMed

    Porpiglia, Ermelinda; Hidalgo, Daniel; Koulnis, Miroslav; Tzafriri, Abraham R; Socolovsky, Merav

    2012-08-01

    Erythropoietin (Epo)-induced Stat5 phosphorylation (p-Stat5) is essential for both basal erythropoiesis and for its acceleration during hypoxic stress. A key challenge lies in understanding how Stat5 signaling elicits distinct functions during basal and stress erythropoiesis. Here we asked whether these distinct functions might be specified by the dynamic behavior of the Stat5 signal. We used flow cytometry to analyze Stat5 phosphorylation dynamics in primary erythropoietic tissue in vivo and in vitro, identifying two signaling modalities. In later (basophilic) erythroblasts, Epo stimulation triggers a low intensity but decisive, binary (digital) p-Stat5 signal. In early erythroblasts the binary signal is superseded by a high-intensity graded (analog) p-Stat5 response. We elucidated the biological functions of binary and graded Stat5 signaling using the EpoR-HM mice, which express a "knocked-in" EpoR mutant lacking cytoplasmic phosphotyrosines. Strikingly, EpoR-HM mice are restricted to the binary signaling mode, which rescues these mice from fatal perinatal anemia by promoting binary survival decisions in erythroblasts. However, the absence of the graded p-Stat5 response in the EpoR-HM mice prevents them from accelerating red cell production in response to stress, including a failure to upregulate the transferrin receptor, which we show is a novel stress target. We found that Stat5 protein levels decline with erythroblast differentiation, governing the transition from high-intensity graded signaling in early erythroblasts to low-intensity binary signaling in later erythroblasts. Thus, using exogenous Stat5, we converted later erythroblasts into high-intensity graded signal transducers capable of eliciting a downstream stress response. Unlike the Stat5 protein, EpoR expression in erythroblasts does not limit the Stat5 signaling response, a non-Michaelian paradigm with therapeutic implications in myeloproliferative disease. Our findings show how the binary and

  7. Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: characterization of post-translational modifications and zinc binding.

    PubMed Central

    Raftery, M J; Harrison, C A; Alewood, P; Jones, A; Geczy, C L

    1996-01-01

    MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972 +/- 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 85Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100 beta. Ca2+ decreased Zn2+ binding in S100 beta but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains. PMID:8645219

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

    PubMed Central

    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. PMID:23774320

  9. Potential regulation of human muscle plasticity by MLC2 post-translational modifications during bed rest and countermeasures.

    PubMed

    Stevens, Laurence; Bastide, Bruno; Hedou, Julie; Cieniewski-Bernard, Caroline; Montel, Valérie; Cochon, Laetitia; Dupont, Erwan; Mounier, Yvonne

    2013-12-01

    This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR+Ex subjects were submitted to combined aerobic and resistive exercises, and BR+Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS-PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR+Nut while BR+Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosylation observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atrophy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading.

  10. Thiazolides, a New Class of Anti-influenza Molecules Targeting Viral Hemagglutinin at the Post-translational Level*

    PubMed Central

    Rossignol, Jean François; La Frazia, Simone; Chiappa, Lucia; Ciucci, Alessandra; Santoro, M. Gabriella

    2009-01-01

    The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection. PMID:19638339

  11. Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling.

    PubMed

    Gutierrez, Danielle B; Garland, Donita; Schey, Kevin L

    2011-12-01

    Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.

  12. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa.

    PubMed

    Whitney, John C; Whitfield, Gregory B; Marmont, Lindsey S; Yip, Patrick; Neculai, A Mirela; Lobsanov, Yuri D; Robinson, Howard; Ohman, Dennis E; Howell, P Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  13. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

  14. Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples.

    PubMed

    Noberini, Roberta; Uggetti, Andrea; Pruneri, Giancarlo; Minucci, Saverio; Bonaldi, Tiziana

    2016-03-01

    Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.

  15. Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

    PubMed Central

    Song, Byoung-Joon; Akbar, Mohammed; Abdelmegeed, Mohamed A.; Byun, Kyunghee; Lee, Bonghee; Yoon, Seung Kew; Hardwick, James P.

    2014-01-01

    Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury. PMID:25465468

  16. Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification?

    PubMed

    Perdivara, Irina; Deterding, Leesa J; Przybylski, Michael; Tomer, Kenneth B

    2010-07-01

    Oxidative modification of tryptophan to kynurenine (KYN) and N-formyl kynurenine (NFK) has been described in mitochondrial proteins associated with redox metabolism, and in human cataract lenses. To a large extent, however, previously reported identifications of these modifications were performed using peptide mass fingerprinting and/or tandem-MS data of proteins separated by gel electrophoresis. To date, it is uncertain whether NFK and KYN may represent sample handling artifacts or exclusively post-translational events. To address the problem of the origin of tryptophan oxidation, we characterized several antibodies by liquid chromatography-tandem mass spectrometry, with and without the use of electrophoretic separation of heavy and light chains. Antibodies are not normally expected to undergo oxidative modifications, however, several tryptophan (Trp) residues on both heavy and light chains were found extensively modified to both doubly oxidized Trp and KYN following SDS-PAGE separation and in-gel digestion. In contrast, those residues were observed as non-modified upon in-solution digestion. These results indicate that Trp oxidation may occur as an artifact in proteins separated by SDS-PAGE, and their presence should be carefully interpreted, especially when gel electrophoretic separation methods are employed.

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

    PubMed

    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.

  18. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  19. GAPP: A Proteogenomic Software for Genome Annotation and Global Profiling of Post-translational Modifications in Prokaryotes.

    PubMed

    Zhang, Jia; Yang, Ming-Kun; Zeng, Honghui; Ge, Feng

    2016-11-01

    Although the number of sequenced prokaryotic genomes is growing rapidly, experimentally verified annotation of prokaryotic genome remains patchy and challenging. To facilitate genome annotation efforts for prokaryotes, we developed an open source software called GAPP for genome annotation and global profiling of post-translational modifications (PTMs) in prokaryotes. With a single command, it provides a standard workflow to validate and refine predicted genetic models and discover diverse PTM events. We demonstrated the utility of GAPP using proteomic data from Helicobacter pylori, one of the major human pathogens that is responsible for many gastric diseases. Our results confirmed 84.9% of the existing predicted H. pylori proteins, identified 20 novel protein coding genes, and corrected four existing gene models with regard to translation initiation sites. In particular, GAPP revealed a large repertoire of PTMs using the same proteomic data and provided a rich resource that can be used to examine the functions of reversible modifications in this human pathogen. This software is a powerful tool for genome annotation and global discovery of PTMs and is applicable to any sequenced prokaryotic organism; we expect that it will become an integral part of ongoing genome annotation efforts for prokaryotes. GAPP is freely available at https://sourceforge.net/projects/gappproteogenomic/.

  20. Rethinking gene regulatory networks in light of alternative splicing, intrinsically disordered protein domains, and post-translational modifications

    PubMed Central

    Niklas, Karl J.; Bondos, Sarah E.; Dunker, A. Keith; Newman, Stuart A.

    2015-01-01

    Models for genetic regulation and cell fate specification characteristically assume that gene regulatory networks (GRNs) are essentially deterministic and exhibit multiple stable states specifying alternative, but pre-figured cell fates. Mounting evidence shows, however, that most eukaryotic precursor RNAs undergo alternative splicing (AS) and that the majority of transcription factors contain intrinsically disordered protein (IDP) domains whose functionalities are context dependent as well as subject to post-translational modification (PTM). Consequently, many transcription factors do not have fixed cis-acting regulatory targets, and developmental determination by GRNs alone is untenable. Modeling these phenomena requires a multi-scale approach to explain how GRNs operationally interact with the intra- and intercellular environments. Evidence shows that AS, IDP, and PTM complicate gene expression and act synergistically to facilitate and promote time- and cell-specific protein modifications involved in cell signaling and cell fate specification and thereby disrupt a strict deterministic GRN-phenotype mapping. The combined effects of AS, IDP, and PTM give proteomes physiological plasticity, adaptive responsiveness, and developmental versatility without inefficiently expanding genome size. They also help us understand how protein functionalities can undergo major evolutionary changes by buffering mutational consequences. PMID:25767796

  1. Post-translational modifications of plant cell wall proteins and peptides: A survey from a proteomics point of view.

    PubMed

    Canut, Hervé; Albenne, Cécile; Jamet, Elisabeth

    2016-08-01

    Plant cell wall proteins (CWPs) and peptides are important players in cell walls contributing to their assembly and their remodeling during development and in response to environmental constraints. Since the rise of proteomics technologies at the beginning of the 2000's, the knowledge of CWPs has greatly increased leading to the discovery of new CWP families and to the description of the cell wall proteomes of different organs of many plants. Conversely, cell wall peptidomics data are still lacking. In addition to the identification of CWPs and peptides by mass spectrometry (MS) and bioinformatics, proteomics has allowed to describe their post-translational modifications (PTMs). At present, the best known PTMs consist in proteolytic cleavage, N-glycosylation, hydroxylation of P residues into hydroxyproline residues (O), O-glycosylation and glypiation. In this review, the methods allowing the capture of the modified proteins based on the specific properties of their PTMs as well as the MS technologies used for their characterization are briefly described. A focus is done on proteolytic cleavage leading to protein maturation or release of signaling peptides and on O-glycosylation. Some new technologies, like top-down proteomics and terminomics, are described. They aim at a finer description of proteoforms resulting from PTMs or degradation mechanisms. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

  2. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; ...

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

  3. Characterization and putative post-translational regulation of α- and β-tubulin gene families in Salix arbutifolia.

    PubMed

    Rao, Guodong; Zeng, Yanfei; He, Caiyun; Zhang, Jianguo

    2016-01-12

    Microtubules, which are composed of heterodimers of α-tubulin (TUA) and β-tubulin (TUB) proteins, are closely associated with cellulose microfibril deposition and play pivotal roles in plant secondary cell wall development. In the present study, we identified eight TUA and twenty TUB genes in willow (Salix arbutifolia). Quantitative real-time PCR analysis showed that the small number of TUA gene family members relative to that of TUBs was complemented by a higher transcript copy number for each TUA gene, which is essential to the maintenance of the tubulin 1:1 heterodimer assembly. In Salix, five of eight TUAs were determined to be unusual because these contained a C-terminal methionine acid, leucine acid, glutamic acid, and glutamine acid, instead of the more typical tyrosine residue, which in turn generated the hypothesis of post-translational modifications (PTMs) that included deleucylation, demethiolation, deglutamynation, and deaspartylation. These PTMs are responsible for the removal of additional amino acid residues from TUAs prior to detyrosination, which is the first step of C-terminal PTMs. The additional PTMs of the TUA gene family might be responsible for the formation of different tubulin heterodimers that may have diverse functions for the adaptation of the woody perennial growth for Salix.

  4. Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment.

    PubMed

    Xia, Hui; Murray, Kermit; Soper, Steven; Feng, June

    2012-02-01

    Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

  5. Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

    PubMed

    Andrade-Silva, Débora; Zelanis, André; Kitano, Eduardo S; Junqueira-de-Azevedo, Inácio L M; Reis, Marcelo S; Lopes, Aline S; Serrano, Solange M T

    2016-08-05

    Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

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

    PubMed

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

    2017-02-03

    As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (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 a search engine, and LcMsSpectator as a data visualization tool. 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.

  7. Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

    PubMed Central

    Herren, Anthony W.; Bers, Donald M.

    2013-01-01

    The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies. PMID:23771687

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

    PubMed Central

    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. PMID:25403473

  9. Characterization of individual histone post-translational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies

    PubMed Central

    Sidoli, Simone; Garcia, Benjamin A.

    2017-01-01

    Summary Histone post-translational modifications (PTMs) play an essential role in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Such PTMs are mostly localized on histone N-terminal tails where, as single units or in a combinatorial manner, influence chromatin reader protein binding and fine-tune the abovementioned activities. Mass spectrometry (MS) is currently the most adopted strategy to characterize proteins and protein PTMs. We hereby describe the protocols to identify and quantify histone PTMs and their patterns using either bottom-up or middle-down proteomics. In the bottom-up strategy we obtain 5–20 aa peptides by derivatization with propionylation followed by trypsin digestion. The newly generated N-termini of histone peptides can be further derivatized with light or isotopically heavy propionyl groups to increase chromatographic retention and allow multiplexed analyses. Moreover, we describe how to perform derivatization and trypsin digestion of histones loaded into a gel, which is usually the final step of immunoprecipitation experiments. In the middle-down strategy we obtain intact histone tails of 50–60 aa by digestion with the enzyme GluC. This allows characterization of combinatorial histone PTMs on N-terminal tails. PMID:27854019

  10. MODi: a powerful and convenient web server for identifying multiple post-translational peptide modifications from tandem mass spectra.

    PubMed

    Kim, Sangtae; Na, Seungjin; Sim, Ji Woong; Park, Heejin; Jeong, Jaeho; Kim, Hokeun; Seo, Younghwan; Seo, Jawon; Lee, Kong-Joo; Paek, Eunok

    2006-07-01

    MOD(i) (http://modi.uos.ac.kr/modi/) is a powerful and convenient web service that facilitates the interpretation of tandem mass spectra for identifying post-translational modifications (PTMs) in a peptide. It is powerful in that it can interpret a tandem mass spectrum even when hundreds of modification types are considered and the number of potential PTMs in a peptide is large, in contrast to most of the methods currently available for spectra interpretation that limit the number of PTM sites and types being used for PTM analysis. For example, using MOD(i), one can consider for analysis both the entire PTM list published on the unimod webpage (http://www.unimod.org) and user-defined PTMs simultaneously, and one can also identify multiple PTM sites in a spectrum. MOD(i) is convenient in that it can take various input file formats such as .mzXML, .dta, .pkl and .mgf files, and it is equipped with a graphical tool called MassPective developed to display MOD(i)'s output in a user-friendly manner and helps users understand MOD(i)'s output quickly. In addition, one can perform manual de novo sequencing using MassPective.

  11. A Systems Biology Overview on Human Diabetic Nephropathy: From Genetic Susceptibility to Post-Transcriptional and Post-Translational Modifications

    PubMed Central

    Conserva, Francesca; Gesualdo, Loreto; Papale, Massimo

    2016-01-01

    Diabetic nephropathy (DN), a microvascular complication occurring in approximately 20–40% of patients with type 2 diabetes mellitus (T2DM), is characterized by the progressive impairment of glomerular filtration and the development of Kimmelstiel-Wilson lesions leading to end-stage renal failure (ESRD). The causes and molecular mechanisms mediating the onset of T2DM chronic complications are yet sketchy and it is not clear why disease progression occurs only in some patients. We performed a systematic analysis of the most relevant studies investigating genetic susceptibility and specific transcriptomic, epigenetic, proteomic, and metabolomic patterns in order to summarize the most significant traits associated with the disease onset and progression. The picture that emerges is complex and fascinating as it includes the regulation/dysregulation of numerous biological processes, converging toward the activation of inflammatory processes, oxidative stress, remodeling of cellular function and morphology, and disturbance of metabolic pathways. The growing interest in the characterization of protein post-translational modifications and the importance of handling large datasets using a systems biology approach are also discussed. PMID:26798653

  12. Characterization and putative post-translational regulation of α- and β-tubulin gene families in Salix arbutifolia

    PubMed Central

    Rao, Guodong; Zeng, Yanfei; He, Caiyun; Zhang, Jianguo

    2016-01-01

    Microtubules, which are composed of heterodimers of α-tubulin (TUA) and β-tubulin (TUB) proteins, are closely associated with cellulose microfibril deposition and play pivotal roles in plant secondary cell wall development. In the present study, we identified eight TUA and twenty TUB genes in willow (Salix arbutifolia). Quantitative real-time PCR analysis showed that the small number of TUA gene family members relative to that of TUBs was complemented by a higher transcript copy number for each TUA gene, which is essential to the maintenance of the tubulin 1:1 heterodimer assembly. In Salix, five of eight TUAs were determined to be unusual because these contained a C-terminal methionine acid, leucine acid, glutamic acid, and glutamine acid, instead of the more typical tyrosine residue, which in turn generated the hypothesis of post-translational modifications (PTMs) that included deleucylation, demethiolation, deglutamynation, and deaspartylation. These PTMs are responsible for the removal of additional amino acid residues from TUAs prior to detyrosination, which is the first step of C-terminal PTMs. The additional PTMs of the TUA gene family might be responsible for the formation of different tubulin heterodimers that may have diverse functions for the adaptation of the woody perennial growth for Salix. PMID:26753794

  13. A Systems Biology Overview on Human Diabetic Nephropathy: From Genetic Susceptibility to Post-Transcriptional and Post-Translational Modifications.

    PubMed

    Conserva, Francesca; Gesualdo, Loreto; Papale, Massimo

    2016-01-01

    Diabetic nephropathy (DN), a microvascular complication occurring in approximately 20-40% of patients with type 2 diabetes mellitus (T2DM), is characterized by the progressive impairment of glomerular filtration and the development of Kimmelstiel-Wilson lesions leading to end-stage renal failure (ESRD). The causes and molecular mechanisms mediating the onset of T2DM chronic complications are yet sketchy and it is not clear why disease progression occurs only in some patients. We performed a systematic analysis of the most relevant studies investigating genetic susceptibility and specific transcriptomic, epigenetic, proteomic, and metabolomic patterns in order to summarize the most significant traits associated with the disease onset and progression. The picture that emerges is complex and fascinating as it includes the regulation/dysregulation of numerous biological processes, converging toward the activation of inflammatory processes, oxidative stress, remodeling of cellular function and morphology, and disturbance of metabolic pathways. The growing interest in the characterization of protein post-translational modifications and the importance of handling large datasets using a systems biology approach are also discussed.

  14. The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence

    PubMed Central

    Faulds-Pain, Alexandra; Twine, Susan M; Vinogradov, Evgeny; Strong, Philippa C R; Dell, Anne; Buckley, Anthony M; Douce, Gillian R; Valiente, Esmeralda; Logan, Susan M; Wren, Brendan W

    2014-01-01

    Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete. PMID:25135277

  15. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  16. Dynamic and distinct histone modifications modulate the expression of key adipogenesis regulatory genes.

    PubMed

    Zhang, Qiongyi; Ramlee, Muhammad Khairul; Brunmeir, Reinhard; Villanueva, Claudio J; Halperin, Daniel; Xu, Feng

    2012-12-01

    Histone modifications and their modifying enzymes are fundamentally involved in the epigenetic regulation of adipogenesis. This study aimed to define the roles of various histone modifications and their "division of labor" in fat cell differentiation. To achieve these goals, we examined the distribution patterns of eight core histone modifications at five key adipogenic regulatory genes, Pref-1, C/EBPβ, C/EBPα, PPARγ2 and aP2, during the adipogenesis of C3H 10T1/2 mouse mesenchymal stem cells (MSCs) and 3T3-L1 preadipocytes. We found that the examined histone modifications are globally stable throughout adipogenesis but show distinct and highly dynamic distribution patterns at specific genes. For example, the Pref-1 gene has lower levels of active chromatin markers and significantly higher H3 K27 tri-methylation in MSCs compared with committed preadipocytes; the C/EBPβ gene is enriched in active chromatin markers at its 3'-UTR; the C/EBPα gene is predominantly marked by H3 K27 tri-methylation in adipogenic precursor cells, and this repressive marker decreases dramatically upon induction; the PPARγ2 and aP2 genes show increased histone acetylation on both H3 and H4 tails during adipogenesis. Further functional studies revealed that the decreased level of H3 K27 tri-methylation leads to de-repression of Pref-1 gene, while the increased level of histone acetylation activates the transcription of PPARγ2 and aP2 genes. Moreover, the active histone modification-marked 3'-UTR of C/EBPβ gene was demonstrated as a strong enhancer element by luciferase assay. Our results indicate that histone modifications are gene-specific at adipogenic regulator genes, and they play distinct roles in regulating the transcriptional network during adipogenesis.

  17. Cortical Layer 1 and Layer 2/3 Astrocytes Exhibit Distinct Calcium Dynamics In Vivo

    PubMed Central

    Takata, Norio; Hirase, Hajime

    2008-01-01

    Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca2+) concentration is important for astrocytes as Ca2+ surges co-occur with gliotransmission and neurotransmitter reception. Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca2+ activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca2+ activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca2+ dynamics by investigating two distinct EEG states (“synchronized” vs. “de-synchronized” states). We found that astrocytes in L1 had nearly twice higher Ca2+ activity than L2/3. Furthermore, Ca2+ fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca2+ activity. These results suggest that spontaneous astrocytic Ca2+ surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity. PMID:18575586

  18. Ecological Dynamics of Two Distinct Viruses Infecting Marine Eukaryotic Decomposer Thraustochytrids (Labyrinthulomycetes, Stramenopiles).

    PubMed

    Takao, Yoshitake; Tomaru, Yuji; Nagasaki, Keizo; Honda, Daiske

    2015-01-01

    Thraustochytrids are cosmopolitan osmotrophic or heterotrophic microorganisms that are considered as important decomposers in coastal ecosystems. However, because of a lack of estimation method for each genus or systematic group of them, relatively little is known about their ecology in situ. Previously, we reported two distinct types of virus infecting thraustochytrids (AuRNAV: reported as SssRNAV, and SmDNAV) suggesting they have wide distributions in the host-virus systems of coastal environments. Here we conducted a field survey from 2004 through 2005 to show the fluctuation pattern of thraustochytrids and their viruses in Hiroshima Bay, Japan. During the field survey, we monitored the dynamics of the two types of thraustochytrid-infecting virus: small viruses causing lysis of Aurantiochytrium sp. NIBH N1-27 (identified as AuRNAV) and the large viruses of Sicyoidochytrium minutum NBRC 102975 (similar to SmDNAV in physiology and morphology). Fluctuation patterns of the two distinct types of virus were different from each other. This may reflect the difference in the preference of organic substrates; i.e., it may be likely the host of AuRNAV (Aurantiochytrium sp.) increases utilizing algal dead bodies or feeble cells as the virus shows a large increase in abundance following raphidophyte blooms; whereas, the trophic nutrient supply for S. minutum may primarily depend on other constantly-supplied organic compounds because it did not show any significant change in abundance throughout the survey. Further study concerning the population composition of thraustochytrids and their viruses may demonstrate the microbial ecology (especially concerning the detrital food web) of marine environments.

  19. Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

    PubMed Central

    Petrenko, Natalia; Chereji, Raˇzvan V.; McClean, Megan N.; Morozov, Alexandre V.; Broach, James R.

    2013-01-01

    All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to “hedge their bets” in responding to an uncertain future, and to balance growth and survival in an unpredictable environment. PMID:23615444

  20. Distinct Element Modelling in Static and Dynamic Conditions with Application to an Underground Archaeological Site

    NASA Astrophysics Data System (ADS)

    Barla, G.; Monacis, G.; Perino, A.; Hatzor, Y. H.

    2010-11-01

    This paper considers the seismic response of the underground water storage cavern at the archaeological site of Tel Beer Sheva, excavated about 3,000 years ago in a highly jointed chalk region in the Negev Desert, in Israel. By using the distinct element method and the UDEC code, the stability conditions of the cavern are analysed. Close attention is given during rock mass modelling to the presence of discontinuities including horizontal bedding and a vertical joint set. The results of the static analyses performed confirm, as illustrated with archaeological researches, that the cavern roof had collapsed, probably during the time of construction, when a massive support pillar in the centre of the opening was constructed to support the remaining roof. The results of the dynamic analyses, which considered the recorded accelerations of the 1995 Nuweiba earthquake, demonstrate that the cavern in its present configuration, with the massive pillar in its centre, did not undergo any significant damage throughout the years, even when subjected to seismic events.

  1. Modification of distinct ion channels differentially modulates Ca2+ dynamics in primary cultured rat ventricular cardiomyocytes

    PubMed Central

    Li, Xichun; Shen, Liping; Zhao, Fang; Zou, Xiaohan; He, Yuwei; Zhang, Fan; Zhang, Chunlei; Yu, Boyang; Cao, Zhengyu

    2017-01-01

    Primary cultured cardiomyocytes show spontaneous Ca2+ oscillations (SCOs) which not only govern contractile events, but undergo derangements that promote arrhythmogenesis through Ca2+ -dependent mechanism. We systematically examined influence on SCOs of an array of ion channel modifiers by recording intracellular Ca2+ dynamics in rat ventricular cardiomyocytes using Ca2+ specific fluorescence dye, Fluo-8/AM. Voltage-gated sodium channels (VGSCs) activation elongates SCO duration and reduces SCO frequency while inhibition of VGSCs decreases SCO frequency without affecting amplitude and duration. Inhibition of voltage-gated potassium channel increases SCO duration. Direct activation of L-type Ca2+ channels (LTCCs) induces SCO bursts while suppressing LTCCs decreases SCO amplitude and slightly increases SCO frequency. Activation of ryanodine receptors (RyRs) increases SCO duration and decreases both SCO amplitude and frequency while inhibiting RyRs decreases SCO frequency without affecting amplitude and duration. The potencies of these ion channel modifiers on SCO responses are generally consistent with their affinities in respective targets demonstrating that modification of distinct targets produces different SCO profiles. We further demonstrate that clinically-used drugs that produce Long-QT syndrome including cisapride, dofetilide, sotalol, and quinidine all induce SCO bursts while verapamil has no effect. Therefore, occurrence of SCO bursts may have a translational value to predict cardiotoxicants causing Long-QT syndrome. PMID:28102360

  2. Post-translational processing of surfactant protein-C proprotein: targeting motifs in the NH(2)-terminal flanking domain are cleaved in late compartments.

    PubMed

    Johnson, A L; Braidotti, P; Pietra, G G; Russo, S J; Kabore, A; Wang, W J; Beers, M F

    2001-03-01

    Rat surfactant protein (SP)-C is a 3.7-kD hydrophobic lung-specific protein generated from proteolytic processing of a 21-kD propeptide (SP-C(21)). We have demonstrated that initial post-translational processing of SP-C(21) involves two cleavages of the COOH-terminus (Beers and colleagues, J. Biol. Chem. 1994;269:20,318--20,328). The goal of the current study was to define processing and function of the NH(2)-terminal flanking domain. Epitope-specific antisera directed against spatially distinct regions of the NH(2) terminus, NPROSP-C(2-9) (epitope = D(2)-L(9)) and NPROSP-C(11-23) (= E(11)-Q(23)) were produced. By Western blotting, both antisera identified SP-C(21) in microsomes. A 6-kD form (SP-C(6)), enriched in lamellar bodies (LBs), was detected only by NPROSP-C(11-23) and not extractable with NaCO(3) treatment. Immunogold staining of ultrathin lung sections with NPROSP-C(11-23) identified proSP-C in both multivesicular bodies (mvb) and LBs whereas NPROSP-C(2-9) labeled only mvb. (35)S-pulse chase analysis demonstrated synthesis of SP-C(21) and three intermediate forms (SP-C(16), SP-C(7), and SP-C(6)). Complete processing involved four separate cleavages with a precursor- product relationship between the low molecular weight forms SP-C(7) and SP-C(6). Fluorescence microscopy of A549 cells expressing fusion proteins of enhanced green fluorescent protein (EGFP) and proSP-C NH(2)-terminal deletion mutants showed targeting of EGFP/SP-C(1-194) and EGFP/SP-C(10-194) to early endosomal antigen-1-negative, CD-63-positive cytoplasmic vesicles whereas EGFP/SP-C(19-194), EGFP/SP-C(Delta 10-18), and EGFP/SP-C(24-194) were restricted to the endoplasmic reticulum (ER). We conclude that synthetic processing includes a previously unrecognized cleavage of the proximal NH(2) terminus (M(1)-L(9)), which occurs after removal of COOH-flanking domains (H(59)-I(194)) but before packaging in LBs, and that the region M(10)-T(18) is required for targeting of proSP-C to post-ER vesicular

  3. Poly r(C) binding protein (PCBP) 1 expression is regulated at the post-translation level in thyroid carcinoma

    PubMed Central

    Zhang, Ming-Peng; Zhang, Wei-San; Tan, Jin; Zhao, Ming-Hui; Lian, Lin-Juan; Cai, Jie

    2017-01-01

    Poly r(C) binding protein (PCBP) 1 or heterogeneous ribonucleoprotein (hnRNP) E1 is a RNA binding protein that plays a vital role in a wide variety of biological processes. PCBP1 has been shown to function as a tumor suppressor by negatively regulating translation of pro-metastatic proteins in different cancers. Loss of PCBP1 expression or its Akt2-mediated phosphorylation at serine 43 residue has both been indicated to de-repress its regulation of EMT inducer proteins. Our previous work has established that PCBP1 functions as a tumor suppressor in thyroid cancer, where its translation is inhibited by microRNA-490-3p. Here we show that thyroid cancer patients can be divided into 2 cohorts based on miR-490-3p expression and PCBP1 mRNA expression-one cohort with high PCBP1 mRNA expression and basal miR-490-3p expression and a second cohort with low PCBP1 mRNA expression and high miR-490-3p expression. However, PCBP1 protein expression is also downregulated in the cohort with high PCBP1 mRNA expression, with expression levels similar to what is observed in patients with the low PCBP1 mRNA expression. Our analysis shows that PCBP1 mRNA is actively translated in patients with high PCBP1 mRNA expression, but that the protein is post translationally degraded by the proteasome machinery. Our results thus elucidate a novel mechanism responsible for down regulation of PCBP1 expression in thyroid cancer. It will be important in future to identify the mechanism that causes degradation of PCBP1 protein and to identify if similar mechanisms are active in other tumors characterized by low PCBP1 protein expression.

  4. dbPTM 2016: 10-year anniversary of a resource for post-translational modification of proteins

    PubMed Central

    Huang, Kai-Yao; Su, Min-Gang; Kao, Hui-Ju; Hsieh, Yun-Chung; Jhong, Jhih-Hua; Cheng, Kuang-Hao; Huang, Hsien-Da; Lee, Tzong-Yi

    2016-01-01

    Owing to the importance of the post-translational modifications (PTMs) of proteins in regulating biological processes, the dbPTM (http://dbPTM.mbc.nctu.edu.tw/) was developed as a comprehensive database of experimentally verified PTMs from several databases with annotations of potential PTMs for all UniProtKB protein entries. For this 10th anniversary of dbPTM, the updated resource provides not only a comprehensive dataset of experimentally verified PTMs, supported by the literature, but also an integrative interface for accessing all available databases and tools that are associated with PTM analysis. As well as collecting experimental PTM data from 14 public databases, this update manually curates over 12 000 modified peptides, including the emerging S-nitrosylation, S-glutathionylation and succinylation, from approximately 500 research articles, which were retrieved by text mining. As the number of available PTM prediction methods increases, this work compiles a non-homologous benchmark dataset to evaluate the predictive power of online PTM prediction tools. An increasing interest in the structural investigation of PTM substrate sites motivated the mapping of all experimental PTM peptides to protein entries of Protein Data Bank (PDB) based on database identifier and sequence identity, which enables users to examine spatially neighboring amino acids, solvent-accessible surface area and side-chain orientations for PTM substrate sites on tertiary structures. Since drug binding in PDB is annotated, this update identified over 1100 PTM sites that are associated with drug binding. The update also integrates metabolic pathways and protein–protein interactions to support the PTM network analysis for a group of proteins. Finally, the web interface is redesigned and enhanced to facilitate access to this resource. PMID:26578568

  5. topPTM: a new module of dbPTM for identifying functional post-translational modifications in transmembrane proteins

    PubMed Central

    Su, Min-Gang; Huang, Kai-Yao; Lu, Cheng-Tsung; Kao, Hui-Ju; Chang, Ya-Han; Lee, Tzong-Yi

    2014-01-01

    Transmembrane (TM) proteins have crucial roles in various cellular processes. The location of post-translational modifications (PTMs) on TM proteins is associated with their functional roles in various cellular processes. Given the importance of PTMs in the functioning of TM proteins, this study developed topPTM (available online at http://topPTM.cse.yzu.edu.tw), a new dbPTM module that provides a public resource for identifying the functional PTM sites on TM proteins with structural topology. Experimentally verified TM topology data were integrated from TMPad, TOPDB, PDBTM and OPM. In addition to the PTMs obtained from dbPTM, experimentally verified PTM sites were manually extracted from research articles by text mining. In an attempt to provide a full investigation of PTM sites on TM proteins, all UniProtKB protein entries containing annotations related to membrane localization and TM topology were considered potential TM proteins. Two effective tools were then used to annotate the structural topology of the potential TM proteins. The TM topology of TM proteins is represented by graphical visualization, as well as by the PTM sites. To delineate the structural correlation between the PTM sites and TM topologies, the tertiary structure of PTM sites on TM proteins was visualized by Jmol program. Given the support of research articles by manual curation and the investigation of domain–domain interactions in Protein Data Bank, 1347 PTM substrate sites are associated with protein–protein interactions for 773 TM proteins. The database content is regularly updated on publication of new data by continuous surveys of research articles and available resources. PMID:24302577

  6. A Novel Approach for Untargeted Post-translational Modification Identification Using Integer Linear Optimization and Tandem Mass Spectrometry*

    PubMed Central

    Baliban, Richard C.; DiMaggio, Peter A.; Plazas-Mayorca, Mariana D.; Young, Nicolas L.; Garcia, Benjamin A.; Floudas, Christodoulos A.

    2010-01-01

    A novel algorithm, PILOT_PTM, has been developed for the untargeted identification of post-translational modifications (PTMs) on a template sequence. The algorithm consists of an analysis of an MS/MS spectrum via an integer linear optimization model to output a rank-ordered list of PTMs that best match the experimental data. Each MS/MS spectrum is analyzed by a preprocessing algorithm to reduce spectral noise and label potential complimentary, offset, isotope, and multiply charged peaks. Postprocessing of the rank-ordered list from the integer linear optimization model will resolve fragment mass errors and will reorder the list of PTMs based on the cross-correlation between the experimental and theoretical MS/MS spectrum. PILOT_PTM is instrument-independent, capable of handling multiple fragmentation technologies, and can address the universe of PTMs for every amino acid on the template sequence. The various features of PILOT_PTM are presented, and it is tested on several modified and unmodified data sets including chemically synthesized phosphopeptides, histone H3-(1–50) polypeptides, histone H3-(1–50) tryptic fragments, and peptides generated from proteins extracted from chromatin-enriched fractions. The data sets consist of spectra derived from fragmentation via collision-induced dissociation, electron transfer dissociation, and electron capture dissociation. The capability of PILOT_PTM is then benchmarked using five state-of-the-art methods, InsPecT, Virtual Expert Mass Spectrometrist (VEMS), Modi, Mascot, and X!Tandem. PILOT_PTM demonstrates superior accuracy on both the small and large scale proteome experiments. A protocol is finally developed for the analysis of a complete LC-MS/MS scan using template sequences generated from SEQUEST and is demonstrated on over 270,000 MS/MS spectra collected from a total chromatin digest. PMID:20103568

  7. Tongxinluo inhibits neointimal formation by regulating the expression and post-translational modification of KLF5 in macrophages

    PubMed Central

    Jiang, Wen; Zheng, Bin; Zhang, Xin-Hua; Yue, Ling-Yan; Liu, Chan; Ma, Dong; Yang, Zhan; Wen, Jin-Kun

    2016-01-01

    Neointimal hyperplasia is a common pathological characteristic in diverse vascular remodeling diseases. The inflammatory response that follows vascular injury plays an important role in intimal hyperplasia. Tongxinluo (TXL), a traditional Chinese medicine, can ameliorate neointimal formation via suppressing vascular inflammatory response induced by vascular injury. However, the mechanisms underlying anti-inflammatory and anti-intimal hyperplasia of TXL are still not fully understood. The aim of present study was to examine whether the expression and post-translational modification of KLF5 were involved in the vasoprotective effects of TXL. In vivo, TXL inhibited neointimal formation induced by carotid artery injury. In vitro, TNF-α treatment of macrophages resulted in the increased proliferation and migration, but the effects of TNF-α on macrophages were blocked by TXL treatment. Next, KLF5 expression was up-regulated by carotid artery injury in vivo, as well as by exposure of macrophages to TNF-α in vitro, whereas TXL treatment abrogated the up-regulation of KLF5 by TNF-α or vascular injury. Intimal hyperplasia was strongly reduced in macrophage-specific KLF5 knockout (KLF5ly-/-) mice, indicating that TXL inhibits intimal hyperplasia by suppression of KLF5 expression. Furthermore, besides down-regulating KLF5 expression in macrophages, TXL also regulated KLF5 stability by ubiquitination and sumoylation of KLF5. Finally, TNF-α induced KLF5 sumoylation via PI3K/Akt signaling, whereas TXL inhibited Akt phosphorylation induced by TNF-α. We conclude that the multiple ingredients in TXL may act on different targets, which in turn generates a range of actions that manifest as a comprehensively vasoprotective effect. PMID:27904679

  8. Agents that Stabilize Mutated von Hippel Lindau Protein Result in Differential Post-Translational Modification and Subcellular Localization

    PubMed Central

    Ding, Zhiyong; German, Peter; Bai, Shanshan; Feng, Zhehui; Gao, Meng; Si, Wendy; Sobieski, Mary M.; Stephan, Clifford C.; Mills, Gordon B.; Jonasch, Eric

    2014-01-01

    Background von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL. Methods The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed. Results Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds. Conclusion 786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL. PMID:22357874

  9. Regulation and role of post-translational modifications of enhancer of zeste homologue 2 in cancer development

    PubMed Central

    Lu, Haiqi; Li, Guangliang; Zhou, Chenyi; Jin, Wei; Qian, Xiaoling; Wang, Zhuo; Pan, Hongming; Jin, Hongchuan; Wang, Xian

    2016-01-01

    Post-translational modifications (PTMs) are critical molecular events which alter protein conformation after their synthesis and diversity protein properties by modulating their stability, localization, interacting partners or the activity of their substrates, consequently exerting pivotal roles in regulating the functions of many important eukaryotic proteins. It has been well acknowledged that PTMs are of great importance in a broad range of biological processes such as gene regulation, cell proliferation, differentiation and apoptosis, tissue development, diseases, tumor progression and drug resistance. As the core and contributing catalytic subunit of Polycomb repressive complex 2(PRC2), Enhancer of zeste homolog 2 (EZH2) is a master epigenetic regulator, often serving as a highly conserved histone methyltransferase (HMTase) to induce histone H3 lysine 27 trimethylation (H3K27me3) and repress gene transcription and expression. Dysregulated EZH2 expression is frequently associated with cancer development and poor prognosis in a wide variety of cancers. Considered its essential role in carcinogenesis, EZH2 is a potential candidate for cancer targeted therapy. Remarkably, mounting evidence highlights that EZH2 expression, activity and stability can be regulated by PTMs including phosphorylation, acetylation, ubiquitination, sumoylation and GlcNAcylation aside from its well-validated modifications in transcriptional and post-transcriptional levels. However, the precise regulatory mechanisms underlying EZH2 PTMs and whether other types of PTMs orchestrate in EZH2 remain largely unclear. In this review, we summarize current advances in the understanding of EZH2 regulation by PTMs and their associated biological functions during tumorigenesis. PMID:28042497

  10. USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level

    PubMed Central

    Pouly, Daniel; Chenaux, Sébastien; Martin, Virginie; Babis, Maja; Koch, Rafael; Nagoshi, Emi; Katanaev, Vladimir L.; Gachon, Frédéric; Staub, Olivier

    2016-01-01

    The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs) oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2), whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd), predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO), rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+. PMID:26756164

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

    PubMed

    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.

  12. Reconcile Mantle Dynamic Models with Compositionally Distinct and Stable LLSVPs with the Observations of the Geoid and Dynamic Topography

    NASA Astrophysics Data System (ADS)

    Liu, X.; Zhong, S.

    2015-12-01

    The geoid has been well explained in mantle flow models with the buoyancy inferred from seismic models that in turn place constraints on mantle viscosity structure (e.g., Hager & Richards, 1989). These models often assume a whole-mantle convection with uniform composition and 1-D viscosity. However, seismic and geochemical observations suggest possible existence of chemically distinct piles under Africa and Pacific which extends hundreds of kilometers above the CMB (i.e., LLSVPs). As compositional heterogeneity would significantly alter the interpretation of seismic anomalies as buoyancy structure, important questions are whether a thermochemical mantle model based on seismic velocity anomalies can reconcile the geoid and how this may impact inference of mantle viscosity structure. In this study, we formulate mantle flow models that use buoyancy derived from seismic model S40RTS (Ritsema et al., 2011), assuming that the LLSVPs are stable with negative buoyancy. The models use temperature-, depth- and composition-dependent viscosity and are computed for the geoid, dynamic topography and flow velocity using CitcomS. Seismic anomalies are converted to buoyancy using thermal conversion factor cT for the whole mantle materials and composition conversion factor cc for the chemical piles defined as the domains with seismic slow anomaly <-0.5% and a maximum height of 500 km. The temperature-dependence viscosity gives rise to 3 orders of magnitude variations in viscosity, and horizontally averaged viscosity profile is consistent with the inferred 1-D viscosity from the geoid. The viscosity in the chemical piles is further reduced by a factor of Cvisc to represent the compositional effect. We measure the stability of the chemical piles by the RMS vertical velocities on the piles boundary. Our preferred thermochemical models with stable chemical piles reach similar variance reduction of geoid at ~64% to that for the uniform composition models. In the preferred model, cT is ~0

  13. Canavanine Alters ROS/RNS Level and Leads to Post-translational Modification of Proteins in Roots of Tomato Seedlings

    PubMed Central

    Krasuska, Urszula; Andrzejczak, Olga; Staszek, Paweł; Bogatek, Renata; Gniazdowska, Agnieszka

    2016-01-01

    Canavanine (CAN), a structural analog of arginine (Arg), is used as a selective inhibitor of inducible NOS in mammals. CAN is incorporated into proteins’ structure in the place of Arg, leading to the formation of aberrant compounds. This non-protein amino acid is found in legumes, e.g., Canavalia ensiformis (L.) DC. or Sutherlandia frutescens (L.) R.Br. and acts as a strong toxin against herbivores or plants. Tomato (Solanum lycopersicum L.) seedlings were treated for 24–72 h with CAN (10 or 50 μM) inhibiting root growth by 50 or 100%, without lethal effect. We determined ROS level/production in root extracts, fluorescence of DAF-FM and APF derivatives corresponding to RNS level in roots of tomato seedlings and linked CAN-induced restriction of root growth to the post-translational modifications (PTMs) of proteins: carbonylation and nitration. Both PTMs are stable markers of nitro-oxidative stress, regarded as the plant’s secondary response to phytotoxins. CAN enhanced H2O2 content and superoxide radicals generation in extracts of tomato roots and stimulated formation of protein carbonyl groups. An elevated level of carbonylated proteins was characteristic for the plants after 72 h of the culture, mainly for the roots exposed to 10 μM CAN. The proteolytic activity was stimulated by tested non-protein amino acid. CAN treatment led to decline of fluorescence of DAF-FM derivatives, and transiently stimulated fluorescence of APF derivatives. Short-term exposure of tomato seedlings to CAN lowered the protein nitration level. Activity of peroxidase, polyamine oxidase and NADPH oxidase, enzymes acting as modulators of H2O2 concentration and governing root architecture and growth were determined. Activities of all enzymes were stimulated by CAN, but no strict CAN concentration dependence was observed. We conclude, that although CAN treatment led to a decline in the nitric oxide level, PTMs observed in roots of plants exposed to CAN are linked rather to the

  14. Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris).

    PubMed

    Kitazaki, Kazuyoshi; Arakawa, Takumi; Matsunaga, Muneyuki; Yui-Kurino, Rika; Matsuhira, Hiroaki; Mikami, Tetsuo; Kubo, Tomohiko

    2015-07-01

    Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet.

  15. Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

    PubMed

    Ott, Wolfgang; Nicolaus, Thomas; Gaub, Hermann E; Nash, Michael A

    2016-04-11

    Repetitive protein-based polymers are important for many applications in biotechnology and biomaterials development. Here we describe the sequential additive ligation of highly repetitive DNA sequences, their assembly into genes encoding protein-polymers with precisely tunable lengths and compositions, and their end-specific post-translational modification with organic dyes and fluorescent protein domains. Our new Golden Gate-based cloning approach relies on incorporation of only type IIS BsaI restriction enzyme recognition sites using PCR, which allowed us to install ybbR-peptide tags, Sortase c-tags, and cysteine residues onto either end of the repetitive gene polymers without leaving residual cloning scars. The assembled genes were expressed in Escherichia coli and purified using inverse transition cycling (ITC). Characterization by cloud point spectrophotometry, and denaturing polyacrylamide gel electrophoresis with fluorescence detection confirmed successful phosphopantetheinyl transferase (Sfp)-mediated post-translational N-terminal labeling of the protein-polymers with a coenzyme A-647 dye (CoA-647) and simultaneous sortase-mediated C-terminal labeling with a GFP domain containing an N-terminal GG-motif in a one-pot reaction. In a further demonstration, we installed an N-terminal cysteine residue into an elastin-like polypeptide (ELP) that was subsequently conjugated to a single chain poly(ethylene glycol)-maleimide (PEG-maleimide) synthetic polymer, noticeably shifting the ELP cloud point. The ability to straightforwardly assemble repetitive DNA sequences encoding ELPs of precisely tunable length and to post-translationally modify them specifically at the N- and C- termini provides a versatile platform for the design and production of multifunctional smart protein-polymeric materials.

  16. Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145

    PubMed Central

    Amin, Rafat; Franz-Wachtel, Mirita; Tiffert, Yvonne; Heberer, Martin; Meky, Mohamed; Ahmed, Yousra; Matthews, Arne; Krysenko, Sergii; Jakobi, Marco; Hinder, Markus; Moore, Jane; Okoniewski, Nicole; Maček, Boris; Wohlleben, Wolfgang; Bera, Agnieszka

    2016-01-01

    Soil-dwelling Streptomyces bacteria such as S.coelicolor have to constantly adapt to the nitrogen (N) availability in their habitat. Thus, strict transcriptional and post-translational control of the N-assimilation is fundamental for survival of this species. GlnR is a global response regulator that controls transcription of the genes related to the N-assimilation in S. coelicolor and other members of the Actinomycetales. GlnR represents an atypical orphan response regulator that is not activated by the phosphorylation of the conserved aspartate residue (Asp 50). We have applied transcriptional analysis, LC-MS/MS analysis and electrophoretic mobility shift assays (EMSAs) to understand the regulation of GlnR in S. coelicolor M145. The expression of glnR and GlnR-target genes was revisited under four different N-defined conditions and a complex N-rich condition. Although, the expression of selected GlnR-target genes was strongly responsive to changing N-concentrations, the glnR expression itself was independent of the N-availability. Using LC-MS/MSanalysis we demonstrated that GlnR was post-translationally modified. The post-translational modifications of GlnR comprise phosphorylation of the serine/threonine residues and acetylation of lysine residues. In the complex N-rich medium GlnR was phosphorylated on six serine/threonine residues and acetylated on one lysine residue. Under defined N-excess conditions only two phosphorylated residues were detected whereas under defined N-limiting conditions no phosphorylation was observed. GlnR phosphorylation is thus clearly correlated with N-rich conditions. Furthermore, GlnR was acetylated on four lysine residues independently of the N-concentration in the defined media and on only one lysine residue in the complex N-rich medium. Using EMSAs we demonstrated that phosphorylation inhibited the binding of GlnR to its targets genes, whereas acetylation had little influence on the formation of GlnR-DNA complex. This study clearly

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

    PubMed

    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.

  18. Dynamic facial expressions evoke distinct activation in the face perception network: a connectivity analysis study.

    PubMed

    Foley, Elaine; Rippon, Gina; Thai, Ngoc Jade; Longe, Olivia; Senior, Carl

    2012-02-01

    Very little is known about the neural structures involved in the perception of realistic dynamic facial expressions. In the present study, a unique set of naturalistic dynamic facial emotional expressions was created. Through fMRI and connectivity analysis, a dynamic face perception network was identified, which is demonstrated to extend Haxby et al.'s [Haxby, J. V., Hoffman, E. A., & Gobbini, M. I. The distributed human neural system for face perception. Trends in Cognitive Science, 4, 223-233, 2000] distributed neural system for face perception. This network includes early visual regions, such as the inferior occipital gyrus, which is identified as insensitive to motion or affect but sensitive to the visual stimulus, the STS, identified as specifically sensitive to motion, and the amygdala, recruited to process affect. Measures of effective connectivity between these regions revealed that dynamic facial stimuli were associated with specific increases in connectivity between early visual regions, such as the inferior occipital gyrus and the STS, along with coupling between the STS and the amygdala, as well as the inferior frontal gyrus. These findings support the presence of a distributed network of cortical regions that mediate the perception of different dynamic facial expressions.

  19. Dynamic Analysis of an SEIR Model with Distinct Incidence for Exposed and Infectives

    PubMed Central

    Li, Junhong; Cui, Ning

    2013-01-01

    An SEIR model with vaccination strategy that incorporates distinct incidence rates for the exposed and the infected populations is studied. By means of Lyapunov function and LaSalle's invariant set theorem, we proved the global asymptotical stable results of the disease-free equilibrium. The sufficient conditions for the global stability of the endemic equilibrium are obtained using the compound matrix theory. Furthermore, the method of direct numerical simulation of the system shows that there is a periodic solution, when the system has three equilibrium points. PMID:23766718

  20. Crenomytilus grayanus 40kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications.

    PubMed

    Matusovsky, Oleg S; Dobrzhanskaya, Anna V; Pankova, Victoria V; Kiselev, Konstantin V; Girich, Ulyana V; Shelud'ko, Nikolay S

    2017-03-02

    Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca(2+)-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis. The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

  1. Insulin induced alteration in post-translational modifications of histone H3 under a hyperglycemic condition in L6 skeletal muscle myoblasts.

    PubMed

    Kabra, Dhiraj G; Gupta, Jeena; Tikoo, Kulbhushan

    2009-06-01

    Chromatin remodelling events, especially histone modifications are proposed to form the mainstay for most of the biological processes. However, the role of these histone modifications in the progression of diabetes is still unknown. Hyperglycemia plays a major role in diabetes and its complications. The present study was undertaken to check the effect of insulin on alterations in post-translational modifications of histone H3 in L6 myoblasts under a hyperglycemic condition. We provide first evidence that insulin under hyperglycemic condition alters multiple histone modifications by enhanced production of reactive oxygen species. Insulin induces dose dependent changes in Lysine 4 and 9 methylation, Ser 10 phosphorylation and acetylation of histone H3. Interestingly, insulin induced generation of reactive oxygen species induces dephosphorylation and deacetylation of histone H3. Preincubation with catalase and DPI prevents these changes in post-translational modifications of histone H3. Furthermore, changes in histone H3 phosphorylation was found to be independent of ERK, p38, RSK2 and MSK1. Moreover, serine/threonine phosphatase inhibitor, okadaic acid attenuates insulin induced dephosphorylation and deacetylation of histone H3, suggesting a role of serine/threonine phosphatases in altering modifications of histone H3. These changes in epigenetic modifications can provide new insights into pathogenesis of diabetes.

  2. Partial Immunoblotting of 2D-Gels: A Novel Method to Identify Post-Translationally Modified Proteins Exemplified for the Myelin Acetylome

    PubMed Central

    Kusch, Kathrin; Uecker, Marina; Liepold, Thomas; Möbius, Wiebke; Hoffmann, Christian; Neumann, Heinz; Werner, Hauke B.; Jahn, Olaf

    2017-01-01

    Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions. PMID:28248254

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

    PubMed Central

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

    2016-01-01

    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, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 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 Cys158 and Cys172 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. PMID:27048590

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

    SciTech Connect

    Dykstra, Andrew B; Rodriguez, Jr., Miguel; Raman, Babu; Cook, Kelsey; Hettich, Robert {Bob} L

    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. Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia

    PubMed Central

    Li, Sheng; Garrett-Bakelman, Francine E.; Chung, Stephen S.; Sanders, Mathijs A.; Hricik, Todd; Rapaport, Franck; Patel, Jay; Dillon, Richard; Vijay, Priyanka; Brown, Anna L.; Perl, Alexander E.; Cannon, Joy; Bullinger, Lars; Luger, Selina; Becker, Michael; Lewis, Ian D.; To, Luen Bik; Delwel, Ruud; Löwenberg, Bob; Döhner, Hartmut; Döhner, Konstanze; Guzman, Monica L.; Hassane, Duane C.; Roboz, Gail J.; Grimwade, David; Valk, Peter J.M.; D’Andrea, Richard J.; Carroll, Martin; Park, Christopher Y.; Neuberg, Donna; Levine, Ross; Melnick, Ari M.; Mason, Christopher E.

    2016-01-01

    Genetic heterogeneity contributes to clinical outcome and progression of most tumors. Yet, little is known regarding allelic diversity for epigenetic compartments and almost no data exists for acute myeloid leukemia (AML). Here we examined epigenetic heterogeneity as assessed by cytosine methylation within defined genomic loci with four CpGs (epigenetic alleles), somatic mutations and transcriptomes of AML patient samples at serial time points. We observe that epigenetic allele burden is linked to inferior outcome and varies considerably during disease progression. Epigenetic and genetic allelic burden and patterning follow different patterns and kinetics during disease progression. We observed a subset of AMLs with high epiallele and low somatic mutation burden at diagnosis, a subset with high somatic mutation and lower epiallele burdens at diagnosis, and a subset with a mixed profile, suggesting distinct modes of tumor heterogeneity. Genes linked to promoter-associated epiallele shifts during tumor progression display increased single-cell transcriptional variance and differential expression, suggesting functional impact on gene regulation. Thus, genetic and epigenetic heterogeneity can occur with distinct kinetics, each likely able to impact biological and clinical features of tumors. PMID:27322744

  6. Dynamic microtubule organization and mitochondrial transport are regulated by distinct Kinesin-1 pathways

    PubMed Central

    Melkov, Anna; Simchoni, Yasmin; Alcalay, Yehonatan; Abdu, Uri

    2015-01-01

    ABSTRACT The microtubule (MT) plus-end motor kinesin heavy chain (Khc) is well known for its role in long distance cargo transport. Recent evidence showed that Khc is also required for the organization of the cellular MT network by mediating MT sliding. We found that mutations in Khc and the gene of its adaptor protein, kinesin light chain (Klc) resulted in identical bristle morphology defects, with the upper part of the bristle being thinner and flatter than normal and failing to taper towards the bristle tip. We demonstrate that bristle mitochondria transport requires Khc but not Klc as a competing force to dynein heavy chain (Dhc). Surprisingly, we demonstrate for the first time that Dhc is the primary motor for both anterograde and retrograde fast mitochondria transport. We found that the upper part of Khc and Klc mutant bristles lacked stable MTs. When following dynamic MT polymerization via the use of GFP-tagged end-binding protein 1 (EB1), it was noted that at Khc and Klc mutant bristle tips, dynamic MTs significantly deviated from the bristle parallel growth axis, relative to wild-type bristles. We also observed that GFP-EB1 failed to concentrate as a focus at the tip of Khc and Klc mutant bristles. We propose that the failure of bristle tapering is due to defects in directing dynamic MTs at the growing tip. Thus, we reveal a new function for Khc and Klc in directing dynamic MTs during polarized cell growth. Moreover, we also demonstrate a novel mode of coordination in mitochondrial transport between Khc and Dhc. PMID:26581590

  7. On reconstruction of dynamic permeability and tortuosity from data at distinct frequencies

    NASA Astrophysics Data System (ADS)

    Ou, Miao-Jung Yvonne

    2014-09-01

    This article focuses on the mathematical problem of reconstructing the dynamic permeability K(\\omega ) and dynamic tortuosity of poroelastic composites from permeability data at different frequencies, utilizing the analytic structure of the Stieltjes function representation of K(\\omega ) derived by Avellaneda and Tortquato (1991 Phys. Fluids A 3 2529), which is valid for all pore space geometry. The integral representation formula (IRF) for dynamic tortuosity is derived and its analytic structure exploited for reconstructing the function from a finite data set. All information of pore-space microstructure is contained in the measure of the IRF. The theory of multipoint Padé approximates for Stieltjes functions guarantees the existence of relaxation kernels that can approximate the dynamic permeability function and the dynamic tortuosity function with high accuracy. In this paper, a numerical algorithm is proposed for computing the relaxation time and the corresponding strength for each element in the relaxation kernels. In the frequency domain, this approximation can be regarded as approximating the Stieltjes function by rational functions with simple poles and positive residues. The main difference between this approach and the curve fitting approach is that the relaxation times and the strengths are computed from the partial fraction decomposition of the multipoint Padé approximates, which is the main subject of the proposed approximation scheme. With the idea from dehomogenization, we also established the exact relations between the moments of the positive measures in the IRFs of permeability and tortuosity with two important parameters in the theory of poroelasticity: the infinite-frequency tortuosity {{\\alpha }_{\\infty }} for the general case and the weighted volume-to-surface ratio Λ for the JKD model, which is regarded as a special case of the general model. From these relations, we suggest a new way for evaluating these two microstructure

  8. Parallel regulation of a modulator-activated current via distinct dynamics underlies comodulation of motor circuit output.

    PubMed

    DeLong, Nicholas D; Kirby, Matthew S; Blitz, Dawn M; Nusbaum, Michael P

    2009-09-30

    The cellular mechanisms underlying comodulation of neuronal networks are not elucidated in most systems. We are addressing this issue by determining the mechanism by which a peptide hormone, crustacean cardioactive peptide (CCAP), modulates the biphasic (protraction/retraction) gastric mill (chewing) rhythm driven by the projection neuron MCN1 in the crab stomatogastric ganglion. MCN1 activates this rhythm by slow peptidergic (CabTRP Ia) and fast GABAergic excitation of the reciprocally inhibitory central pattern generator neurons LG (protraction) and Int1 (retraction), respectively. MCN1 synaptic transmission is limited to the retraction phase, because LG inhibits MCN1 during protraction. Bath-applied CCAP also excites both LG and Int1, but selectively prolongs protraction. Here, we use computational modeling and dynamic-clamp manipulations to establish that CCAP prolongs the gastric mill protractor (LG) phase and maintains the retractor (Int1) phase duration by activating the same modulator-activated inward current (I(MI)) in LG as MCN1-released CabTRP Ia. However, the CCAP-activated current (I(MI-CCAP)) and MCN1-activated current (I(MI-MCN1)) exhibit distinct time courses in LG during protraction. This distinction results from I(MI-CCAP) being regulated only by postsynaptic voltage, whereas I(MI-MCN1) is also regulated by LG presynaptic inhibition of MCN1. Hence, without CCAP, retraction and protraction duration are determined by the time course of I(MI-MCN1) buildup and feedback inhibition-mediated decay, respectively, in LG. With I(MI-CCAP) continually present, the impact of the feedback inhibition is reduced, prolonging protraction and maintaining retraction duration. Thus, comodulation of rhythmic motor activity can result from convergent activation, via distinct dynamics, of a single voltage-dependent current.

  9. Lateral Hypothalamus Contains Two Types of Palatability-Related Taste Responses with Distinct Dynamics

    PubMed Central

    Yoshida, Takashi; Monk, Kevin J.; Katz, Donald B.

    2013-01-01

    The taste of foods, in particular the palatability of these tastes, exerts a powerful influence on our feeding choices. Although the lateral hypothalamus (LH) has long been known to regulate feeding behavior, taste processing in LH remains relatively understudied. Here, we examined single-unit LH responses in rats subjected to a battery of taste stimuli that differed in both chemical composition and palatability. Like neurons in cortex and amygdala, LH neurons produced a brief epoch of nonspecific responses followed by a protracted period of taste-specific firing. Unlike in cortex, however, where palatability-related information only appears 500 ms after the onset of taste-specific firing, taste specificity in LH was dominated by palatability-related firing, consistent with LH's role as a feeding center. Upon closer inspection, taste-specific LH neurons fell reliably into one of two subtypes: the first type showed a reliable affinity for palatable tastes, low spontaneous firing rates, phasic responses, and relatively narrow tuning; the second type showed strongest modulation to aversive tastes, high spontaneous firing rates, protracted responses, and broader tuning. Although neurons producing both types of responses were found within the same regions of LH, cross-correlation analyses suggest that they may participate in distinct functional networks. Our data shed light on the implementation of palatability processing both within LH and throughout the taste circuit, and may ultimately have implications for LH's role in the formation and maintenance of taste preferences and aversions. PMID:23719813

  10. Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

    PubMed Central

    Saffarian, Saveez; Cocucci, Emanuele; Kirchhausen, Tomas

    2009-01-01

    Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as “coated pits” and “coated plaques.” Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane—classical coated pits and coated plaques—and that these two assemblies interact quite differently with other intracellular structures. PMID:19809571

  11. Dynamics of centrosome translocation and microtubule organization in neocortical neurons during distinct modes of polarization.

    PubMed

    Sakakibara, Akira; Sato, Toshiyuki; Ando, Ryota; Noguchi, Namiko; Masaoka, Makoto; Miyata, Takaki

    2014-05-01

    Neuronal migration and process formation require cytoskeletal organization and remodeling. Recent studies suggest that centrosome translocation is involved in initial axon outgrowth, while the role of centrosomal positioning is not clear. Here, we examine relations between centrosomal positioning, axonogenesis, and microtubule (MT) polarization in multipolar and bipolar neocortical neurons. We monitored dynamic movements of centrosomes and MT plus ends in migratory neurons in embryonic mouse cerebral slices. In locomoting bipolar neurons, the centrosome oriented toward the pia-directed leading process. Bipolar neurons displayed dense MT plus end dynamics in leading processes, while trailing processes showed clear bidirectional MTs. In migrating multipolar neurons, new processes emerged irrespective of centrosome localization, followed by centrosome reorientations toward the dominant process. Anterograde movements of MT plus ends occurred in growing processes and retrograde movements were observed after retraction of the distal tip. In multipolar neurons, axon formed by tangential extension of a dominant process and the centrosome oriented toward the growing axon, while in locomoting neurons, an axon formed opposite to the direction of migration and the centrosome localized to the base of the leading process. Our data suggest that MT organization may alter centrosomal localization and that centrosomal positioning does not necessarily direct process formation.

  12. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.

    PubMed

    Shav-Tal, Yaron; Blechman, Janna; Darzacq, Xavier; Montagna, Cristina; Dye, Billy T; Patton, James G; Singer, Robert H; Zipori, Dov

    2005-05-01

    Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

  13. Face processing regions are sensitive to distinct aspects of temporal sequence in facial dynamics.

    PubMed

    Reinl, Maren; Bartels, Andreas

    2014-11-15

    Facial movement conveys important information for social interactions, yet its neural processing is poorly understood. Computational models propose that shape- and temporal sequence sensitive mechanisms interact in processing dynamic faces. While face processing regions are known to respond to facial movement, their sensitivity to particular temporal sequences has barely been studied. Here we used fMRI to examine the sensitivity of human face-processing regions to two aspects of directionality in facial movement trajectories. We presented genuine movie recordings of increasing and decreasing fear expressions, each of which were played in natural or reversed frame order. This two-by-two factorial design matched low-level visual properties, static content and motion energy within each factor, emotion-direction (increasing or decreasing emotion) and timeline (natural versus artificial). The results showed sensitivity for emotion-direction in FFA, which was timeline-dependent as it only occurred within the natural frame order, and sensitivity to timeline in the STS, which was emotion-direction-dependent as it only occurred for decreased fear. The occipital face area (OFA) was sensitive to the factor timeline. These findings reveal interacting temporal sequence sensitive mechanisms that are responsive to both ecological meaning and to prototypical unfolding of facial dynamics. These mechanisms are temporally directional, provide socially relevant information regarding emotional state or naturalness of behavior, and agree with predictions from modeling and predictive coding theory.

  14. Post-translational modifications of integral membrane proteins resolved by top-down Fourier transform mass spectrometry with collisionally activated dissociation.

    PubMed

    Ryan, Christopher M; Souda, Puneet; Bassilian, Sara; Ujwal, Rachna; Zhang, Jun; Abramson, Jeff; Ping, Peipei; Durazo, Armando; Bowie, James U; Hasan, S Saif; Baniulis, Danas; Cramer, William A; Faull, Kym F; Whitelegge, Julian P

    2010-05-01

    Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to today's bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14-261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (-17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N(6)-(retinylidene) modification (266.20 Da) between residues 225-248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b(6)f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane beta-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some

  15. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics

    PubMed Central

    Drori, Ran; Celik, Yeliz; Davies, Peter L.; Braslavsky, Ido

    2014-01-01

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH. PMID:25008081

  16. Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.

    PubMed

    Drori, Ran; Celik, Yeliz; Davies, Peter L; Braslavsky, Ido

    2014-09-06

    Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organisms by inhibiting the growth of endogenous ice crystals are called antifreeze proteins (AFPs). The binding of AFPs to ice causes a separation between the melting point and the freezing point of the ice crystal (thermal hysteresis, TH). TH produced by hyperactive AFPs is an order of magnitude higher than that produced by a typical fish AFP. The basis for this difference in activity remains unclear. Here, we have compared the time dependence of TH activity for both hyperactive and moderately active AFPs using a custom-made nanolitre osmometer and a novel microfluidics system. We found that the TH activities of hyperactive AFPs were time-dependent, and that the TH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopy measurement revealed that despite their higher TH activity, hyperactive AFPs from two insects (moth and beetle) took far longer to accumulate on the ice surface than did a moderately active fish AFP. An ice-binding protein from a bacterium that functions as an ice adhesin rather than as an antifreeze had intermediate TH properties. Nevertheless, the accumulation of this ice adhesion protein and the two hyperactive AFPs on the basal plane of ice is distinct and extensive, but not detectable for moderately active AFPs. Basal ice plane binding is the distinguishing feature of antifreeze hyperactivity, which is not strictly needed in fish that require only approximately 1°C of TH. Here, we found a correlation between the accumulation kinetics of the hyperactive AFP at the basal plane and the time sensitivity of the measured TH.

  17. Prefrontal neurons of opposite spatial preference display distinct target selection dynamics.

    PubMed

    Lennert, Therese; Martinez-Trujillo, Julio C

    2013-05-29

    Neurons in the primate dorsolateral prefrontal cortex (dlPFC) of one hemisphere are selective for the location of attended targets in both visual hemifields. Whether dlPFC neurons with selectivity for opposite hemifields directly compete with each other for target selection or instead play distinct roles during the allocation of attention remains unclear. We explored this issue by recording neuronal responses in the right dlPFC of two macaques while they allocated attention to a target in one hemifield and ignored a distracter on the opposite side. Forty-nine percent of the recorded neurons were target location selective. Neurons selective for contralateral targets (58%) systematically discriminated targets from distracters faster than neurons selective for ipsilateral targets (42%). Additionally, during trials in which sensory stimulation remained the same but both stimuli were task irrelevant and animals were required to detect a change in the color of a fixation spot, contralateral neurons still reliably discriminated the putative target from the distracter, whereas ipsilateral neurons did not. The latter result indicates that target-distracter discrimination by contralateral neurons could occur independently of discrimination by ipsilateral cells; thus, the two cell types may represent two different components of the prefrontal circuitry underlying the allocation of attention to targets in the presence of distracters. Moreover, the response of both contralateral and ipsilateral neurons to a single target was substantially reduced by the presence of a distracter in the contralateral hemifield. This result suggests that the presence of the distracter triggered inhibitory interactions within the dlPFC circuitry that suppressed responses to the attended target.

  18. Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity.

    PubMed

    Hugues, Stéphanie; Fetler, Luc; Bonifaz, Laura; Helft, Julie; Amblard, François; Amigorena, Sebastian

    2004-12-01

    Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8(+) T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15-20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC-T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.

  19. Distinct temporal filtering mechanisms are engaged during dynamic increases and decreases of noxious stimulus intensity.

    PubMed

    Mørch, Carsten Dahl; Frahm, Ken Steffen; Coghill, Robert C; Arendt-Nielsen, Lars; Andersen, Ole Kæseler

    2015-10-01

    Physical stimuli are subject to pronounced temporal filtering during afferent processing such that changes occurring at certain rates are amplified and others are diminished. Temporal filtering of nociceptive information remains poorly understood. However, the phenomenon of offset analgesia, where a disproportional drop in perceived pain intensity is caused by a slight drop in noxious heat stimulation, indicates potent temporal filtering in the pain pathways. To develop a better understanding of how dynamic changes in a physical stimulus are constructed into an experience of pain, a transfer function between the skin temperature and the perceived pain intensity was modeled. Ten seconds of temperature-controlled near-infrared (970 nm) laser stimulations above the pain threshold with a 1°C increment, decrement, or constant temperature were applied to the dorsum of the hand of healthy human volunteers. The skin temperature was assessed by an infrared camera. Offset analgesia was evoked by laser heat stimulation. The estimated transfer functions showed shorter latencies when the temperature was increased by 1°C (0.53 seconds [0.52-0.54 seconds]) than when decreased by 1°C (1.15 seconds [1.12-1.18 seconds]) and smaller gains (increase: 0.89 [0.82-0.97]; decrease: 2.61 [1.91-3.31]). The maximal gain was observed at rates around 0.06 Hz. These results show that temperature changes occurring around 0.06 Hz are best perceived and that a temperature decrease is associated with a larger but slower change in pain perception than a comparable temperature increase. These psychophysical findings confirm the existence of differential mechanisms involved in temporal filtering of dynamic increases and decreases in noxious stimulus intensity.

  20. Distinct temporal filtering mechanisms are engaged during dynamic increases and decreases of noxious stimulus intensity

    PubMed Central

    Mørch, Carsten Dahl; Frahm, Ken Steffen; Coghill, Robert C.; Arendt-Nielsen, Lars; Andersen, Ole Kæseler

    2015-01-01

    Abstract Physical stimuli are subject to pronounced temporal filtering during afferent processing such that changes occurring at certain rates are amplified and others are diminished. Temporal filtering of nociceptive information remains poorly understood. However, the phenomenon of offset analgesia, where a disproportional drop in perceived pain intensity is caused by a slight drop in noxious heat stimulation, indicates potent temporal filtering in the pain pathways. To develop a better understanding of how dynamic changes in a physical stimulus are constructed into an experience of pain, a transfer function between the skin temperature and the perceived pain intensity was modeled. Ten seconds of temperature-controlled near-infrared (970 nm) laser stimulations above the pain threshold with a 1°C increment, decrement, or constant temperature were applied to the dorsum of the hand of healthy human volunteers. The skin temperature was assessed by an infrared camera. Offset analgesia was evoked by laser heat stimulation. The estimated transfer functions showed shorter latencies when the temperature was increased by 1°C (0.53 seconds [0.52-0.54 seconds]) than when decreased by 1°C (1.15 seconds [1.12-1.18 seconds]) and smaller gains (increase: 0.89 [0.82-0.97]; decrease: 2.61 [1.91-3.31]). The maximal gain was observed at rates around 0.06 Hz. These results show that temperature changes occurring around 0.06 Hz are best perceived and that a temperature decrease is associated with a larger but slower change in pain perception than a comparable temperature increase. These psychophysical findings confirm the existence of differential mechanisms involved in temporal filtering of dynamic increases and decreases in noxious stimulus intensity. PMID:26035254

  1. Distinct Litter Stabilization Dynamics Pathways for Decomposition of Pine Needle and Fine Root Within Soil

    NASA Astrophysics Data System (ADS)

    Mambelli, S.; Filley, T. R.; Bird, J.; Dawson, T.; Torn, M. S.

    2008-12-01

    experiment while most lignin phenols for the humin from the root addition had greater 13C content than SFA. This indicates that molecular fragments of plant biopolymers can readily associate with both labile and stabilized SOM fractions. At the same time, these results suggest that distinct decomposition and stabilization pathways exist for litters, such as needles vs. roots, of different chemical quality.

  2. Delta(9)-tetrahydrocannabinol regulates the p53 post-translational modifiers Murine double minute 2 and the Small Ubiquitin MOdifier protein in the rat brain.

    PubMed

    Gowran, Aoife; Murphy, Carrie E; Campbell, Veronica A

    2009-11-03

    The phytocannabinoid Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), the main psychoactive cannabinoid in cannabis, activates a number of signalling cascades including p53. This study examines the role of Delta(9)-THC in regulating the p53 post-translational modifier proteins, Murine double minute (Mdm2) and Small Ubquitin-like MOdifier protein 1 (SUMO-1) in cortical neurons. Delta(9)-THC increased both Mdm2 and SUMO-1 protein expression and induced the deSUMOylation of p53 in a cannabinoid receptor type 1 (CB(1))-receptor dependent manner. We demonstrate that Delta(9)-THC decreased the SUMOylation of the CB(1) receptor. The data reveal a novel role for cannabinoid receptor activation in modulating the SUMO regulatory system.

  3. Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry.

    PubMed Central

    Aplin, R T; Baldwin, J E; Roach, P L; Robinson, C V; Schofield, C J

    1993-01-01

    Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. Images Figure 1 PMID:8396910

  4. The Solanum chacoense ovary receptor kinase 11 (ScORK11) undergoes tissue-dependent transcriptional, translational and post-translational regulation.

    PubMed

    Germain, Hugo; Gray-Mitsumune, Madoka; Houde, Josée; Benhamman, Rachid; Sawasaki, Tatsuya; Endo, Yaeta; Matton, Daniel P

    2013-09-01

    Using a subtraction screen to isolate weakly expressed transcripts from ovule and ovary libraries, we uncovered 30 receptor-like kinases that were predominantly expressed in ovary and fruit tissues following fertilization [1]. Here we describe the analysis of Solanum chacoense ovule receptor kinase 11 (ScORK11), a member of the large LRR III receptor kinase subfamily that localizes to the plasma membrane. In situ analyses demonstrated that ScORK11 gene expression was mainly restricted to the ovule integument, the embryo sac and the pericarp of the fruit. Tight regulation of ScORK11 expression at the mRNA level was also accompanied by both translational and post-translational regulation of protein levels.

  5. Clinical and environmental genotypes of Vibrio vulnificus display distinct, quorum-sensing-mediated, chitin detachment dynamics

    PubMed Central

    Phippen, Britney L.; Oliver, James D.

    2015-01-01

    The ability for bacteria to attach to and detach from various substrata is important for colonization, survival and transitioning to new environments. An opportunistic human pathogen, Vibrio vulnificus, can cause potentially fatal septicemia after ingestion of undercooked seafood. Based on genetic polymorphisms, strains of this species are subtyped into clinical (C) and environmental (E) genotypes. Vibrio vulnificus readily associates with chitin, thus we investigated chitin detachment dynamics in these disparate genotypes. We found that C-genotypes detach significantly more than E-genotypes after 24 hours in aerobic as well as anaerobic conditions. Furthermore, expression of genes involved in type IV pilin production was significantly downregulated in C-genotypes compared to E-genotypes, suggesting an importance in detachment. Interestingly, gbpA, a gene that has been shown to be important in host colonization in V. cholerae, was upregulated in the C-genotypes during detachment. Additionally, we found that C-genotypes detached to a greater extent, and produced more quorum-sensing (QS) autoinducer-2 molecules relative to E-genotypes, which suggests a role for QS in detachment. These findings suggest that for V. vulnificus, QS-mediated detachment may be a potential mechanism for transitioning into a human host for C-genotypes, while facilitating E-genotype maintenance in the estuarine environment. PMID:26377182

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

    PubMed Central

    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

  7. Systematic Assessment of the Benefits and Caveats in Mining Microbial Post-Translational Modifications from Shotgun Proteomic Data; Response of Shewanella oneidensis to Chromate Exposure

    SciTech Connect

    Thompson, Melissa R; Thompson, Dorothea K; Hettich, Robert {Bob} L

    2008-01-01

    Microbes are known to regulate both gene expression and protein activity through the use of post-translational modifications (PTMs). Common PTMs involved in cellular signaling and gene control include methylations, acetylations, and phosphorylations; whereas oxidations have been implicated as an indicator for stress. Shewanella oneidensis MR-1 is a gram-negative bacterium that demonstrates both respiratory versatility and the ability to sense and adapt to diverse environmental conditions. The dataset used in this study consisted of tandem mass spectra derived from mid-log phase aerobic cultures of S. oneidensis shocked either with or without 1 mM chromate [Cr(VI)]. In this study, three algorithms (DBDigger, Sequest, and InsPecT) were evaluated for their ability to scrutinize shotgun proteomic data for evidence of PTMs. The use of conservative scoring filters for peptides or proteins versus creating a sub-database first from a non-modification search was evaluated with DBDigger. The use of higher scoring filters for peptide identifications was found to result in optimal identifications of PTM peptides with a 2% false discovery rate (FDR) for the total dataset using the DBDigger algorithm. However, the FDR climbs to about 50% when considering PTM peptides only. Sequest was evaluated as a method for confirming PTM peptides putatively identified using DBDigger; however, there was a low identification rate (~25%) for the searched spectra. InsPecT was found to have a lower FDR (~9%) than DBDigger for PTM peptides. Comparisons between InsPecT and DBDigger were made with respect to both the FDR and PTM peptide identifications. As a demonstration of this approach, a number of S. oneidensis chemotaxis proteins as well as low-abundance signal transduction proteins were identified as being post-translationally modified in response to chromate challenge.

  8. Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels

    PubMed Central

    Portelli, Michael A.; Siedlinski, Mateusz; Stewart, Ceri E.; Postma, Dirkje S.; Nieuwenhuis, Maartje A.; Vonk, Judith M.; Nurnberg, Peter; Altmuller, Janine; Moffatt, Miriam F.; Wardlaw, Andrew J.; Parker, Stuart G.; Connolly, Martin J.; Koppelman, Gerard H.; Sayers, Ian

    2014-01-01

    The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17×10−7), which was also observed in a COPD population (combined P=5.04×10−12). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.—Portelli, M. A., Siedlinski, M., Stewart, C. E., Postma, D. S., Nieuwenhuis, M. A., Vonk, J. M., Nurnberg, P., Altmuller, J., Moffatt, M. F., Wardlaw, A. J., Parker, S. G., Connolly, M. J., Koppelman, G. H., Sayers, I. Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels. PMID:24249636

  9. EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.

    PubMed

    Chiang, Yihan; Chou, Cheng-Yang; Hsu, Keng-Fu; Huang, Yu-Fang; Shen, Meng-Ru

    2008-03-01

    Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.

  10. Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites

    PubMed Central

    Foth, Bernardo J; Zhang, Neng; Mok, Sachel; Preiser, Peter R; Bozdech, Zbynek

    2008-01-01

    Background Malaria is a one of the most important infectious diseases and is caused by parasitic protozoa of the genus Plasmodium. Previously, quantitative characterization of the P. falciparum transcriptome demonstrated that the strictly controlled progression of these parasites through their intra-erythrocytic developmental cycle is accompanied by a continuous cascade of gene expression. Although such analyses have proven immensely useful, the correlations between abundance of transcripts and their cognate proteins remain poorly characterized. Results Here, we present a quantitative time-course analysis of relative protein abundance for schizont-stage parasites (34 to 46 hours after invasion) based on two-dimensional differential gel electrophoresis of protein samples labeled with fluorescent dyes. For this purpose we analyzed parasite samples taken at 4-hour intervals from a tightly synchronized culture and established more than 500 individual protein abundance profiles with high temporal resolution and quantitative reproducibility. Approximately half of all profiles exhibit a significant change in abundance and 12% display an expression peak during the observed 12-hour time interval. Intriguingly, identification of 54 protein spots by mass spectrometry revealed that 58% of the corresponding proteins - including actin-I, enolase, eukaryotic initiation factor (eIF)4A, eIF5A, and several heat shock proteins - are represented by more than one isoform, presumably caused by post-translational modifications, with the various isoforms of a given protein frequently showing different expression patterns. Furthermore, comparisons with transcriptome data generated from the same parasite samples reveal evidence of significant post-transcriptional gene expression regulation. Conclusions Together, our data indicate that both post-transcriptional and post-translational events are widespread and of presumably great biological significance during the intra

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

    PubMed

    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.

  12. Collapsin response mediator protein 2: high-resolution crystal structure sheds light on small-molecule binding, post-translational modifications, and conformational flexibility.

    PubMed

    Myllykoski, Matti; Baumann, Anne; Hensley, Kenneth; Kursula, Petri

    2017-01-02

    Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.

  13. Nitric oxide modulates Lycopersicon esculentum C-repeat binding factor 1 (LeCBF1) transcriptionally as well as post-translationally by nitrosylation.

    PubMed

    Kashyap, Prakriti; Sehrawat, Ankita; Deswal, Renu

    2015-11-01

    Nitric oxide (NO) production increases in the cold stress. This cold enhanced NO manifests its effect either by regulating the gene expression or by modulating proteins by NO based post-translational modifications (PTMs) including S-nitrosylation. CBF (C-repeat binding factor) dependent cold stress signaling is most studied cold stress-signaling pathway in plants. SNP (sodium nitroprusside, a NO donor) treatment to tomato seedlings showed four fold induction of LeCBF1 (a cold inducible CBF) transcript in cold stress. S-nitrosylation as PTM of CBF has not been analyzed till date. In silico analysis using GPS-SNO 1.0 software predicted Cys 68 as the probable site for nitrosylation in LeCBF1. The 3D structure and motif prediction showed it to be present in the beta hairpin loop and hence available for S-nitrosylation. LeCBF1 was cloned and expressed in Escherichia coli. LeCBF1 accumulated in the inclusion bodies, which were solubilized under denaturing conditions and purified after on column refolding by Ni-NTA His tag affinity chromatography. Purified LeCBF1 resolved as a 34 kDa spot with a slightly basic pI (8.3) on a 2-D gel. MALDI-TOF mass spectrometry identified it as LeCBF1 and western blotting using anti-LeCBF1 antibodies confirmed its purification. Biotin switch assay and neutravidin affinity chromatography showed LeCBF1 to be S-nitrosylated in presence of GSNO (NO donor) as well as endogenously (without donor) in cold stress treated tomato seedlings. Dual regulation of LeCBF1 by NO at both transcriptional as well as post-translational level (by S-nitrosylation) is shown for the first time.

  14. Phosphorus dynamics in biogeochemically distinct regions of the southeast subtropical Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Duhamel, Solange; Björkman, Karin M.; Repeta, Daniel J.; Karl, David M.

    2017-02-01

    The southeast subtropical Pacific Ocean was sampled along a zonal transect between the coasts of Chile and Easter Island. This remote area of the world's ocean presents strong gradients in physical (e.g., temperature, density and light), chemical (e.g., salinity and nutrient concentrations) and microbiological (e.g., cell abundances, biomass and specific growth rates) properties. The goal of this study was to describe the phosphorus (P) dynamics in three main ecosystems along this transect: the upwelling regime off the northern Chilean coast, the oligotrophic area associated with the southeast subtropical Pacific gyre and the transitional area in between these two biomes. We found that inorganic phosphate (Pi) concentrations were high and turnover times were long (>210 nmol l-1 and >31 d, respectively) in the upper water column, along the entire transect. Pi uptake rates in the gyre were low (euphotic layer integrated rates were 0.26 mmol m-2 d-1 in the gyre and 1.28 mmol m-2 d-1 in the upwelling region), yet not only driven by decreases in particle mass or cell abundance (particulate P- and cell- normalized Pi uptake rates in the euphotic layer were ∼1-4 times and ∼3-15 times lower in the gyre than in the upwelling, respectively). However these Pi uptake rates were at or near the maximum Pi uptake velocity (i.e., uptake rates in Pi amended samples were not significantly different from those at ambient concentration: 1.5 and 23.7 nmol l-1 d-1 at 50% PAR in the gyre and upwelling, respectively). Despite the apparent Pi replete conditions, selected dissolved organic P (DOP) compounds were readily hydrolyzed. Nucleotides were the most bioavailable of the DOP substrates tested. Microbes actively assimilated adenosine-5‧-triphosphate (ATP) leading to Pi and adenosine incorporation as well as Pi release to the environment. The southeast subtropical Pacific Ocean is a Pi-sufficient environment, yet DOP hydrolytic processes are maintained and contribute to P

  15. Effect of Post-Translational Amidation on Islet Amyloid Polypeptide Conformational Ensemble: Implications for Its Aggregation Early Steps

    PubMed Central

    Tran, Linh; Ha-Duong, Tâp

    2016-01-01

    The human islet amyloid polypeptide (hIAPP) is an intrinsically disordered protein that can self-assemble into fibrillar aggregates that play a key role in the pathogenesis of the type II diabetes mellitus. hIAPP can transiently adopt α-helix and β-strand conformations that could be important intermediate species on the fibrillization pathway. However, experimental studies of the monomeric peptide conformations are limited due to its high aggregation propensity, and the early steps of the hIAPP association are not clearly characterized. In particular, the question of whether the aggregation-prone conformation is α-helical or β-strand-rich is still debated. In this study, combining extensive all-atom molecular dynamics (MD) and replica exchange molecular dynamics (REMD) simulations in explicit water, we shed some light on the differences between the amidated and non-amidated hIAPP conformational ensembles. Our study shows that, when compared to the amidated monomer, the non-amidation of hIAPP induces a significantly lower propensity to form β-strands, especially aggregation-prone β-hairpins. Since the fibrillization of the non-amidated hIAPP is significantly slower than that of the amidated peptide, this indicates that the early steps of the peptide oligomerization involve the association of β-hairpins or β-strands structures. PMID:27854243

  16. Distinct charge dynamics in battery electrodes revealed by in situ and operando soft X-ray spectroscopy

    PubMed Central

    Liu, Xiaosong; Wang, Dongdong; Liu, Gao; Srinivasan, Venkat; Liu, Zhi; Hussain, Zahid; Yang, Wanli

    2013-01-01

    Developing high-performance batteries relies on material breakthroughs. During the past few years, various in situ characterization tools have been developed and have become indispensible in studying and the eventual optimization of battery materials. However, soft X-ray spectroscopy, one of the most sensitive probes of electronic states, has been mainly limited to ex situ experiments for battery research. Here we achieve in situ and operando soft X-ray absorption spectroscopy of lithium-ion battery cathodes. Taking advantage of the elemental, chemical and surface sensitivities of soft X-rays, we discover distinct lithium-ion and electron dynamics in Li(Co1/3Ni1/3Mn1/3)O2 and LiFePO4 cathodes in polymer electrolytes. The contrast between the two systems and the relaxation effect in LiFePO4 is attributed to a phase transformation mechanism, and the mesoscale morphology and charge conductivity of the electrodes. These discoveries demonstrate feasibility and power of in situ soft X-ray spectroscopy for studying integrated and dynamic effects in batteries. PMID:24100759

  17. Distinct charge dynamics in battery electrodes revealed by in situ and operando soft X-ray spectroscopy.

    PubMed

    Liu, Xiaosong; Wang, Dongdong; Liu, Gao; Srinivasan, Venkat; Liu, Zhi; Hussain, Zahid; Yang, Wanli

    2013-01-01

    Developing high-performance batteries relies on material breakthroughs. During the past few years, various in situ characterization tools have been developed and have become indispensible in studying and the eventual optimization of battery materials. However, soft X-ray spectroscopy, one of the most sensitive probes of electronic states, has been mainly limited to ex situ experiments for battery research. Here we achieve in situ and operando soft X-ray absorption spectroscopy of lithium-ion battery cathodes. Taking advantage of the elemental, chemical and surface sensitivities of soft X-rays, we discover distinct lithium-ion and electron dynamics in Li(Co(¹/₃)Ni(¹/₃)Mn(¹/₃))O₂ and LiFePO₄ cathodes in polymer electrolytes. The contrast between the two systems and the relaxation effect in LiFePO₄ is attributed to a phase transformation mechanism, and the mesoscale morphology and charge conductivity of the electrodes. These discoveries demonstrate feasibility and power of in situ soft X-ray spectroscopy for studying integrated and dynamic effects in batteries.

  18. Potato virus Y HCPro localization at distinct, dynamically related and environment-influenced structures in the cell cytoplasm.

    PubMed

    del Toro, Francisco; Fernández, Fátima Tena; Tilsner, Jens; Wright, Kathryn M; Tenllado, Francisco; Chung, Bong Nam; Praveen, Shelly; Canto, Tomas

    2014-12-01

    Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.

  19. Distinct roles for Cav2.1–2.3 in activity-dependent synaptic dynamics

    PubMed Central

    Ricoy, Ulises M.

    2014-01-01

    Synaptic transmission throughout most of the CNS is steeply dependent on presynaptic calcium influx through the voltage-gated calcium channels Cav2.1–Cav2.3. In addition to triggering exocytosis, this calcium influx also recruits short-term synaptic plasticity. During the complex patterns of presynaptic activity that occur in vivo, several forms of plasticity combine to generate a synaptic output that is dynamic, in which the size of a given excitatory postsynaptic potential (EPSP) in response to a given spike depends on the short-term history of presynaptic activity. It remains unclear whether the different Cav2 channels play distinct roles in defining these synaptic dynamics and, if so, under what conditions different Cav2 family members most effectively determine synaptic output. We examined these questions by measuring the effects of calcium channel-selective toxins on synaptic transmission at the Schaffer collateral synapse in hippocampal slices from adult mice in response to both low-frequency stimulation and complex stimulus trains derived from in vivo recordings. Blockade of Cav2.1 had a greater inhibitory effect on synaptic transmission during low-frequency components of the stimulus train than on synaptic transmission during high-frequency components of the train, indicating that Cav2.1 had a greater fractional contribution to synaptic transmission at low frequencies than at high frequencies. Relative to Cav2.1, Cav2.2 had a disproportionately reduced contribution to synaptic transmission at frequencies >20 Hz, while Cav2.3 had a disproportionately increased contribution to synaptic transmission at frequencies >1 Hz. These activity-dependent effects of different Cav2 family members shape the filtering characteristics of GABAB receptor-mediated presynaptic inhibition. Thus different Cav2 channels vary in their coupling to synaptic transmission over different frequency ranges, with consequences for the frequency tuning of both synaptic dynamics and

  20. Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community

    SciTech Connect

    Li, Zhou; Wang, Yingfeng; Yao, Qiuming; Justice, Nicholas B.; Ahn, Tae-Hyuk; Xu, Dong; Hettich, Robert {Bob} L; Banfield, Jillian F.; Pan, Chongle

    2014-01-01

    Detailed characterization of posttranslational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.

  1. Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair

    PubMed Central

    Pipalia, Tapan G.; Koth, Jana; Roy, Shukolpa D.; Hammond, Christina L.; Kawakami, Koichi

    2016-01-01

    ABSTRACT Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7+ cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular

  2. Pre- and post-translational negative effect of beta-adrenoceptor agonists on adiponectin secretion: in vitro and in vivo studies.

    PubMed Central

    Delporte, Marie-Laure; Funahashi, Tohru; Takahashi, Masahiko; Matsuzawa, Yuji; Brichard, Sonia M

    2002-01-01

    The adipose-derived hormone, adiponectin (ApN), has a role in fuel homoeostasis, insulin action and atherosclerosis. Regulation of ApN by catecholamines has scarcely been investigated. We examined the effects of beta-adrenergic agonists (and their second messenger, cAMP) on ApN gene expression, production and secretion in mouse in vitro and in vivo; their effects in human fat were also briefly studied in vitro. beta-Adrenergic agonists and cAMP inhibited ApN gene expression in human visceral adipose tissue. Likewise, cAMP down-regulated ApN mRNAs in cultured mouse explants from visceral and subcutaneous regions. The amount of ApN released into the medium decreased concomitantly. cAMP also caused qualitative changes in ApN secretion. Under basal conditions, ApN was secreted as a single 32 kDa species. In the presence of cAMP, an additional and probably immature (not modified post-translationally) 30 kDa species was also sorted. This altered secretion resulted from cAMP-induced quantitative and qualitative changes of ApN within the adipocyte. Under basal conditions, the 32 kDa form of ApN was mainly associated with high-density microsomes (HDMs), while the 30 kDa species was confined to a pool recovered with the cytosol fraction. cAMP depleted intracellular ApN at the expense of both HDM and cytosol fractions, and abnormally targeted ApN species to the different subcellular compartments as a result of impaired maturation. beta-Adrenergic agonists mimicked the inhibitory effects of cAMP on ApN mRNA and secretion, the beta(3)-agonist BRL37344 being the most potent. Administration of BRL37344 to mice reduced ApN mRNAs in both adipose regions, and ApN levels in plasma. In conclusion, beta-agonists inhibited ApN production and maturation, and thus exerted a dual (pre- and post-translational) negative effect on ApN secretion by cultured mouse adipose explants. ApN inhibition by beta-agonists was reproduced in mouse in vivo and in humans in vitro. ApN down-regulation may

  3. Bioinformatics analysis of a Saccharomyces cerevisiae N-terminal proteome provides evidence of alternative translation initiation and post-translational N-terminal acetylation.

    PubMed

    Helsens, Kenny; Van Damme, Petra; Degroeve, Sven; Martens, Lennart; Arnesen, Thomas; Vandekerckhove, Joël; Gevaert, Kris

    2011-08-05

    Initiation of protein translation is a well-studied fundamental process, albeit high-throughput and more comprehensive determination of the exact translation initiation sites (TIS) was only recently made possible following the introduction of positional proteomics techniques that target protein N-termini. Precise translation initiation is of crucial importance, as truncated or extended proteins might fold, function, and locate erroneously. Still, as already shown for some proteins, alternative translation initiation can also serve as a regulatory mechanism. By applying N-terminal COFRADIC (combined fractional diagonal chromatography), we here isolated N-terminal peptides of a Saccharomyces cerevisiae proteome and analyzed both annotated and alternative TIS. We analyzed this N-terminome of S. cerevisiae which resulted in the identification of 650 unique N-terminal peptides corresponding to database annotated TIS. Furthermore, 56 unique N(α)-acetylated peptides were identified that suggest alternative TIS (MS/MS-based), while MS-based evidence of N(α)-acetylation led to an additional 33 such peptides. To improve the overall sensitivity of the analysis, we also included the 5' UTR (untranslated region) in-frame translations together with the yeast protein sequences in UniProtKB/Swiss-Prot. To ensure the quality of the individual peptide identifications, peptide-to-spectrum matches were only accepted at a 99% probability threshold and were subsequently analyzed in detail by the Peptizer tool to automatically ascertain their compliance with several expert criteria. Furthermore, we have also identified 60 MS/MS-based and 117 MS-based N(α)-acetylated peptides that point to N(α)-acetylation as a post-translational modification since these peptides did not start nor were preceded (in their corresponding protein sequence) by a methionine residue. Next, we evaluated consensus sequence features of nucleic acids and amino acids across each of these groups of peptides and

  4. Comparing and combining capillary electrophoresis electrospray ionization mass spectrometry and nano-liquid chromatography electrospray ionization mass spectrometry for the characterization of post-translationally modified histones.

    PubMed

    Sarg, Bettina; Faserl, Klaus; Kremser, Leopold; Halfinger, Bernhard; Sebastiano, Roberto; Lindner, Herbert H

    2013-09-01

    We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques

  5. Cytosolic Ca2+ and Ca2+-activated Cl− current dynamics: insights from two functionally distinct mouse exocrine cells

    PubMed Central

    Giovannucci, David R; Bruce, Jason I. E; Straub, Stephen V; Arreola, Jorge; Sneyd, James; Shuttleworth, Trevor J; Yule, David I

    2002-01-01

    The dynamics of Ca2+ release and Ca2+-activated Cl− currents in two related, but functionally distinct exocrine cells, were studied to gain insight into how the molecular specialization of Ca2+ signalling machinery are utilized to produce different physiological endpoints: in this case, fluid or exocytotic secretion. Digital imaging and patch-clamp methods were used to monitor the temporal and spatial properties of changes in cytosolic Ca2+ concentration ([Ca2+]c) and Cl− currents following the controlled photolytic release of caged-InsP3 or caged-Ca2+. In parotid and pancreatic acinar cells, changes in [Ca2+]c and activation of a Ca2+-activated Cl− current occurred with close temporal coincidence. In parotid, a rapid global Ca2+ signal was invariably induced, even with low-level photolytic release of threshold amounts of InsP3. In pancreas, threshold stimulation generated an apically delimited [Ca2+]c signal, while a stronger stimulus induced a global [Ca2+]c signal which exhibited characteristics of a propagating wave. InsP3 was more effective in parotid, where [Ca2+]c signals initiated with shorter latency and exhibited a faster time-to-peak than in pancreas. The increased potency of InsP3 in parotid probably results from a four-fold higher number of InsP3 receptors as measured by radiolabelled InsP3 binding and western blot analysis. The Ca2+ sensitivity of the Cl− channels in parotid and pancreas was determined from the [Ca2+]-current relationship measured during a dynamic ‘Ca2+ ramp’ produced by the continuous, low-level photolysis of caged-Ca2+. In addition to a greater number of InsP3 receptors, the Cl− current density of parotid acinar cells was more than four-fold greater than that of pancreatic cells. Whereas activation of the current was tightly coupled to increases in Ca2+ in both cell types, local Ca2+ clearance was found to contribute substantially to the deactivation of the current in parotid. These data reveal specializations of

  6. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components*

    PubMed Central

    Soldi, Monica; Bonaldi, Tiziana

    2013-01-01

    Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status. PMID:23319141

  7. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus.

    PubMed

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-10-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine(23) and arginine(24)) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins.

  8. Post-translational enzyme modification by the phosphopantetheinyl transferase is required for lysine and penicillin biosynthesis but not for roquefortine or fatty acid formation in Penicillium chrysogenum.

    PubMed

    García-Estrada, Carlos; Ullán, Ricardo V; Velasco-Conde, Tania; Godio, Ramiro P; Teijeira, Fernando; Vaca, Inmaculada; Feltrer, Raúl; Kosalková, Katarina; Mauriz, Elba; Martín, Juan F

    2008-10-15

    NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4'-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT(-)) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT(-) mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT(-) with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.

  9. An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins.

    PubMed

    Aksu, Sevil; Scheler, Christian; Focks, Nicole; Leenders, Frauke; Theuring, Franz; Salnikow, Johann; Jungblut, Peter R

    2002-10-01

    Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.

  10. Hsp90 transcriptionally and post-translationally regulates the expression of NDRG1 and maintains the stability of its modifying kinase GSK3beta.

    PubMed

    Banz, Vanessa M; Medová, Michaela; Keogh, Adrian; Furer, Cynthia; Zimmer, Yitzhak; Candinas, Daniel; Stroka, Deborah

    2009-10-01

    N-myc downstream-regulated gene 1 (NRDG1) is a stress-induced protein whose putative function is suppression of tumor metastasis. A recent proteonomic study showed NDRG1 interacts with the molecular chaperone heat shock protein 90 (Hsp90). From their reported association, we investigated if NDRG1 is dependent on Hsp90 for its stability and is therefore a yet unidentified Hsp90 client protein. Here, we demonstrate that endogenous NDRG1 and Hsp90 physically associate in hepatocellular cancer cell lines. However, geldanamycin (GA)-mediated inhibition of Hsp90 did not disrupt their interaction or result in NDRG1 protein destabilization. On the contrary, inhibition of Hsp90 led to a transcriptional increase of NDRG1 protein which was associated with cell growth arrest. We also observed that GA inhibited the phosphorylation of NDRG1 by targeting its regulating kinases, serum- and glucocorticoid-induced kinase 1 (SGK1) and glycogen synthase kinase 3 beta (GSK3beta). We demonstrate that in the presence of GA, GSK3beta protein and activity were decreased thus indicating that Hsp90 is necessary for GSK3beta stability. Taken together, our data demonstrate that NDRG1 is not a classic client protein but interacts with Hsp90 and is still dually regulated by Hsp90 at a transcriptional and post-translational level. Finally, we suggest for the first time GSK3beta as a new client protein of Hsp90.

  11. Post-translational regulation of rice MADS29 function: homodimerization or binary interactions with other seed-expressed MADS proteins modulate its translocation into the nucleus

    PubMed Central

    Nayar, Saraswati; Kapoor, Meenu; Kapoor, Sanjay

    2014-01-01

    OsMADS29 is a seed-specific MADS-box transcription factor that affects embryo development and grain filling by maintaining hormone homeostasis and degradation of cells in the nucellus and nucellar projection. Although it has a bipartite nuclear localization signal (NLS) sequence, the transiently expressed OsMADS29 monomer does not localize specifically in the nucleus. Dimerization of the monomers alters the intracellular localization fate of the resulting OsMADS29 homodimer, which then translocates into the nucleus. By generating domain-specific deletions/mutations, we show that two conserved amino acids (lysine23 and arginine24) in the NLS are important for nuclear localization of the OsMADS29 homodimer. Furthermore, the analyses involving interaction of OsMADS29 with 30 seed-expressed rice MADS proteins revealed 19 more MADS-box proteins, including five E-class proteins, which interacted with OsMADS29. Eleven of these complexes were observed to be localized in the nucleus. Deletion analysis revealed that the KC region (K-box and C-terminal domain) plays a pivotal role in homodimerization. These data suggest that the biological function of OsMADS29 may not only be regulated at the level of transcription and translation as reported earlier, but also at the post-translational level by way of the interaction between OsMADS29 monomers, and between OsMADS29 and other MADS-box proteins. PMID:25096923

  12. Similarities of omega gliadins from Triticum urartu to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing.

    PubMed

    DuPont, F M; Vensel, W; Encarnacao, T; Chan, R; Kasarda, D D

    2004-05-01

    The omega-gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6 x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2 x=14, AA ). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified omega-gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded omega-gliadins were smaller than 1B- or 1D-encoded omega-gliadins. The N-terminal amino acid sequences for 1A omega-gliadin mature peptides were nearly identical to those for the T. urartu omega-gliadins and were more similar to 1D omega-gliadin sequences than to sequences for T. monococum omega-gliadins, barley C-hordeins, or rye omega-secalins. They diverged greatly from the N-terminal sequences for the 1B omega-gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those omega-gliadins with N-terminal sequences beginning with KEL.

  13. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-01-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8+ T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses. PMID:23941868

  14. Genome-wide protein QTL mapping identifies human plasma kallikrein as a post-translational regulator of serum uPAR levels.

    PubMed

    Portelli, Michael A; Siedlinski, Mateusz; Stewart, Ceri E; Postma, Dirkje S; Nieuwenhuis, Maartje A; Vonk, Judith M; Nurnberg, Peter; Altmuller, Janine; Moffatt, Miriam F; Wardlaw, Andrew J; Parker, Stuart G; Connolly, Martin J; Koppelman, Gerard H; Sayers, Ian

    2014-02-01

    The soluble cleaved urokinase plasminogen activator receptor (scuPAR) is a circulating protein detected in multiple diseases, including various cancers, cardiovascular disease, and kidney disease, where elevated levels of scuPAR have been associated with worsening prognosis and increased disease aggressiveness. We aimed to identify novel genetic and biomolecular mechanisms regulating scuPAR levels. Elevated serum scuPAR levels were identified in asthma (n=514) and chronic obstructive pulmonary disease (COPD; n=219) cohorts when compared to controls (n=96). In these cohorts, a genome-wide association study of serum scuPAR levels identified a human plasma kallikrein gene (KLKB1) promoter polymorphism (rs4253238) associated with serum scuPAR levels in a control/asthma population (P=1.17 × 10(-7)), which was also observed in a COPD population (combined P=5.04 × 10(-12)). Using a fluorescent assay, we demonstrated that serum KLKB1 enzymatic activity was driven by rs4253238 and is inverse to scuPAR levels. Biochemical analysis identified that KLKB1 cleaves scuPAR and negates scuPAR's effects on primary human bronchial epithelial cells (HBECs) in vitro. Chymotrypsin was used as a proproteolytic control, while basal HBECs were used as a control to define scuPAR-driven effects. In summary, we reveal a novel post-translational regulatory mechanism for scuPAR using a hypothesis-free approach with implications for multiple human diseases.

  15. Synthesis and post-translational assembly of intermediate filaments in avian erythroid cells: vimentin assembly limits the rate of synemin assembly.

    PubMed

    Moon, R T; Lazarides, E

    1983-09-01

    The assembly of vimentin intermediate filaments and the high molecular weight filament crosslinking protein, synemin, was studied in erythroid cells from 10-day chicken embryos. Pulse labeling studies show that newly synthesized vimentin is present both in a Triton X-100-insoluble form and in a soluble form. The incorporation of labeled vimentin into the insoluble fraction increases linearly with time, while the soluble pool of labeled vimentin saturates quickly. In contrast, synemin accumulates rapidly in the Triton X-100-soluble fraction and begins to accumulate in the insoluble fraction only after a considerable lag of time. Pulse-chase studies reveal that the detergent-soluble pools of both vimentin and synemin contain precursors for their post-translational assembly into detergent-insoluble filaments and that the half-life of soluble synemin is about twice as long as that of soluble vimentin. Immunoprecipitation of solubilized filaments with synemin antiserum precipitates vimentin with synemin. On the other hand, soluble vimentin does not coimmunoprecipitate with soluble synemin. These results suggest that, in the assembly of vimentin and synemin into intermediate filaments, vimentin filament elongation generates synemin binding sites, and thus the rate of vimentin filament elongation limits the rate of synemin assembly.

  16. Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

    PubMed Central

    Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

    2016-01-01

    Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

  17. Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase.

    PubMed

    Lin, Yuan; Liu, Jun; Liu, Xun; Ou, Yongbin; Li, Meng; Zhang, Huiling; Song, Botao; Xie, Conghua

    2013-12-01

    The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

  18. Post-translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold-induced sweetening of potato tubers.

    PubMed

    McKenzie, Marian J; Chen, Ronan K Y; Harris, John C; Ashworth, Matthew J; Brummell, David A

    2013-01-01

    Cold-induced sweetening (CIS) is a serious post-harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark-coloured and bitter-tasting product and generating the probable carcinogen acrylamide as a by-product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS-susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS-resistant line increased susceptibility to CIS. The results show that post-translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.

  19. The Saccharomyces cerevisiae poly(A)-binding protein is subject to multiple post-translational modifications, including the methylation of glutamic acid.

    PubMed

    Low, Jason K K; Hart-Smith, Gene; Erce, Melissa A; Wilkins, Marc R

    2014-01-10

    Poly(A)-binding protein in mouse and man was recently found to be highly post-translationally modified. Here we analysed an ortholog of this protein, Pab1 from Saccharomyces cerevisiae, to assess the conservation and thus likely importance of these modifications. Pab1 showed the presence of six sites of methylated glutamate, five sites of lysine acetylation, and one phosphorylation of serine. Many modifications on Pab1 showed either complete conservation with those on human or mouse PABPC1, were present on nearby residues and/or were present in the same domain(s). The conservation of methylated glutamate, an unusual modification, was of particular note and suggests a conserved function. Comparison of methylated glutamate sites in human, mouse and yeast poly(A)-binding protein, along with methylation sites catalysed by CheR L-glutamyl protein methyltransferase from Salmonella typhimurium, revealed that the methylation of glutamate preferentially occurs in EE and DE motifs or other small regions of acidic amino acids. The conservation of methylated glutamate in the same protein between mouse, man and yeast suggests the presence of a eukaryotic l-glutamyl protein methyltransferase and that the modification is of functional significance.

  20. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

    PubMed

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-10-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

  1. Expression of RALT, a feedback inhibitor of ErbB receptors, is subjected to an integrated transcriptional and post-translational control.

    PubMed

    Fiorini, Monia; Ballarò, Costanza; Sala, Gianluca; Falcone, Germana; Alemà, Stefano; Segatto, Oreste

    2002-09-19

    Over-expression studies have demonstrated that RALT (receptor associated late transducer) is a feedback inhibitor of ErbB-2 mitogenic and transforming signals. In growth-arrested cells, expression of endogenous RALT is induced by mitogenic stimuli, is high throughout mid to late G1 and returns to baseline as cells move into S phase. Here, we show that physiological levels of RALT effectively suppress ErbB-2 mitogenic signals. We also investigate the regulatory mechanisms that preside to the control of RALT expression. We demonstrate that pharmacological ablation of extracellular signal-regulated kinase (ERK) activation leads to blockade of RALT expression, unlike genetic and/or pharmacological interference with the activities of PKC, Src family kinases, p38 SAPK and PI-3K. Tamoxifen-dependent activation of an inducible Raf : ER chimera was sufficient to induce RALT expression. Thus, activation of the Ras-Raf-ERK pathway is necessary and sufficient to drive RALT expression. The RALT protein is labile and was found to accumulate robustly upon pharmacological inhibition of the proteasome. We were able to detect ubiquitin-conjugated RALT species in living cells, suggesting that ubiquitinylation targets RALT for proteasome-dependent degradation. Such an integrated transcriptional and post-translational control is likely to provide RALT with the ability to fluctuate timely in order to tune ErbB signals.

  2. Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy.

    PubMed

    Cesnik, Anthony J; Shortreed, Michael R; Sheynkman, Gloria M; Frey, Brian L; Smith, Lloyd M

    2016-03-04

    Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines.

  3. A conserved type IV pilin signal peptide H-domain is critical for the post-translational regulation of flagella-dependent motility.

    PubMed

    Esquivel, Rianne N; Pohlschroder, Mechthild

    2014-08-01

    In many bacteria and archaea, type IV pili facilitate surface adhesion, the initial step in biofilm formation. Haloferax volcanii has a specific set of adhesion pilins (PilA1-A6) that, although diverse, contain an absolutely conserved signal peptide hydrophobic (H) domain. Data presented here demonstrate that these pilins (PilA1-A6) also play an important role in regulating flagella-dependent motility, which allows cells to rapidly transition between planktonic and sessile states. Cells lacking adhesion pilins exhibit a severe motility defect, however, expression of any one of the adhesion pilins in trans can rescue the motility and adhesion. Conversely, while deleting pilB3-C3, genes required for PilA pilus biosynthesis, results in cells lacking pili and having an adhesion defect, it does not affect motility, indicating that motility regulation requires the presence of pilins, but not assembled pili. Mutagenesis studies revealed that the pilin-dependent motility regulatory mechanism does not require the diverse C-terminal region of the PilA pilins but specifically involves the conserved H-domain. This novel post-translational regulatory mechanism, which employs components that promote biofilm formation to inhibit motility, can provide a rapid response to changing environmental conditions. A model for this regulatory mechanism, which may also be present in other prokaryotes, is discussed.

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

    PubMed Central

    Hijazi, May; Velasquez, Silvia M.; Jamet, Elisabeth; Estevez, José M.; Albenne, Cécile

    2014-01-01

    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. PMID:25177325

  5. Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity

    PubMed Central

    Funk, Kristen E.; Thomas, Stefani N.; Schafer, Kelsey N.; Cooper, Grace L.; Liao, Zhongping; Clark, David J.; Yang, Austin J.; Kuret, Jeff

    2015-01-01

    In Alzheimer disease, the microtubule-associated protein tau dissociates from the neuronal cytoskeleton and aggregates to form cytoplasmic inclusions. Although hyper-phosphorylation of tau Ser and Thr residues is an established trigger of tau misfunction and aggregation, tau modifications extend to Lys residues as well, raising the possibility that different modification signatures depress or promote aggregation propensity depending on site occupancy. To identify Lys-residue modifications associated with normal tau function, soluble tau proteins isolated from four cognitively normal human brains were characterized by mass spectrometry methods. The major detectable Lys modification was found to be methylation, which appeared in the form of mono- and di-methyl Lys residues distributed among at least eleven sites. Unlike tau phosphorylation sites, the frequency of Lys methylation was highest in the microtubule binding repeat region that mediates both microtubule binding and homotypic interactions. When purified recombinant human tau was modified in vitro through reductive methylation, its ability to promote tubulin polymerization was retained, whereas its aggregation propensity was greatly attenuated at both nucleation and extension steps. These data establish Lys methylation as part of the normal tau post-translational modification signature in human brain, and suggest that it can function in part to protect against pathological tau aggregation. PMID:24869773

  6. New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans.

    PubMed

    Kim, Hyun-Min; Colaiácovo, Monica P

    2015-06-01

    Although a growing number of studies have reported the importance of SUMOylation in genome maintenance and DNA double-strand break repair (DSBR), relevant target proteins and how this modification regulates their functions are yet to be clarified. Here, we analyzed SUMOylation of ZTF-8, the homolog of mammalian RHINO, to test the functional significance of this protein modification in the DSBR and DNA damage response (DDR) pathways in the Caenorhabditis elegans germline. We found that ZTF-8 is a direct target for SUMOylation in vivo and that its modification is required for DNA damage checkpoint induced apoptosis and DSBR. Non-SUMOylatable mutants of ZTF-8 mimic the phenotypes observed in ztf-8 null mutants, including reduced fertility, impaired DNA damage repair, and defective DNA damage checkpoint activation. However, while mutants for components acting in the SUMOylation pathway fail to properly localize ZTF-8, its localization is not altered in the ZTF-8 non-SUMOylatable mutants. Taken together, these data show that direct SUMOylation of ZTF-8 is required for its function in DSBR as well as DDR but not its localization. ZTF-8's human ortholog is enriched in the germline, but its meiotic role as well as its post-translational modification has never been explored. Therefore, our discovery may assist in understanding the regulatory mechanism of this protein in DSBR and DDR in the germline.

  7. Arabidopsis PARG1 is the key factor promoting cell survival among the enzymes regulating post-translational poly(ADP-ribosyl)ation

    PubMed Central

    Zhang, Hailei; Gu, Zongying; Wu, Qiao; Yang, Lifeng; Liu, Caifeng; Ma, Hong; Xia, Yiji; Ge, Xiaochun

    2015-01-01

    Poly(ADP-ribosyl)ation is a reversible post-translational modification of proteins, characterized by the addition of poly(ADP-ribose) (PAR) to proteins by poly(ADP-ribose) polymerase (PARP), and removal of PAR by poly(ADP-ribose) glycohydrolase (PARG). Three PARPs and two PARGs have been found in Arabidopsis, but their respective roles are not fully understood. In this study, the functions of each PARP and PARG in DNA repair were analyzed based on their mutant phenotypes under genotoxic stresses. Double or triple mutant analysis revealed that PARP1 and PARP2, but not PARP3, play a similar but not critical role in DNA repair in Arabidopsis seedlings. PARG1 and PARG2 play an essential and a minor role, respectively under the same conditions. Mutation of PARG1 results in increased DNA damage level and enhanced cell death in plants after bleomycin treatment. PARG1 expression is induced primarily in root and shoot meristems by bleomycin and induction of PARG1 is dependent on ATM and ATR kinases. PARG1 also antagonistically modulates the DNA repair process by preventing the over-induction of DNA repair genes. Our study determined the contribution of each PARP and PARG member in DNA repair and indicated that PARG1 plays a critical role in this process. PMID:26516022

  8. Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry.

    PubMed

    Sidoli, Simone; Lin, Shu; Karch, Kelly R; Garcia, Benjamin A

    2015-03-17

    Histone proteins are key components of chromatin. Their N-terminal tails are enriched in combinatorial post-translational modifications (PTMs), which influence gene regulation, DNA repair, and chromosome condensation. Mass spectrometry (MS)-based middle-down proteomics has emerged as a technique to analyze co-occurring PTMs, as it allows for the characterization of intact histone tails (>50 aa) rather than short (<20 aa) peptides analyzed by bottom-up. However, a demonstration of its reliability is still lacking. We compared results obtained with the middle-down and the bottom-up strategy in calculating PTM relative abundance and stoichiometry. Since bottom-up was proven to have biases in peptide signal detection such as uneven ionization efficiency, we performed an external correction using a synthetic peptide library with known peptide relative abundance. Corrected bottom-up data were used as reference. Calculated abundances of single PTMs showed similar deviations from the reference when comparing middle-down and uncorrected bottom-up results. Moreover, we show that the two strategies provided similar performance in defining accurate PTM stoichiometry. Collectively, we evidenced that the middle-down strategy is at least equally reliable to bottom-up in quantifying histone PTMs.

  9. Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125

    PubMed Central

    Hejjaoui, Mirva; Butterfield, Sara; Fauvet, Bruno; Vercruysse, Filip; Cui, Jia; Dikiy, Igor; Prudent, Michel; Olschewski, Diana; Zhang, Yan; Eliezer, David; Lashuel, Hilal A.

    2013-01-01

    Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of α-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine → glutamate or aspartate substitutions, there are no natural amino acids that can mimic phosphor-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of α-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding and subcellular localization of α-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in α-syn structure, aggregation and functions in heath and disease. PMID:22339654

  10. Human Proteomic Variation Revealed by Combining RNA-Seq Proteogenomics and Global Post-Translational Modification (G-PTM) Search Strategy

    PubMed Central

    2015-01-01

    Mass-spectrometry-based proteomic analysis underestimates proteomic variation due to the absence of variant peptides and posttranslational modifications (PTMs) from standard protein databases. Each individual carries thousands of missense mutations that lead to single amino acid variants, but these are missed because they are absent from generic proteomic search databases. Myriad types of protein PTMs play essential roles in biological processes but remain undetected because of increased false discovery rates in variable modification searches. We address these two fundamental shortcomings of bottom-up proteomics with two recently developed software tools. The first consists of workflows in Galaxy that mine RNA sequencing data to generate sample-specific databases containing variant peptides and products of alternative splicing events. The second tool applies a new strategy that alters the variable modification approach to consider only curated PTMs at specific positions, thereby avoiding the combinatorial explosion that traditionally leads to high false discovery rates. Using RNA-sequencing-derived databases with this Global Post-Translational Modification (G-PTM) search strategy revealed hundreds of single amino acid variant peptides, tens of novel splice junction peptides, and several hundred posttranslationally modified peptides in each of ten human cell lines. PMID:26704769

  11. Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

    PubMed Central

    Sidoli, Simone; Bhanu, Natarajan V.; Karch, Kelly R.; Wang, Xiaoshi; Garcia, Benjamin A.

    2016-01-01

    Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude. PMID:27286567

  12. Linking structural dynamics and functional diversity in asymmetric catalysis.

    PubMed

    Nojiri, Akihiro; Kumagai, Naoya; Shibasaki, Masakatsu

    2009-03-18

    Proteins, the functional molecules in biological systems, are sophisticated chemical devices that have evolved over billions of years. Their function is intimately related to their three-dimensional structure and elegantly regulated by conformational changes through allosteric regulators and a number of reversible or unidirectional post-translational modifications. This functional diversification in response to external stimuli allows for an orderly and timely progression of intra- and extracellular events. In contrast, enantioselective catalysts generally exhibit limited conformational flexibility and thereby exert a single specific function. Exploiting the features of conformationally flexible asymmetric ligands and the variable coordination patterns of rare earth metals, we demonstrate dynamic structural and functional changes of a catalyst in asymmetric catalysis, leading to two distinct reaction outcomes in a single flask.

  13. The Metamorphic Nature of the Tau Protein: Dynamic Flexibility Comes at a Cost

    PubMed Central

    Sabbagh, Jonathan J.; Dickey, Chad A.

    2016-01-01

    Accumulation of the microtubule associated protein tau occurs in several neurodegenerative diseases including Alzheimer's disease (AD). The tau protein is intrinsically disordered, giving it unique structural properties that can be dynamically altered by post-translational modifications such as phosphorylation and cleavage. Over the last decade, technological advances in nuclear magnetic resonance (NMR) spectroscopy and structural modeling have permitted more in-depth insights into the nature of tau. These studies have helped elucidate how metamorphism of tau makes it ideally suited for dynamic microtubule regulation, but how it also facilitates tau self-assembly, oligomerization, and neurotoxicity. This review will focus on how the distinct structure of tau governs its function, accumulation, and toxicity as well as how other cellular factors such as molecular chaperones control these processes. PMID:26834532

  14. The Metamorphic Nature of the Tau Protein: Dynamic Flexibility Comes at a Cost.

    PubMed

    Sabbagh, Jonathan J; Dickey, Chad A

    2016-01-01

    Accumulation of the microtubule associated protein tau occurs in several neurodegenerative diseases including Alzheimer's disease (AD). The tau protein is intrinsically disordered, giving it unique structural properties that can be dynamically altered by post-translational modifications such as phosphorylation and cleavage. Over the last decade, technological advances in nuclear magnetic resonance (NMR) spectroscopy and structural modeling have permitted more in-depth insights into the nature of tau. These studies have helped elucidate how metamorphism of tau makes it ideally suited for dynamic microtubule regulation, but how it also facilitates tau self-assembly, oligomerization, and neurotoxicity. This review will focus on how the distinct structure of tau governs its function, accumulation, and toxicity as well as how other cellular factors such as molecular chaperones control these processes.

  15. Interindividual differences in the dynamics of the homeostatic process are trait-like and distinct for sleep versus wakefulness.

    PubMed

    Rusterholz, Thomas; Tarokh, Leila; Van Dongen, Hans P A; Achermann, Peter

    2017-04-01

    The sleep homeostatic Process S reflects the build-up of sleep pressure during waking and its dissipation during sleep. Process S is modelled as a saturating exponential function during waking and a decreasing exponential function during sleep. Slow wave activity is a physiological marker for non-rapid eye movement (non-REM) sleep intensity and serves as an index of Process S. There is considerable interindividual variability in the sleep homeostatic responses to sleep and sleep deprivation. The aim of this study was to investigate whether interindividual differences in Process S are trait-like. Polysomnographic recordings of 8 nights (12-h sleep opportunities, 22:00-10:00 hours) interspersed with three 36-h periods of sustained wakefulness were performed in 11 healthy young adults. Empirical mean slow wave activity per non-REM sleep episode at episode mid-points were used for parameter estimation. Parameters of Process S were estimated using different combinations of consecutive sleep recordings, resulting in two to three sets of parameters per subject. Intraclass correlation coefficients were calculated to assess whether the parameters were stable across the study protocol and they showed trait-like variability among individuals. We found that the group-average time constants of the build-up and dissipation of Process S were 19.2 and 2.7 h, respectively. Intraclass correlation coefficients ranged from 0.48 to 0.56, which reflects moderate trait variability. The time constants of the build-up and dissipation varied independently among subjects, indicating two distinct traits. We conclude that interindividual differences in the parameters of the dynamics of the sleep homeostatic Process S are trait-like.

  16. Sodium arsenite down-regulates the expression of X-linked inhibitor of apoptosis protein via translational and post-translational mechanisms in hepatocellular carcinoma

    SciTech Connect

    Chen, Hong; Hao, Yuqing; Wang, Lijing; Jia, Dongwei; Ruan, Yuanyuan; Gu, Jianxin

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer Sodium arsenite down-regulates the protein expression level of XIAP in HCC. Black-Right-Pointing-Pointer Sodium arsenite inhibits the de novo XIAP synthesis and its IRES activity. Black-Right-Pointing-Pointer Sodium arsenite decreases XIAP stability and promotes its proteasomal degradation. Black-Right-Pointing-Pointer Overexpression of XIAP attenuates the pro-apoptotic effect of sodium arsenite. -- Abstract: X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitors of apoptosis protein (IAP) family, and has been reported to exhibit elevated expression levels in hepatocellular carcinoma (HCC) and promote cell survival, metastasis and tumor recurrence. Targeting XIAP has proven effective for the inhibition of cancer cell proliferation and restoration of cancer cell chemosensitivity. Arsenic (or sodium arsenite) is a potent anti-tumor agent used to treat patients with acute promyelocytic leukemia (APL). Additionally, arsenic induces cell growth inhibition, cell cycle arrest and apoptosis in human HCC cells. In this study, we identified XIAP as a target for sodium arsenite-induced cytotoxicity in HCC. The exposure of HCC cell lines to sodium arsenite resulted in inhibition of XIAP expression in both a dose- and time-dependent manner. Sodium arsenite blocked the de novo XIAP synthesis and the activity of its internal ribosome entry site (IRES) element. Moreover, treatment with sodium arsenite decreased the protein stability of XIAP and induced its ubiquitin-proteasomal degradation. Overexpression of XIAP attenuated the pro-apoptotic effect of sodium arsenite in HCC. Taken together, our data demonstrate that sodium arsenite suppresses XIAP expression via translational and post-translational mechanisms in HCC.

  17. Post-translational modification of the NKG2D ligand RAET1G leads to cell surface expression of a glycosylphosphatidylinositol-linked isoform.

    PubMed

    Ohashi, Maki; Eagle, Robert A; Trowsdale, John

    2010-05-28

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform.

  18. Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform*

    PubMed Central

    Ohashi, Maki; Eagle, Robert A.; Trowsdale, John

    2010-01-01

    NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform. PMID:20304922

  19. Post-translational deregulation of YAP1 is genetically controlled in rat liver cancer and determines the fate and stem-like behavior of the human disease

    PubMed Central

    Demartis, Maria I.; Brozzetti, Stefania; Calvisi, Diego F.; Porcu, Alberto; Feo, Claudio F.; Seddaiu, Maria A.; Daino, Lucia; Berasain, Carmen; Tomasi, Maria L.; Avila, Matias A.; Feo, Francesco; Pascale, Rosa M.

    2016-01-01

    Previous studies showed that YAP1 is over-expressed in hepatocellular carcinoma (HCC). Here we observed higher expression of Yap1/Ctgf axis in dysplastic nodules and HCC chemically-induced in F344 rats, genetically susceptible to hepatocarcinogenesis, than in lesions induced in resistant BN rats. In BN rats, highest increase in Yap1-tyr357, p73 phosphorylation and Caspase 3 cleavage occurred. In human HCCs with poorer prognosis (< 3 years survival after partial liver resection, HCCP), levels of YAP1, CTGF, 14–3–3, and TEAD proteins, and YAP1-14-3-3 and YAP1-TEAD complexes were higher than in HCCs with better outcome (> 3 years survival; HCCB). In the latter, higher levels of phosphorylated YAP1-ser127, YAP1-tyr357 and p73, YAP1 ubiquitination, and Caspase 3 cleavage occurred. Expression of stemness markers NANOG, OCT-3/4, and CD133 were highest in HCCP and correlated with YAP1 and YAP1-TEAD levels. In HepG2, Huh7, and Hep3B cells, forced YAP1 over-expression led to stem cell markers expression and increased cell viability, whereas inhibition of YAP1 expression by specific siRNA, or transfection of mutant YAP1 which does not bind to TEAD, induced opposite alterations. These changes were associated, in Huh7 cells transfected with YAP1 or YAP1 siRNA, with stimulation or inhibition of cell migration and invasivity, respectively. Furthermore, transcriptome analysis showed that YAP1 transfection in Huh7 cells induces over-expression of genes involved in tumor stemness. In conclusion, Yap1 post-translational modifications favoring its ubiquitination and apoptosis characterize HCC with better prognosis, whereas conditions favoring the formation of YAP1-TEAD complexes are associated with aggressiveness and acquisition of stemness features by HCC cells. PMID:27359056

  20. Pancreatic β-cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.1 delayed rectifier channels

    PubMed Central

    Kim, S-J; Widenmaier, S B; Choi, W S; Nian, C; Ao, Z; Warnock, G; McIntosh, C H S

    2012-01-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic β-cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in β-cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 β-cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of β-cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the β-cell prosurvival effects of incretins and of 7-transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function. PMID:21818121

  1. Comparative study of the post-translational processing of the mannose-binding lectins in the bulbs of garlic (Allium sativum L.) and ramsons (Allium ursinum L.).

    PubMed

    Smeets, K; Van Damme, E J; Peumans, W J

    1994-08-01

    The biosynthesis and processing of the homodimeric and heterodimeric lectins from the bulbs of garlic (Allium sativum) and ramsons (wild garlic; Allium ursinum) were studied using pulse and pulse-chase labelling experiments on developing bulbs. By combining the results of the in vivo biosynthesis studies and the cDNA cloning of the respective lectins, the sequence of events leading from the primary translation products into the mature lectin polypeptides could be reconstructed. From this it is demonstrated that garlic and ramsons use different schemes of post-translational modifications in order to synthesize apparently similar lectins from totally different precursors. Both the homomeric garlic lectin (ASAII) and its homologue in ramsons (AUAII) are synthesized on the endoplasmic reticulum (ER) as nonglycosylated 13.5 kDa precursors, which, after their transport out of the ER are converted into the mature 12.0 kDa lectin polypeptides by the cleavage of a C-terminal peptide. The heterodimeric garlic lectin ASAI is synthesized on the ER as a single glycosylated precursor of 38 kDa, which after its transport out of the ER undergoes a complex processing which gives rise to two mature lectin subunits of 11.5 and 12.5 kDa. In contrast, both subunits of the heterodimeric ramsons lectin AUAI are synthesized separately on the ER as glycosylated precursors, which after their transport out of the ER are deglycosylated and further processed into the mature lectin polypeptides by the cleavage of a C-terminal peptide.

  2. Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan*

    PubMed Central

    Wang, Lauren W.; Leonhard-Melief, Christina; Haltiwanger, Robert S.; Apte, Suneel S.

    2009-01-01

    Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the 36WDAWGPWSECSRTC49 sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp39 and Trp42. C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-3H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion. PMID:19671700

  3. Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan.

    PubMed

    Wang, Lauren W; Leonhard-Melief, Christina; Haltiwanger, Robert S; Apte, Suneel S

    2009-10-30

    Protein C-mannosylation is the attachment of alpha-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the (36)WDAWGPWSECSRTC(49) sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp(39) and Trp(42). C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-(3)H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.

  4. New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination

    PubMed Central

    Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

    2016-01-01

    Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV–V when coleoptiles initiate the formation of the photosynthetic tissues. PMID:27194739

  5. ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

    PubMed

    Xing, Shufan; van Deenen, Nicole; Magliano, Pasqualina; Frahm, Lea; Forestier, Edith; Nawrath, Christiane; Schaller, Hubert; Gronover, Christian S; Prüfer, Dirk; Poirier, Yves

    2014-07-01

    Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

  6. Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1.

    PubMed

    Bonet-Costa, Carles; Vilaseca, Marta; Diema, Claudio; Vujatovic, Olivera; Vaquero, Alejandro; Omeñaca, Núria; Castejón, Lucía; Bernués, Jordi; Giralt, Ernest; Azorín, Fernando

    2012-07-16

    Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

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

    PubMed Central

    Mandrich, Luigi; Cerreta, Mariangela; Manco, Giuseppe

    2015-01-01

    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. PMID:26656916

  8. In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

    PubMed

    Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

    2016-01-15

    Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression.

  9. Post-translational Modification of Ribosomal Proteins: Structural and Functional Characterization of RimO from Thermotoga maritima, a Radical S-adenosylmethionine methylthiotransferase

    SciTech Connect

    Arragain, S.; Latour, J; Forouhar, F; Neely, H; Montelione, G; Hunt, J; Mulliez, E; Fontecave, M; Atta, M; et al.

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 {angstrom} crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

  10. Post-translational Modification of Ribosomal Proteins - Structural and Functional Characterization of RimO from Thermotoga Maritima, A Radiacal S-Adenosylmethionine Methylthiotransferase

    SciTech Connect

    Arragain, S.; Garcia-Serres, R; Blondin, G; Douki, T; Clemancey, M; Latour, J; Forouhar, F; Neely, H; Montelione, G; et. al.

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826-1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 {angstrom} crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

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

    PubMed Central

    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

  12. Structure of a Putative Lipoate Protein Ligase from Thermoplasma acidophilum and the Mechanism of Target Selection for Post-Translational Modification

    SciTech Connect

    McManus,E.; Luisi, B.; Perham, R.

    2006-01-01

    Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 Angstroms resolution. The protein was found to fall into the a/{beta} class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T. acidophilum LplA and limited proteolysis of E. coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T. acidophilumligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T. acidophilum structure; tryptic cleavage of the corresponding loop in the E. coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T. acidophilum lacks a C-terminal domain found in its counterparts in E. coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E. coli and S. pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T. acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.

  13. Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post-translational processing that removes immunogenicity while retaining fibronectin binding.

    PubMed

    Scott, Nichollas E; Marzook, N Bishara; Deutscher, Ania; Falconer, Linda; Crossett, Ben; Djordjevic, Steven P; Cordwell, Stuart J

    2010-01-01

    Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.

  14. Modulation of farnesoid X receptor results in post-translational modification of poly (ADP-ribose) polymerase 1 in the liver

    SciTech Connect

    Zhu, Yan; Li, Guodong; Dong, Yafeng; Zhou, Helen H.; Kong, Bo; Aleksunes, Lauren M.; Richardson, Jason R.; Li, Fei; Guo, Grace L.

    2013-01-15

    The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in mice leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3 month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18 months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver. -- Highlights: ► Proteomic analysis identified novel FXR targets. ► FXR modification altered post-translational modification of the Parp1 protein. ► Altered Parp1 function may contribute to mechanisms of FXR regulation of liver functions.

  15. Human (α2→6) and Avian (α2→3) Sialylated Receptors of Influenza A Virus Show Distinct Conformations and Dynamics in Solution

    PubMed Central

    Macchi, Eleonora; Yates, Edwin A.; Naggi, Annamaria; Shriver, Zachary; Raman, Rahul; Sasisekharan, R.; Torri, Giangiacomo; Guerrini, Marco

    2013-01-01

    Differential interactions between influenza A virus protein hemagglutinin (HA) and α2→3 (avian) or α2→6 (human) sialylated glycan receptors play an important role in governing host specificity and adaptation of the virus. Previous analysis of HA–glycan interactions with trisaccharides showed that, in addition to the terminal sialic acid linkage, the conformation and topology of the glycans, while they are bound to HA, are key factors in regulating these interactions. Here, the solution conformation and dynamics of two representative avian and human glycan pentasaccharide receptors [LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc; LSTc, (Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc] have been explored using nuclear magnetic resonance and molecular dynamics simulation. Analyses demonstrate that, in solution, human and avian receptors sample distinct conformations, topologies, and dynamics. These unique features of avian and human receptors in solution could represent distinct molecular characteristics for recognition by HA, thereby providing the HA–glycan interaction specificity in influenza. PMID:24015903

  16. Human (α2→6) and avian (α2→3) sialylated receptors of influenza A virus show distinct conformations and dynamics in solution.

    PubMed

    Sassaki, Guilherme L; Elli, Stefano; Rudd, Timothy R; Macchi, Eleonora; Yates, Edwin A; Naggi, Annamaria; Shriver, Zachary; Raman, Rahul; Sasisekharan, R; Torri, Giangiacomo; Guerrini, Marco

    2013-10-15

    Differential interactions between influenza A virus protein hemagglutinin (HA) and α2→3 (avian) or α2→6 (human) sialylated glycan receptors play an important role in governing host specificity and adaptation of the virus. Previous analysis of HA-glycan interactions with trisaccharides showed that, in addition to the terminal sialic acid linkage, the conformation and topology of the glycans, while they are bound to HA, are key factors in regulating these interactions. Here, the solution conformation and dynamics of two representative avian and human glycan pentasaccharide receptors [LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc; LSTc, (Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc] have been explored using nuclear magnetic resonance and molecular dynamics simulation. Analyses demonstrate that, in solution, human and avian receptors sample distinct conformations, topologies, and dynamics. These unique features of avian and human receptors in solution could represent distinct molecular characteristics for recognition by HA, thereby providing the HA-glycan interaction specificity in influenza.

  17. Gait dynamics in Parkinson's disease: common and distinct behavior among stride length, gait variability, and fractal-like scaling.

    PubMed

    Hausdorff, Jeffrey M

    2009-06-01

    Parkinson's disease (PD) is a common, debilitating neurodegenerative disease. Gait disturbances are a frequent cause of disability and impairment for patients with PD. This article provides a brief introduction to PD and describes the gait changes typically seen in patients with this disease. A major focus of this report is an update on the study of the fractal properties of gait in PD, the relationship between this feature of gait and stride length and gait variability, and the effects of different experimental conditions on these three gait properties. Implications of these findings are also briefly described. This update highlights the idea that while stride length, gait variability, and fractal scaling of gait are all impaired in PD, distinct mechanisms likely contribute to and are responsible for the regulation of these disparate gait properties.

  18. Gait dynamics in Parkinson's disease: Common and distinct behavior among stride length, gait variability, and fractal-like scaling

    NASA Astrophysics Data System (ADS)

    Hausdorff, Jeffrey M.

    2009-06-01

    Parkinson's disease (PD) is a common, debilitating neurodegenerative disease. Gait disturbances are a frequent cause of disability and impairment for patients with PD. This article provides a brief introduction to PD and describes the gait changes typically seen in patients with this disease. A major focus of this report is an update on the study of the fractal properties of gait in PD, the relationship between this feature of gait and stride length and gait variability, and the effects of different experimental conditions on these three gait properties. Implications of these findings are also briefly described. This update highlights the idea that while stride length, gait variability, and fractal scaling of gait are all impaired in PD, distinct mechanisms likely contribute to and are responsible for the regulation of these disparate gait properties.

  19. Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants

    PubMed Central

    Kotliński, Maciej; Rutowicz, Kinga; Kniżewski, Łukasz; Palusiński, Antoni; Olędzki, Jacek; Fogtman, Anna; Rubel, Tymon; Koblowska, Marta; Dadlez, Michał; Ginalski, Krzysztof; Jerzmanowski, Andrzej

    2016-01-01

    Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones. PMID:26820416

  20. Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis.

    PubMed

    Van den Steen, Philippe E; Proost, Paul; Grillet, Bernard; Brand, David D; Kang, Andrew H; Van Damme, Jo; Opdenakker, Ghislain

    2002-03-01

    During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin-8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high-performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O-linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post-translational modifications at P2' and P5', respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T-cell activation. These data lend support to the thesis that extracellular proteolysis and other post-translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes.

  1. Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants.

    PubMed

    Kotliński, Maciej; Rutowicz, Kinga; Kniżewski, Łukasz; Palusiński, Antoni; Olędzki, Jacek; Fogtman, Anna; Rubel, Tymon; Koblowska, Marta; Dadlez, Michał; Ginalski, Krzysztof; Jerzmanowski, Andrzej

    2016-01-01

    Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

  2. The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

    PubMed Central

    Leronni, Daniela

    2013-01-01

    Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures. PMID:23897891

  3. Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants

    PubMed Central

    Gieldon, Artur; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Wenta, Tomasz; Golik, Przemyslaw; Dubin, Grzegorz; Lipinska, Barbara; Ciarkowski, Jerzy

    2016-01-01

    HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not

  4. TccP2-mediated subversion of actin dynamics by EPEC 2 - a distinct evolutionary lineage of enteropathogenic Escherichia coli.

    PubMed

    Whale, Andrew D; Hernandes, Rodrigo T; Ooka, Tadasuke; Beutin, Lothar; Schüller, Stephanie; Garmendia, Junkal; Crowther, Lynette; Vieira, Mônica A M; Ogura, Yoshitoshi; Krause, Gladys; Phillips, Alan D; Gomes, Tania A T; Hayashi, Tetsuya; Frankel, Gad

    2007-06-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspF(U) (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H-), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck-/- cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin

  5. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis.

    PubMed

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F; Gorfe, Alemayehu A

    2016-03-08

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15-20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1-3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.

  6. Transcriptome analysis of different developmental stages of amphioxus reveals dynamic changes of distinct classes of genes during development.

    PubMed

    Yang, Kevin Yi; Chen, Yuan; Zhang, Zuming; Ng, Patrick Kwok-Shing; Zhou, Wayne Junwei; Zhang, Yinfeng; Liu, Minghua; Chen, Junyuan; Mao, Bingyu; Tsui, Stephen Kwok-Wing

    2016-03-16

    Vertebrates diverged from other chordates approximately 500 million years ago and have adopted several modifications of developmental processes. Amphioxus is widely used in evolutionary developmental biology research, such as on the basic patterning mechanisms involved in the chordate body plan and the origin of vertebrates. The fast development of next-generation sequencing has advanced knowledge of the genomic organization of amphioxus; however, many aspects of gene regulation during amphioxus development have not been fully characterized. In this study, we applied high-throughput sequencing on the transcriptomes of 13 developmental stages of Chinese amphioxus to gain a comprehensive understanding of transcriptional processes occurring from the fertilized egg to the adult stage. The expression levels of 3,423 genes were significantly changed (FDR ≤ 0.01). All of these genes were included in a clustering analysis, and enrichment of biological functions associated with these clusters was determined. Significant changes were observed in several important processes, including the down-regulation of the cell cycle and the up-regulation of translation. These results should build a foundation for identifying developmentally important genes, especially those regulatory factors involved in amphioxus development, and advance understanding of the developmental dynamics in vertebrates.

  7. Transcriptome analysis of different developmental stages of amphioxus reveals dynamic changes of distinct classes of genes during development

    PubMed Central

    Yang, Kevin Yi; Chen, Yuan; Zhang, Zuming; Ng, Patrick Kwok-Shing; Zhou, Wayne Junwei; Zhang, Yinfeng; Liu, Minghua; Chen, Junyuan; Mao, Bingyu; Tsui, Stephen Kwok-Wing

    2016-01-01

    Vertebrates diverged from other chordates approximately 500 million years ago and have adopted several modifications of developmental processes. Amphioxus is widely used in evolutionary developmental biology research, such as on the basic patterning mechanisms involved in the chordate body plan and the origin of vertebrates. The fast development of next-generation sequencing has advanced knowledge of the genomic organization of amphioxus; however, many aspects of gene regulation during amphioxus development have not been fully characterized. In this study, we applied high-throughput sequencing on the transcriptomes of 13 developmental stages of Chinese amphioxus to gain a comprehensive understanding of transcriptional processes occurring from the fertilized egg to the adult stage. The expression levels of 3,423 genes were significantly changed (FDR ≤ 0.01). All of these genes were included in a clustering analysis, and enrichment of biological functions associated with these clusters was determined. Significant changes were observed in several important processes, including the down-regulation of the cell cycle and the up-regulation of translation. These results should build a foundation for identifying developmentally important genes, especially those regulatory factors involved in amphioxus development, and advance understanding of the developmental dynamics in vertebrates. PMID:26979494

  8. Distinct growth factor-induced dynamic mass redistribution (DMR) profiles for monitoring oncogenic signaling pathways in various cancer cells.

    PubMed

    Du, Yuhong; Li, Zijian; Li, Lian; Chen, Zhuo Georgia; Sun, Shi-Yong; Chen, Peifang; Shin, Dong M; Khuri, Fadlo R; Fu, Haian

    2009-01-01

    Targeting dysregulated signaling pathways in tumors has led to the development of a novel class of signal transduction inhibitors, including inhibitors of the epidermal growth factor (EGF) receptor (EGFR). To dissect oncogenic pathways, identify key pathway determinants, and evaluate the efficacy of targeted agents, it is vital to develop technologies that allow the detection of temporal signaling events under physiological conditions. Here we report the application of a label-free optical biosensor to reveal the rapid response of cancer cells to EGF, expressed as a dynamic mass redistribution (DMR) signal. In response to EGF, squamous cell carcinoma of the head and neck cells exhibited a rapid rise in DMR signal, whereas lung adenocarcinoma cells showed a biphasic DMR profile, suggesting a cell type-dependent DMR response. Pharmacological studies suggested the importance of EGFR and the phosphatidylinositol-3 kinase pathway in mediating the EGF-induced DMR response. The defined DMR signatures offer a simple yet sensitive tool for evaluating EGFR-targeted agents, as shown with gefitinib and erlotinib. The assay can also be used for cell-based high-throughput screening of EGF pathway inhibitors, as demonstrated by its robust performance in a 384-well plate format (Z' > 0.5). This technology is applicable to other oncogenic pathways for the discovery of novel therapeutic agents for the treatment of various cancers.

  9. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis

    PubMed Central

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F.; Gorfe, Alemayehu A.

    2016-01-01

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15–20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1–3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy. PMID:26958889

  10. The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

    PubMed Central

    Cobos, Eva S.; Villegas, Sandra; Martinez, Jose C.

    2014-01-01

    The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational

  11. Mapping of Post-translational Modifications of Transition Proteins, TP1 and TP2, and Identification of Protein Arginine Methyltransferase 4 and Lysine Methyltransferase 7 as Methyltransferase for TP2*

    PubMed Central

    Gupta, Nikhil; Madapura, M. Pradeepa; Bhat, U. Anayat; Rao, M. R. Satyanarayana

    2015-01-01

    In a unique global chromatin remodeling process during mammalian spermiogenesis, 90% of the nucleosomal histones are replaced by testis-specific transition proteins, TP1, TP2, and TP4. These proteins are further substituted by sperm-specific protamines, P1 and P2, to form a highly condensed sperm chromatin. In spermatozoa, a small proportion of chromatin, which ranges from 1 to 10% in mammals, retains the nucleosomal architecture and is implicated to play a role in transgenerational inheritance. However, there is still no mechanistic understanding of the interaction of chromatin machinery with histones and transition proteins, which facilitate this selective histone replacement from chromatin. Here, we report the identification of 16 and 19 novel post-translational modifications on rat endogenous transition proteins, TP1 and TP2, respectively, by mass spectrometry. By in vitro assays and mutational analysis, we demonstrate that protein arginine methyltransferase PRMT4 (CARM1) methylates TP2 at Arg71, Arg75, and Arg92 residues, and lysine methyltransferase KMT7 (Set9) methylates TP2 at Lys88 and Lys91 residues. Further studies with modification-specific antibodies that recognize TP2K88me1 and TP2R92me1 modifications showed that they appear in elongating to condensing spermatids and predominantly associated with the chromatin-bound TP2. This work establishes the repertoire of post-translational modifications that occur on TP1 and TP2, which may play a significant role in various chromatin-templated events during spermiogenesis and in the establishment of the sperm epigenome. PMID:25818198

  12. ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

    PubMed Central

    Vaziri, H; West, M D; Allsopp, R C; Davison, T S; Wu, Y S; Arrowsmith, C H; Poirier, G G; Benchimol, S

    1997-01-01

    Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging. PMID:9312059

  13. One-shot LC-MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and β-crystallins induced by γ-irradiation.

    PubMed

    Kim, Ingu; Saito, Takeshi; Fujii, Norihiko; Kanamoto, Takashi; Fujii, Noriko

    2016-12-01

    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 water-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 sites 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 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.

  14. Temperature-sensitive, Post-translational Regulation of Plant Omega-3 Fatty-acid Desaturases is Mediated by the Endoplasmic Reticulum-associated Degradation Pathway

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Changes in ambient temperature represent a major physiological challenge to poikilothermic organisms that requires rapid adjustments in the composition of cellular membranes in order to preserve overall membrane dynamics and integrity. In plants, the endoplasmic reticulum-localized omega-3 fatty ac...

  15. Dynamic regulation of aquaporin-4 water channels in neurological disorders

    PubMed Central

    Hsu, Ying; Tran, Minh; Linninger, Andreas A.

    2015-01-01

    Aquaporin-4 water channels play a central role in brain water regulation in neurological disorders. Aquaporin-4 is abundantly expressed at the astroglial endfeet facing the cerebral vasculature and the pial membrane, and both its expression level and subcellular localization significantly influence brain water transport. However, measurements of aquaporin-4 levels in animal models of brain injury often report opposite trends of change at the injury core and the penumbra. Furthermore, aquaporin-4 channels play a beneficial role in brain water clearance in vasogenic edema, but a detrimental role in cytotoxic edema and exacerbate cell swelling. In light of current evidence, we still do not have a complete understanding of the role of aquaporin-4 in brain water transport. In this review, we propose that the regulatory mechanisms of aquaporin-4 at the transcriptional, translational, and post-translational levels jointly regulate water permeability in the short and long time scale after injury. Furthermore, in order to understand why aquaporin-4 channels play opposing roles in cytotoxic and vasogenic edema, we discuss experimental evidence on the dynamically changing osmotic gradients between blood, extracellular space, and the cytosol during the formation of cytotoxic and vasogenic edema. We conclude with an emerging picture of the distinct osmotic environments in cytotoxic and vasogenic edema, and propose that the directions of aquaporin-4-mediated water clearance in these two types of edema are distinct. The difference in water clearance pathways may provide an explanation for the conflicting observations of the roles of aquaporin-4 in edema resolution. PMID:26526878

  16. Post-translational modification and conformational state of Heat Shock Protein 90 differentially affect binding of chemically diverse small molecule inhibitors

    PubMed Central

    Beebe, Kristin; Mollapour, Mehdi; Scroggins, Bradley; Prodromou, Chrisostomos; Xu, Wanping; Tokita, Mari; Taldone, Tony; Pullen, Lester; Zierer, Bettina K.; Lee, Min-Jung; Trepel, Jane; Buchner, Johannes; Bolon, Daniel; Chiosis, Gabriela; Neckers, Leonard

    2013-01-01

    Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic. PMID:23867252

  17. Oxidative and nitrosative-based signaling and associated post-translational modifications orchestrate the acclimation of citrus plants to salinity stress.

    PubMed

    Tanou, Georgia; Filippou, Panagiota; Belghazi, Maya; Job, Dominique; Diamantidis, Grigorios; Fotopoulos, Vasileios; Molassiotis, Athanassios

    2012-11-01

    Reactive oxygen and nitrogen species are involved in a plethora of cellular responses in plants; however, our knowledge on the outcomes of oxidative and nitrosative signaling is still unclear. To better understand how oxidative and nitrosative signals are integrated to regulate cellular adjustments to external conditions, local and systemic responses were investigated in the roots and leaves of sour orange plants (Citrus aurantium L.) after root treatment with hydrogen peroxide (H(2) O(2) ) or sodium nitroprusside (a nitric oxide donor), followed by NaCl stress for 8 days. Phenotypic and physiological data showed that pre-exposure to these treatments induced an acclimation to subsequent salinity stress that was accompanied by both local and systemic H(2) O(2) and nitric oxide (NO) accumulation. Combined histochemical and fluorescent probe approaches showed the existence of a vascular-driven long-distance reactive oxygen species and NO signaling pathway. Transcriptional analysis of genes diagnostic for H(2) O(2) and NO signaling just after treatments or after 8 days of salt stress revealed tissue- and time-specific mechanisms controlling internal H(2) O(2) and NO homeostasis. Furthermore, evidence is presented showing that protein carbonylation, nitration and S-nitrosylation are involved in acclimation to salinity stress. In addition, this work enabled characterization of potential carbonylated, nitrated and nitrosylated proteins with distinct or overlapping signatures. This work provides a framework to better understand the oxidative and nitrosative priming network in citrus plants subjected to salinity conditions.

  18. Insights into the Distinct Lithiation/Sodiation of Porous Cobalt Oxide by in Operando Synchrotron X-ray Techniques and Ab Initio Molecular Dynamics Simulations.

    PubMed

    Xu, Gui-Liang; Sheng, Tian; Chong, Lina; Ma, Tianyuan; Sun, Cheng-Jun; Zuo, Xiaobing; Liu, Di-Jia; Ren, Yang; Zhang, Xiaoyi; Liu, Yuzi; Heald, Steve M; Sun, Shi-Gang; Chen, Zonghai; Amine, Khalil

    2017-02-08

    Sodium-ion batteries (SIBs) have been considered as one of the promising power source candidates for the stationary storage industries owing to the much lower cost of sodium than lithium. It is well-known that the electrode materials largely determine the energy density of the battery systems. However, recent discoveries on the electrode materials showed that most of them present distinct lithium and sodium storage performance, which is not yet well understood. In this work, we performed a comparative understanding on the structural changes of porous cobalt oxide during its electrochemical lithiation and sodiation process by in operando synchrotron small angel X-ray scattering, X-ray diffraction, and X-ray absorption spectroscopy. It was found that compared to the lithiation process, the porous cobalt oxide undergoes less pore structure changes, oxidation state, and local structure changes as well as crystal structure evolution during its sodiation process, which is attributed to the intrinsic low sodiation activity of cobalt oxide as evidenced by ab initio molecular dynamics simulations. Moreover, it was indicated that the sodiation activity of metal sulfides is higher than that of metal oxides, indicating a better candidate for SIBs. Such understanding is crucial for future design and improvement of high-performance electrode materials for SIBs.

  19. Differential dopamine release dynamics in the nucleus accumbens core and shell track distinct aspects of goal-directed behavior for sucrose.

    PubMed

    Cacciapaglia, Fabio; Saddoris, Michael P; Wightman, R Mark; Carelli, Regina M

    2012-04-01

    Mesolimbic dopamine projections to the nucleus accumbens (NAc) have been implicated in goal-directed behaviors for natural rewards and in learning processes involving cue-reward associations. The NAc has been traditionally subdivided into two anatomically distinct sub-regions with different functional properties: the shell and the core. The aim of the present study was to characterize rapid dopamine transmission across the two NAc sub-regions during cue-signaled operant behavior for a natural (sucrose) reward in rats. Using fast-scan cyclic voltammetry (FSCV) we observed differences in the magnitude and dynamics of dopamine release events between the shell and core. Specifically, although cue-evoked dopamine release was observed in both sub-regions, it was larger and longer lasting in the shell compared with the core. Further, secondary dopamine release events were observed following the lever press response for sucrose in the NAc shell, but not the core. These findings demonstrate that the NAc displays regional specificity in dopamine transmission patterns during cued operant behavior for natural reward.

  20. Relationship of the Topological Distances and Activities between mPGES-1 and COX-2 versus COX-1: Implications of the Different Post-Translational Endoplasmic Reticulum Organizations of COX-1 and COX-2.

    PubMed

    Akasaka, Hironari; So, Shui-Ping; Ruan, Ke-He

    2015-06-16

    In vascular inflammation, prostaglandin E2 (PGE₂) is largely biosynthesized by microsomal PGE₂ synthase-1 (mPGES-1), competing with other downstream eicosanoid-synthesizing enzymes, such as PGIS, a synthase of a vascular protector prostacyclin (PGI₂), to isomerize the cyclooxygenase (COX)-2-derived prostaglandin H2 (PGH₂). In this study, we found that a majority of the product from the cells co-expressing human COX-2, mPGES-1, and PGIS was PGE₂. We hypothesize that the molecular and cellular mechanisms are related to the post-translational endoplasmic reticulum (ER) arrangement of those enzymes. A set of fusion enzymes, COX-2-linker [10 amino acids (aa)]-PGIS and COX-2-linker (22 amino acids)-PGIS, were created as "The Bioruler", in which the 10 and 22 amino acids are defined linkers with known helical structures and distances (14.4 and 30.8 Å, respectively). Our experiments have shown that the efficiency of PGI₂ biosynthesis was reduced when the separation distance increased from 10 to 22 amino acids. When COX-2-10aa-PGIS (with a 14.4 Å separation) was co-expressed with mPGES-1 on the ER membrane, a major product was PGE₂, but not PGI₂. However, expression of COX-2-10aa-PGIS and mPGES-1 on a separated ER with a distance of ≫30.8 Å reduced the level of PGE₂ production. These data indicated that the mPGES-1 is "complex-likely" colocalized with COX-2 within a distance of 14.4 Å. In addition, the cells co-expressing COX-1-10aa-PGIS and mPGES-1 produced PGI₂ mainly, but not PGE₂. This indicates that mPGES-1 is expressed much farther from COX-1. These findings have led to proposed models showing the different post-translational ER organization between COX-2 and COX-1 with respect to the topological arrangement of the mPGES-1 during vascular inflammation.

  1. Post-Translational Control of IL-1β via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

    PubMed Central

    Niebler, Martina; Qian, Xu; Höfler, Daniela; Kogosov, Vlada; Kaewprag, Jittranan; Kaufmann, Andreas M.; Ly, Regina; Böhmer, Gerd; Zawatzky, Rainer; Rösl, Frank; Rincon-Orozco, Bladimiro

    2013-01-01

    Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical

  2. Post-Translational Regulation of the Glucose-6-Phosphatase Complex by Cyclic Adenosine Monophosphate Is a Crucial Determinant of Endogenous Glucose Production and Is Controlled by the Glucose-6-Phosphate Transporter.

    PubMed

    Soty, Maud; Chilloux, Julien; Delalande, François; Zitoun, Carine; Bertile, Fabrice; Mithieux, Gilles; Gautier-Stein, Amandine

    2016-04-01

    The excessive endogenous glucose production (EGP) induced by glucagon participates in the development of type 2 diabetes. To further understand this hormonal control, we studied the short-term regulation by cyclic adenosine monophosphate (cAMP) of the glucose-6-phosphatase (G6Pase) enzyme, which catalyzes the last reaction of EGP. In gluconeogenic cell models, a 1-h treatment by the adenylate cyclase activator forskolin increased G6Pase activity and glucose production independently of any change in enzyme protein amount or G6P content. Using specific inhibitors or protein overexpression, we showed that the stimulation of G6Pase activity involved the protein kinase A (PKA). Results of site-directed mutagenesis, mass spectrometry analyses, and in vitro phosphorylation experiments suggested that the PKA stimulation of G6Pase activity did not depend on a direct phosphorylation of the enzyme. However, the temperature-dependent induction of both G6Pase activity and glucose release suggested a membrane-based mechanism. G6Pase is composed of a G6P transporter (G6PT) and a catalytic unit (G6PC). Surprisingly, we demonstrated that the increase in G6PT activity was required for the stimulation of G6Pase activity by forskolin. Our data demonstrate the existence of a post-translational mechanism that regulates G6Pase activity and reveal the key role of G6PT in the hormonal regulation of G6Pase activity and of EGP.

  3. Quantitative testing critical-taper wedge theory with distinct-element modeling and the role of dynamics in controlling wedge tapers

    NASA Astrophysics Data System (ADS)

    Strayer, Luther; Suppe, John

    2014-05-01

    Critical-taper wedge mechanics (e.g. Davis, et al. 1983, Dahlen 1990) provides fundamental relationships between the observed tapered geometries of fold-and-thrust belts and accretionary wedges and their detachment and wedge strengths. This theory has given diverse insight into kinematics, roles of erosion and sedimentation, and the morphology of compressive mountain belts, much of which has been aided by extensive analog and numerical modeling. The field has grown large, with several thousand papers addressing real-world, analog, and numerical wedges (cf. Buiter 2012). The majority of the insight has been qualitative, but nevertheless quite influential in our current understanding of mountain belts and submarine wedges. In contrast, quantitative applications of wedge theory, either to nature or models, has been rather limited because of the complexity of most wedge equations. It it is easy to become "lost in parameter space" with many strength parameters that are difficult to constrain or have ambiguous meaning, given real-world data and observations. Recently wedge theory has been recast into a very simple form (Suppe 2007; Yeh and Suppe 2014) that provides an unambiguous relationship between the observed covariation of surface slope α with detachment dip β and the wedge W and fault F strengths with few assumptions. In the real world we have limited knowledge of strengths, forces, fluid pressures and earthquake history, or the relationship between strength heterogeneity and structural style, or to what extent the strength of a wedge is an evolving macroscopic property (e.g. folding, imbrications and strain localization) or a material property. The well-defined relationship between wedge taper and global strength makes numerical wedges an ideal tool for the study of compressive mountain belts. In this work: [1] We successfully test this simpler quantitative wedge theory over a very wide range of wedge strengths and structural styles using distinct

  4. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  5. The core 2 beta-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins.

    PubMed

    Markine-Goriaynoff, Nicolas; Gillet, Laurent; Karlsen, Odd A; Haarr, Lars; Minner, Frédéric; Pastoret, Paul-Pierre; Fukuda, Minoru; Vanderplasschen, Alain

    2004-02-01

    The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only virus gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucine type (C2GnT-M). Recently, our phylogenetic study revealed that the Bo17 gene has been acquired from an ancestor of the African buffalo around 1.5 million years ago. Despite this recent origin, the Bo17 sequence has spread to fixation in the virus population possibly by natural selection. Supporting the latter hypothesis, it has been shown by our group for the V. test strain that Bo17 is expressed during BoHV-4 replication in vitro, and that Bo17 expression product (pBo17) has all three enzymic activities exhibited by cellular C2GnT-M, i.e. core 2, core 4 and I branching activities. In the present study, firstly it was investigated whether encoding a functional C2GnT-M is a general property of BoHV-4 strains. Analysis of nine representative strains of the BoHV-4 species revealed that all of them express the Bo17 gene and the associated core 2 branching activity during virus replication in vitro. Secondly, in order to investigate the roles of Bo17, its kinetic class of expression was analysed and a deleted recombinant strain was produced. These experiments revealed that Bo17 is expressed as an early gene which is not essential for virus replication in vitro. However, comparison of the structural proteins, produced by the wild-type, the revertant and the deleted viruses, by 2D gels demonstrated that pBo17 contributes to the post-translational modifications of structural proteins. Possible roles of Bo17 in vivo are discussed.

  6. Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications

    PubMed Central

    Solstad, Runar Gjerp; Li, Chun; Isaksson, Johan; Johansen, Jostein; Svenson, Johan; Stensvåg, Klara; Haug, Tor

    2016-01-01

    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. PMID:27007817

  7. Sub-proteome S-nitrosylation analysis in Brassica juncea hints at the regulation of Brassicaceae specific as well as other vital metabolic pathway(s) by nitric oxide and suggests post-translational modifications cross-talk.

    PubMed

    Sehrawat, Ankita; Deswal, Renu

    2014-12-01

    Abiotic stress affects the normal physiology of the plants and results in crop loss. Brassica juncea is an oil yielding crop affected by abiotic stress. In future, over 30% yield loss by abiotic stress is predicted in India. Understanding the mechanism of plant response to stress would help in developing stress tolerant crops. Nitric oxide (NO) is now viewed as a remarkably important signaling molecule, involved in regulating stress responses. S-Nitrosylation is a NO based post-translational modification (PTM), linked with the regulation of many physiologically relevant targets. In the last decade, over 700 functionally varied S-nitrosylated proteins were identified, which suggested broad-spectrum regulation. To understand the physiological significance of S-nitrosylation, it was analyzed in cold stress. Functional categorization and validation of some of the B. juncea S-nitrosylated targets, suggested that NO produced during stress regulates cellular detoxification by modulating enzymes of ascorbate glutathione cycle, superoxide dismutase, glutathione S-transferase and glyoxalase I by S-nitrosylation in crude, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) depleted and apoplastic fractions. Interestingly, S-nitrosylation of enzymes associated with glucosinolate hydrolysis pathway, suggests a novel regulation of this Brassicaceae specific pathway by NO. Moreover, identification of enzymes of Glycolysis and Calvin cycle in crude and RuBisCO depleted fractions showed the regulation of metabolic as well as photosynthetic pathways by S-nitrosylation. S-Nitrosylation of cell wall modifying and proteolytic enzymes in the apoplast suggested differential and spatial regulation by S-nitrosylation. To have an overview of physiological role(s) of NO, collective information on NO based signaling (mainly by S-nitrosylation) is presented in this review.

  8. Novel Antimicrobial Peptides EeCentrocins 1, 2 and EeStrongylocin 2 from the Edible Sea Urchin Echinus esculentus Have 6-Br-Trp Post-Translational Modifications.

    PubMed

    Solstad, Runar Gjerp; Li, Chun; Isaksson, Johan; Johansen, Jostein; Svenson, Johan; Stensvåg, Klara; Haug, Tor

    2016-01-01

    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.

  9. The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters*

    PubMed Central

    Aleidi, Shereen M.; Howe, Vicky; Sharpe, Laura J.; Yang, Alryel; Rao, Geetha; Brown, Andrew J.; Gelissen, Ingrid C.

    2015-01-01

    The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. PMID:26296893

  10. Microtubule proteins and their post-translational forms in the cerebrospinal fluid of patients with paraparesis associated with HTLV-I infection and in SH-SY5Y cells: an in vitro model of HTLV-I-induced disease.

    PubMed

    Maldonado, Horacio; Ortiz-Riaño, Emilio; Krause, Bernardo; Barriga, Andrés; Medina, Fernando; Pando, M Elsa; Alberti, Carolina; Kettlun, Ana M; Collados, Lucía; García, Lorena; Cartier, Luis; Valenzuela, M Antonieta

    2008-01-01

    HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by axonal degeneration of the corticospinal tracts. The specific requirements for transport of proteins and organelles to the distal part of the long axon are crucial in the corticospinal tracts. Microtubule dysfunction could be involved in this disease, configuring an axonal transport disease. We measured tubulin and its post-translational modified forms (acetylated and tyrosinated) in CSF of patients and controls, as well as tau and its phosphorylated forms. There were no significant differences in the contents of tubulin and acetyl-tubulin between patients and controls; tyrosyl-tubulin was not detected. In HAM/TSP, tau levels were significantly reduced, while the ratio of pT181/total tau was higher in patients than in controls, this being completely different from what is reported in other neurodegenerative diseases. Phosphorylation at T181 was also confirmed by Mass Spectrometry analysis. Western Blotting with monospecific polyclonal antibodies against pS199, pT205, pT231, pS262, pS356, pS396, pS404 and pS422 did not show differences in phosphorylation in these residues between patients and controls. Treating human SH-SY5Y neuroblastoma cells, a well-known in vitro neurite retraction model, with culture supernatant of MT-2 cells (HTLV-I infected cell line that secretes the viral Tax protein) we observed neurite retraction and an increase in tau phosphorylation at T181. A disruption of normal phosphorylation of tau protein in T181 could result in its dysfunction, contributing to axonal damage.

  11. Hydrogen/Deuterium Exchange Reveals Distinct Agonist/Partial Agonist Receptor Dynamics within the intact Vitamin D Receptor/Retinoid X Receptor Heterodimer

    PubMed Central

    Zhang, Jun; Chalmers, Michael J.; Stayrook, Keith R.; Burris, Lorri L.; Garcia-Ordonez, Ruben D.; Pascal, Bruce D.; Burris, Thomas P.; Dodge, Jeffery A.; Griffin, Patrick R.

    2010-01-01

    Summary Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as providing insight into the role of AF-2 dynamics in the action of VDR partial agonists. PMID:20947021

  12. The leader peptide of mutacin 1140 has distinct structural components compared to related class I lantibiotics.

    PubMed

    Escano, Jerome; Stauffer, Byron; Brennan, Jacob; Bullock, Monica; Smith, Leif

    2014-12-01

    Lantibiotics are ribosomally synthesized peptide antibiotics composed of an N-terminal leader peptide that promotes the core peptide's interaction with the post translational modification (PTM) enzymes. Following PTMs, mutacin 1140 is transported out of the cell and the leader peptide is cleaved to yield the antibacterial peptide. Mutacin 1140 leader peptide is structurally unique compared to other class I lantibiotic leader peptides. Herein, we further our understanding of the structural differences of mutacin 1140 leader peptide with regard to other class I leader peptides. We have determined that the length of the leader peptide is important for the biosynthesis of mutacin 1140. We have also determined that mutacin 1140 leader peptide contains a novel four amino acid motif compared to related lantibiotics. PTM enzyme recognition of the leader peptide appears to be evolutionarily distinct from related class I lantibiotics. Our study on mutacin 1140 leader peptide provides a basis for future studies aimed at understanding its interaction with the PTM enzymes.

  13. Phosphorylation induces distinct alpha-synuclein strain formation

    PubMed Central

    Ma, Meng-Rong; Hu, Zhi-Wen; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2016-01-01

    Synucleinopathies are a group of neurodegenerative diseases associated with alpha-synuclein (α-Syn) aggregation. Recently, increasing evidence has demonstrated the existence of different structural characteristics or ‘strains’ of α-Syn, supporting the concept that synucleinopathies share several common features with prion diseases and possibly explaining how a single protein results in different clinical phenotypes within synucleinopathies. In earlier studies, the different strains were generated through the regulation of solution conditions, temperature, or repetitive seeded fibrillization in vitro. Here, we synthesize homogeneous α-Syn phosphorylated at serine 129 (pS129 α-Syn), which is highly associated with the pathological changes, and demonstrate that phosphorylation at Ser129 induces α-Syn to form a distinct strain with different structures, propagation properties, and higher cytotoxicity compared with the wild-type α-Syn. The results are the first demonstration that post-translational modification of α-Syn can induce different strain formation, offering a new mechanism for strain formation. PMID:27853185

  14. Gait dynamics in Parkinson’s disease: Common and distinct behavior among stride length, gait variability, and fractal-like scaling

    PubMed Central

    Hausdorff, Jeffrey M.

    2009-01-01

    Parkinson’s disease (PD) is a common, debilitating neurodegenerative disease. Gait disturbances are a frequent cause of disability and impairment for patients with PD. This article provides a brief introduction to PD and describes the gait changes typically seen in patients with this disease. A major focus of this report is an update on the study of the fractal properties of gait in PD, the relationship between this feature of gait and stride length and gait variability, and the effects of different experimental conditions on these three gait properties. Implications of these findings are also briefly described. This update highlights the idea that while stride length, gait variability, and fractal scaling of gait are all impaired in PD, distinct mechanisms likely contribute to and are responsible for the regulation of these disparate gait properties. PMID:19566273

  15. The lateral membrane organization and dynamics of myelin proteins PLP and MBP are dictated by distinct galactolipids and the extracellular matrix.

    PubMed

    Ozgen, Hande; Schrimpf, Waldemar; Hendrix, Jelle; de Jonge, Jenny C; Lamb, Don C; Hoekstra, Dick; Kahya, Nicoletta; Baron, Wia

    2014-01-01

    In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP's diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.

  16. Self-Catalyzed Assembly of Peptide Scaffolded Nanozyme as a Dynamic Biosensing System.

    PubMed

    Li, Hao; Huang, Yue; Yu, Yue; Li, Genxi; Karamanos, Yannis

    2016-02-03

    In this work, a new strategy of biosensor design is developed based on the assembly of amyloid beta and its multiple interactions with other bioactive species. These interactions can enable amyloid beta peptide as a multifunctional sensing element, so the immobilization of sensing probe and the step-by-step modification of the sensing interface have all been dispensed with. Instead, the kinetics of the assembly of a peptide-based catalytic network serves to convert the quantity of analyte into amplified signal readout. The designed dynamic assembling and biosensing system has also been successfully applied in detecting the activity of polyglutamylation, an essential post translation modification controlling cell skeleton and cell cycle, in biological complex samples. Further studies reveal that the serum abundance of a polyglutamylase, tubulin tyrosine ligase-like protein 12, may show parallel with the degree of development of prostate cancer and the discrimination between early cancerous development and benign conditions. And the obtained result is more distinct than that based on PSA detection, the current gold standard. This study may also point to the prospective of extending this design strategy to broader range of biosensing applications in the future.

  17. Fkh1 and Fkh2 Bind Multiple Chromosomal Elements in the S. cerevisiae Genome with Distinct Specificities and Cell Cycle Dynamics

    PubMed Central

    Knott, Simon R. V.; Fox, Catherine A.; Tavaré, Simon; Aparicio, Oscar M.

    2014-01-01

    Forkhead box (FOX) transcription factors regulate a wide variety of cellular functions in higher eukaryotes, including cell cycle control and developmental regulation. In Saccharomyces cerevisiae, Forkhead proteins Fkh1 and Fkh2 perform analogous functions, regulating genes involved in cell cycle control, while also regulating mating-type silencing and switching involved in gamete development. Recently, we revealed a novel role for Fkh1 and Fkh2 in the regulation of replication origin initiation timing, which, like donor preference in mating-type switching, appears to involve long-range chromosomal interactions, suggesting roles for Fkh1 and Fkh2 in chromatin architecture and organization. To elucidate how Fkh1 and Fkh2 regulate their target DNA elements and potentially regulate the spatial organization of the genome, we undertook a genome-wide analysis of Fkh1 and Fkh2 chromatin binding by ChIP-chip using tiling DNA microarrays. Our results confirm and extend previous findings showing that Fkh1 and Fkh2 control the expression of cell cycle-regulated genes. In addition, the data reveal hundreds of novel loci that bind Fkh1 only and exhibit a distinct chromatin structure from loci that bind both Fkh1 and Fkh2. The findings also show that Fkh1 plays the predominant role in the regulation of a subset of replication origins that initiate replication early, and that Fkh1/2 binding to these loci is cell cycle-regulated. Finally, we demonstrate that Fkh1 and Fkh2 bind proximally to a variety of genetic elements, including centromeres and Pol III-transcribed snoRNAs and tRNAs, greatly expanding their potential repertoire of functional targets, consistent with their recently suggested role in mediating the spatial organization of the genome. PMID:24504085

  18. Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties

    NASA Astrophysics Data System (ADS)

    Agarwal, Shubhangi; Dey, Sanjay; Ghosh, Biplab; Biswas, Maitree; Dasgupta, Jhimli

    2017-02-01

    Molecular mechanisms of xenosiderophore and heme acquisitions using periplasmic binding protein (PBP) dependent ATP-binding cassette transporters to scavenge the essential nutrient iron are elusive yet in Vibrio cholerae. Our current study delineates the structures, dynamics and ligand binding properties of two Type III PBPs of V. cholerae, VcFhuD and VcHutB. Through crystal structures and fluorescence quenching studies we demonstrate unique features of VcFhuD to bind both hydroxamate and catecholate type xenosiderophores. Like E. coli FhuD, VcFhuD binds ferrichrome and ferri-desferal using conserved Tryptophans and R102. However, unlike EcFhuD, slightly basic ligand binding pocket of VcFhuD could favour ferri-enterobactin binding with plausible participation of R203, along with R102, like it happens in catecholate binding PBPs. Structural studies coupled with spectrophotometric and native PAGE analysis indicated parallel binding of two heme molecules to VcHutB in a pH dependent manner, while mutational analysis established the relative importance of Y65 and H164 in heme binding. MD simulation studies exhibited an unforeseen inter-lobe swinging motion in Type III PBPs, magnitude of which is inversely related to the packing of the linker helix with its neighboring helices. Small inter-lobe movement in VcFhuD or dramatic twisting in VcHutB is found to influence ligand binding.

  19. Effect of three distinct recombinant cell-binding domains of FN (rCDFN) and substrate crosslinking density on the morphology and dynamics of Human Dermal Fibroblast cells

    NASA Astrophysics Data System (ADS)

    Ghosh, K.; Shu, Xz; Ge, S.; Fang, X.; Rafailovich, M.; Prestwich, Gd; Clark, Raf

    2004-03-01

    Tissue development depends largely on how cells sense and respond to extracellular matrix (ECM). Coupling of cell integrins with ECM ligands generates both, a ligation-induced signaling and a state of isometric tension, within the cell cytoskeleton (CSK). The extent to which ECM supports this prestress governs cell shape and function. In this study, we formulated hydrogels by coupling the three rCDFN to cross-linked thiolated HA backbone using a PEGDA crosslinker. We investigated cell stiffness and the corresponding actin CSK arrangement as a function of hydrogel stiffness and adhesiveness. Hydrogel stiffness was controlled by the amount of crosslinker added, while the adhesiveness was controlled by the nature and amount of the rFNfs coupled to the hydrogel backbone. AFM was used to determine the surface modulus of live cells. Confocal microscope was used to obtain images of the actin CSK of fixed and stained cells. It was found that cells seeded on either stiffer or more adhesive surfaces had larger surface moduli and more stretched actin CSK than those seeded on softer or less adhesive substrates. This indicated that the cortical tension was able to sense both, the underlying substrate stiffness and adhesiveness. Diffusion dynamics of cells seeded on these substrates was also monitored and correlated with cell morphology, substrate stiffness and nature of rCDFN.

  20. Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties

    PubMed Central

    Agarwal, Shubhangi; Dey, Sanjay; Ghosh, Biplab; Biswas, Maitree; Dasgupta, Jhimli

    2017-01-01

    Molecular mechanisms of xenosiderophore and heme acquisitions using periplasmic binding protein (PBP) dependent ATP-binding cassette transporters to scavenge the essential nutrient iron are elusive yet in Vibrio cholerae. Our current study delineates the structures, dynamics and ligand binding properties of two Type III PBPs of V. cholerae, VcFhuD and VcHutB. Through crystal structures and fluorescence quenching studies we demonstrate unique features of VcFhuD to bind both hydroxamate and catecholate type xenosiderophores. Like E. coli FhuD, VcFhuD binds ferrichrome and ferri-desferal using conserved Tryptophans and R102. However, unlike EcFhuD, slightly basic ligand binding pocket of VcFhuD could favour ferri-enterobactin binding with plausible participation of R203, along with R102, like it happens in catecholate binding PBPs. Structural studies coupled with spectrophotometric and native PAGE analysis indicated parallel binding of two heme molecules to VcHutB in a pH dependent manner, while mutational analysis established the relative importance of Y65 and H164 in heme binding. MD simulation studies exhibited an unforeseen inter-lobe swinging motion in Type III PBPs, magnitude of which is inversely related to the packing of the linker helix with its neighboring helices. Small inter-lobe movement in VcFhuD or dramatic twisting in VcHutB is found to influence ligand binding. PMID:28216648

  1. Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations*

    PubMed Central

    Cabana, Jérôme; Holleran, Brian; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan; Lavigne, Pierre

    2015-01-01

    Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT1) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the Gq/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway), and the D74N-AT1 receptor (biased for the β-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT1, N111G-AT1, and AngII-N111G-AT1 receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT1 receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT1 receptor. The biased agonist [Sar1,Ile8]AngII also showed a preference for the ground state of the WT-AT1 receptor compared with AngII. These results suggest that activation of the Gq/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor. PMID:25934394

  2. Social conformity despite individual preferences for distinctiveness.

    PubMed

    Smaldino, Paul E; Epstein, Joshua M

    2015-03-01

    We demonstrate that individual behaviours directed at the attainment of distinctiveness can in fact produce complete social conformity. We thus offer an unexpected generative mechanism for this central social phenomenon. Specifically, we establish that agents who have fixed needs to be distinct and adapt their positions to achieve distinctiveness goals, can nevertheless self-organize to a limiting state of absolute conformity. This seemingly paradoxical result is deduced formally from a small number of natural assumptions and is then explored at length computationally. Interesting departures from this conformity equilibrium are also possible, including divergence in positions. The effect of extremist minorities on these dynamics is discussed. A simple extension is then introduced, which allows the model to generate and maintain social diversity, including multimodal distinctiveness distributions. The paper contributes formal definitions, analytical deductions and counterintuitive findings to the literature on individual distinctiveness and social conformity.

  3. Human germline and pan-cancer variomes and their distinct functional profiles.

    PubMed

    Pan, Yang; Karagiannis, Konstantinos; Zhang, Haichen; Dingerdissen, Hayley; Shamsaddini, Amirhossein; Wan, Quan; Simonyan, Vahan; Mazumder, Raja

    2014-10-01

    Identification of non-synonymous single nucleotide variations (nsSNVs) has exponentially increased due to advances in Next-Generation Sequencing technologies. The functional impacts of these variations have been difficult to ascertain because the corresponding knowledge about sequence functional sites is quite fragmented. It is clear that mapping of variations to sequence functional features can help us better understand the pathophysiological role of variations. In this study, we investigated the effect of nsSNVs on more than 17 common types of post-translational modification (PTM) sites, active sites and binding sites. Out of 1 705 285 distinct nsSNVs on 259 216 functional sites we identified 38 549 variations that significantly affect 10 major functional sites. Furthermore, we found distinct patterns of site disruptions due to germline and somatic nsSNVs. Pan-cancer analysis across 12 different cancer types led to the identification of 51 genes with 106 nsSNV affected functional sites found in 3 or more cancer types. 13 of the 51 genes overlap with previously identified Significantly Mutated Genes (Nature. 2013 Oct 17;502(7471)). 62 mutations in these 13 genes affecting functional sites such as DNA, ATP binding and various PTM sites occur across several cancers and can be prioritized for additional validation and investigations.

  4. Carnosic acid sensitized TRAIL-mediated apoptosis through down-regulation of c-FLIP and Bcl-2 expression at the post translational levels and CHOP-dependent up-regulation of DR5, Bim, and PUMA expression in human carcinoma caki cells

    PubMed Central

    Bae, Jae Hoon; Kwon, Taeg Kyu

    2015-01-01

    Carnosic acid is a phenolic diterpene from rosmarinus officinalis, and has multiple functions, such as anti-inflammatory, anti-viral, and anti-tumor activity. In this study, we examined whether carnosic acid could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that carnosic acid markedly induced TRAIL-mediated apoptosis in human renal carcinoma (Caki, ACHN, and A498), and human hepatocellular carcinoma (SK-HEP-1), and human breast carcinoma (MDA-MB-231) cells, but not normal cells (TMCK-1 and HSF). Carnosic acid induced down-regulation of c-FLIP and Bcl-2 expression at the post-translational levels, and the over-expression of c-FLIP and Bcl-2 markedly blocked carnosic acid-induced TRAIL sensitization. Furthermore, carnosic acid induced death receptor (DR)5, Bcl-2 interacting mediator of cell death (Bim), and p53 up-regulated modulator of apoptosis (PUMA) expression at the transcriptional levels via CCAAT/enhancer-binding protein-homologous protein (CHOP). Down-regulation of CHOP expression by siRNA inhibited DR5, Bim, and PUMA expression, and attenuated carnosic acid plus TRAIL-induced apoptosis. Taken together, our study demonstrates that carnosic acid enhances sensitization against TRAIL-mediated apoptosis through the down-regulation of c-FLIP and Bcl-2 expression, and up-regulation of ER stress-mediated DR5, Bim, and PUMA expression at the transcriptional levels. PMID:25596735

  5. Neurobeachin Regulates Glutamate- and GABA-Receptor Targeting to Synapses via Distinct Pathways.

    PubMed

    Farzana, F; Zalm, R; Chen, N; Li, K W; Grant, Seth G N; Smit, A B; Toonen, R F; Verhage, M

    2016-05-01

    Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.

  6. Foundations of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Baltaxe, Christiane A. M.

    This treatise on the theoretical and historical foundations of distinctive feature theory traces the evolution of the distinctive features concept in the context of related notions current in linguistic theory, discusses the evolution of individual distinctive features, and criticizes certain acoustic and perceptual correlates attributed to these…

  7. EMdeCODE: a novel algorithm capable of reading words of epigenetic code to predict enhancers and retroviral integration sites and to identify H3R2me1 as a distinctive mark of coding versus non-coding genes

    PubMed Central

    Santoni, Federico Andrea

    2013-01-01

    Existence of some extra-genetic (epigenetic) codes has been postulated since the discovery of the primary genetic code. Evident effects of histone post-translational modifications or DNA methylation over the efficiency and the regulation of DNA processes are supporting this postulation. EMdeCODE is an original algorithm that approximate the genomic distribution of given DNA features (e.g. promoter, enhancer, viral integration) by identifying relevant ChIPSeq profiles of post-translational histone marks or DNA binding proteins and combining them in a supermark. EMdeCODE kernel is essentially a two-step procedure: (i) an expectation-maximization process calculates the mixture of epigenetic factors that maximize the Sensitivity (recall) of the association with the feature under study; (ii) the approximated density is then recursively trimmed with respect to a control dataset to increase the precision by reducing the number of false positives. EMdeCODE densities improve significantly the prediction of enhancer loci and retroviral integration sites with respect to previous methods. Importantly, it can also be used to extract distinctive factors between two arbitrary conditions. Indeed EMdeCODE identifies unexpected epigenetic profiles specific for coding versus non-coding RNA, pointing towards a new role for H3R2me1 in coding regions. PMID:23234700

  8. SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

    SciTech Connect

    Steven C. Huber

    2009-05-12

    Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.

  9. Combined electron transfer dissociation-collision-induced dissociation fragmentation in the mass spectrometric distinction of leucine, isoleucine, and hydroxyproline residues in Peptide natural products.

    PubMed

    Gupta, Kallol; Kumar, Mukesh; Chandrashekara, Krishnappa; Krishnan, Kozhalmannom S; Balaram, Padmanabhan

    2012-02-03

    Distinctions between isobaric residues have been a major challenge in mass spectrometric peptide sequencing. Here, we propose a methodology for distinction among isobaric leucine, isoleucine, and hydroxyproline, a commonly found post-translationally modified amino acid with a nominal mass of 113 Da, through a combined electron transfer dissociation-collision-induced dissociation approach. While the absence of c and z(•) ions, corresponding to the Yyy-Xxx (Xxx = Leu, Ile, or Hyp) segment, is indicative of the presence of hydroxyproline, loss of isopropyl (Δm = 43 Da) or ethyl radicals (Δm = 29 Da), through collisional activation of z radical ions, are characteristic of leucine or isoleucine, respectively. Radical migration processes permit distinctions even in cases where the specific z(•) ions, corresponding to the Yyy-Leu or -Ile segments, are absent or of low intensity. This tandem mass spectrometric (MS(n)) method has been successfully implemented in a liquid chromatography-MS(n) platform to determine the identity of 23 different isobaric residues from a mixture of five different peptides. The approach is convenient for distinction of isobaric residues from any crude peptide mixture, typically encountered in natural peptide libraries or proteomic analysis.

  10. An Asymmetrically Balanced Organization of Kinases versus Phosphatases across Eukaryotes Determines Their Distinct Impacts

    PubMed Central

    Shemesh, Netta; Ziv-Ukelson, Michal; Ben-Zvi, Anat

    2017-01-01

    Protein phosphorylation underlies cellular response pathways across eukaryotes and is governed by the opposing actions of phosphorylating kinases and de-phosphorylating phosphatases. While kinases and phosphatases have been extensively studied, their organization and the mechanisms by which they balance each other are not well understood. To address these questions we performed quantitative analyses of large-scale 'omics' datasets from yeast, fly, plant, mouse and human. We uncovered an asymmetric balance of a previously-hidden scale: Each organism contained many different kinase genes, and these were balanced by a small set of highly abundant phosphatase proteins. Kinases were much more responsive to perturbations at the gene and protein levels. In addition, kinases had diverse scales of phenotypic impact when manipulated. Phosphatases, in contrast, were stable, highly robust and flatly organized, with rather uniform impact downstream. We validated aspects of this organization experimentally in nematode, and supported additional aspects by theoretic analysis of the dynamics of protein phosphorylation. Our analyses explain the empirical bias in the protein phosphorylation field toward characterization and therapeutic targeting of kinases at the expense of phosphatases. We show quantitatively and broadly that this is not only a historical bias, but stems from wide-ranging differences in their organization and impact. The asymmetric balance between these opposing regulators of protein phosphorylation is also common to opposing regulators of two other post-translational modification systems, suggesting its fundamental value. PMID:28135269

  11. Is Face Distinctiveness Gender Based?

    ERIC Educational Resources Information Center

    Baudouin, Jean-Yves; Gallay, Mathieu

    2006-01-01

    Two experiments were carried out to study the role of gender category in evaluations of face distinctiveness. In Experiment 1, participants had to evaluate the distinctiveness and the femininity-masculinity of real or artificial composite faces. The composite faces were created by blending either faces of the same gender (sexed composite faces,…

  12. Counselor Identity: Conformity or Distinction?

    ERIC Educational Resources Information Center

    McLaughlin, Jerry E.; Boettcher, Kathryn

    2009-01-01

    The authors explore 3 debates in other disciplines similar to counseling's identity debate in order to learn about common themes and outcomes. Conformity, distinction, and cohesion emerged as common themes. They conclude that counselors should retain their distinctive, humanistic approach rather than conforming to the dominant, medical approach.

  13. Development of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Meyer, Peggy L.

    Since the beginning of man's awareness of his language capabilities and language structure, he has assumed that speech is composed of discrete entities. The linguist attempts to establish a model of the workings of these distinctive sounds in a language. Utilizing an historical basis for discussion, this general survey of the distinctive feature…

  14. Optimal Distinctiveness Signals Membership Trust.

    PubMed

    Leonardelli, Geoffrey J; Loyd, Denise Lewin

    2016-07-01

    According to optimal distinctiveness theory, sufficiently small minority groups are associated with greater membership trust, even among members otherwise unknown, because the groups are seen as optimally distinctive. This article elaborates on the prediction's motivational and cognitive processes and tests whether sufficiently small minorities (defined by relative size; for example, 20%) are associated with greater membership trust relative to mere minorities (45%), and whether such trust is a function of optimal distinctiveness. Two experiments, examining observers' perceptions of minority and majority groups and using minimal groups and (in Experiment 2) a trust game, revealed greater membership trust in minorities than majorities. In Experiment 2, participants also preferred joining minorities over more powerful majorities. Both effects occurred only when minorities were 20% rather than 45%. In both studies, perceptions of optimal distinctiveness mediated effects. Discussion focuses on the value of relative size and optimal distinctiveness, and when membership trust manifests.

  15. Diverse lamin-dependent mechanisms interact to control chromatin dynamics

    PubMed Central

    Camozzi, Daria; Capanni, Cristina; Cenni, Vittoria; Mattioli, Elisabetta; Columbaro, Marta; Squarzoni, Stefano; Lattanzi, Giovanna

    2014-01-01

    Interconnected functional strategies govern chromatin dynamics in eukaryotic cells. In this context, A and B type lamins, the nuclear intermediate filaments, act on diverse platforms involved in tissue homeostasis. On the nuclear side, lamins elicit large scale or fine chromatin conformational changes, affect DNA damage response factors and transcription factor shuttling. On the cytoplasmic side, bridging-molecules, the LINC complex, associate with lamins to coordinate chromatin dynamics with cytoskeleton and extra-cellular signals.   Consistent with such a fine tuning, lamin mutations and/or defects in their expression or post-translational processing, as well as mutations in lamin partner genes, cause a heterogeneous group of diseases known as laminopathies. They include muscular dystrophies, cardiomyopathy, lipodystrophies, neuropathies, and progeroid syndromes. The study of chromatin dynamics under pathological conditions, which is summarized in this review, is shedding light on the complex and fascinating role of the nuclear lamina in chromatin regulation. PMID:25482195

  16. Distinct patterns of seasonal Greenland glacier velocity.

    PubMed

    Moon, Twila; Joughin, Ian; Smith, Ben; van den Broeke, Michiel R; van de Berg, Willem Jan; Noël, Brice; Usher, Mika

    2014-10-28

    Predicting Greenland Ice Sheet mass loss due to ice dynamics requires a complete understanding of spatiotemporal velocity fluctuations and related control mechanisms. We present a 5 year record of seasonal velocity measurements for 55 marine-terminating glaciers distributed around the ice sheet margin, along with ice-front position and runoff data sets for each glacier. Among glaciers with substantial speed variations, we find three distinct seasonal velocity patterns. One pattern indicates relatively high glacier sensitivity to ice-front position. The other two patterns are more prevalent and appear to be meltwater controlled. These patterns reveal differences in which some subglacial systems likely transition seasonally from inefficient, distributed hydrologic networks to efficient, channelized drainage, while others do not. The difference may be determined by meltwater availability, which in some regions may be influenced by perennial firn aquifers. Our results highlight the need to understand subglacial meltwater availability on an ice sheet-wide scale to predict future dynamic changes.

  17. Distinct types of eigenvector localization in networks

    PubMed Central

    Pastor-Satorras, Romualdo; Castellano, Claudio

    2016-01-01

    The spectral properties of the adjacency matrix provide a trove of information about the structure and function of complex networks. In particular, the largest eigenvalue and its associated principal eigenvector are crucial in the understanding of nodes’ centrality and the unfolding of dynamical processes. Here we show that two distinct types of localization of the principal eigenvector may occur in heterogeneous networks. For synthetic networks with degree distribution P(q) ~ q−γ, localization occurs on the largest hub if γ > 5/2; for γ < 5/2 a new type of localization arises on a mesoscopic subgraph associated with the shell with the largest index in the K-core decomposition. Similar evidence for the existence of distinct localization modes is found in the analysis of real-world networks. Our results open a new perspective on dynamical processes on networks and on a recently proposed alternative measure of node centrality based on the non-backtracking matrix. PMID:26754565

  18. Modeling the dynamics of the nucleosome at various levels.

    NASA Astrophysics Data System (ADS)

    Onufriev, Alexey; Fenley, Andrew; Zmuda-Ruscio, Jory; Adams, David

    2007-03-01

    The primary level of DNA compaction in eukaryotic organisms is the nucleosome, yet details of its dynamics are not fully understood. While the whole nucleosome must be highly stable, protective of its genetic material, at the same time its tightly wrapped DNA should be highly accessible, easily revealing its information content. A combination of atom-level classical molecular dynamics and a course-grained continuum description provide insights into the functioning of the system. In particular, the nucleosomal DNA appears to be considerably more flexible than what can be expected based on its canonical persistence length. A coarse-grained electrostatic model of the nucleosome explains how its stability can be modulated with small environmental changes as well as post-translational modifications. Implications for the nucleosome assembly process in vivo are discussed.

  19. Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis.

    PubMed

    Sun, Rongfang; Qi, Huayu

    2014-01-01

    Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here

  20. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems

    NASA Astrophysics Data System (ADS)

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  1. AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.

    PubMed

    Pressler, Katharina; Vorkapic, Dina; Lichtenegger, Sabine; Malli, Gerald; Barilich, Benjamin P; Cakar, Fatih; Zingl, Franz G; Reidl, Joachim; Schild, Stefan

    2016-09-01

    The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM.

  2. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems.

    PubMed

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  3. Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans.

    PubMed

    Cadiñanos, Juan; Schmidt, Walter K; Fueyo, Antonio; Varela, Ignacio; López-Otín, Carlos; Freije, José M P

    2003-03-15

    Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.

  4. Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans.

    PubMed Central

    Cadiñanos, Juan; Schmidt, Walter K; Fueyo, Antonio; Varela, Ignacio; López-Otín, Carlos; Freije, José M P

    2003-01-01

    Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes. PMID:12487630

  5. China English: Its Distinctive Features

    ERIC Educational Resources Information Center

    Yang, Wei-dong; Dai, Wei-ping

    2011-01-01

    This paper attempts to expound that China English boasting its own distinctive features on the levels of phonology, words, sentences and discourse has been playing an irreplaceable role in intercultural activities, though still in its infancy and in the process of developing and perfecting itself, and it now makes every effort to move towards…

  6. Educational Psychology: The Distinctive Contribution

    ERIC Educational Resources Information Center

    Cameron, R. J.

    2006-01-01

    This paper, written in the twenty-first anniversary year of the journal "Educational Psychology in Practice", attempts to uncover those distinctive aspects of the discipline and the practice of applied psychology in general and educational psychology in particular. After considering some of the reasons for attempting this task at this point in…

  7. Distinctiveness Maps for Image Matching

    NASA Technical Reports Server (NTRS)

    Manduchi, Roberto; Tomasi, Carlo

    2000-01-01

    Stereo correspondence is hard because different image features can look alike. We propose a measure for the ambiguity of image points that allows matching distinctive points first and breaks down the matching task into smaller and separate subproblems. Experiments with an algorithm based on this measure demonstrate the ensuing efficiency and low likelihood of incorrect matches.

  8. Accumulation of distinct prelamin A variants in human diploid fibroblasts differentially affects cell homeostasis

    SciTech Connect

    Candelario, Jose; Borrego, Stacey; Reddy, Sita; Comai, Lucio

    2011-02-01

    Lamin A is a component of the nuclear lamina that plays a major role in the structural organization and function of the nucleus. Lamin A is synthesized as a prelamin A precursor which undergoes four sequential post-translational modifications to generate mature lamin A. Significantly, a large number of point mutations in the LMNA gene cause a range of distinct human disorders collectively known as laminopathies. The mechanisms by which mutations in lamin A affect cell function and cause disease are unclear. Interestingly, recent studies have suggested that alterations in the normal lamin A pathway can contribute to cellular dysfunction. Specifically, we and others have shown, at the cellular level, that in the absence of mutations or altered splicing events, increased expression of wild-type prelamin A results in a growth defective phenotype that resembles that of cells expressing the mutant form of lamin A, termed progerin, associated with Hutchinson-Gilford Progeria syndrome (HGPS). Remarkably, the phenotypes of cells expressing elevated levels of wild-type prelamin A can be reversed by either treatment with farnesyltransferase inhibitors or overexpression of ZMPSTE24, a critical prelamin A processing enzyme, suggesting that minor increases in the steady-state levels of one or more prelamin A intermediates is sufficient to induce cellular toxicity. Here, to investigate the molecular basis of the lamin A pathway toxicity, we characterized the phenotypic changes occurring in cells expressing distinct prelamin A variants mimicking specific prelamin A processing intermediates. This analysis demonstrates that distinct prelamin A variants differentially affect cell growth, nuclear membrane morphology, nuclear distribution of lamin A and the fundamental process of transcription. Expression of prelamin A variants that are constitutively farnesylated induced the formation of lamin A aggregates and dramatic changes in nuclear membrane morphology, which led to reduced

  9. Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

    PubMed Central

    Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

    2014-01-01

    Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660

  10. Non-monotonic Response to Monotonic Stimulus: Regulation of Glyoxylate Shunt Gene-Expression Dynamics in Mycobacterium tuberculosis

    PubMed Central

    Gennaro, Maria L.; Igoshin, Oleg A.

    2016-01-01

    Understanding how dynamical responses of biological networks are constrained by underlying network topology is one of the fundamental goals of systems biology. Here we employ monotone systems theory to formu