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Sample records for distinct dynamic post-translational

  1. Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes

    PubMed Central

    Olausson, Bjoern E.S.; Grossfield, Alan; Pitman, Michael C.; Brown, Michael F.; Feller, Scott E.; Vogel, Alexander

    2012-01-01

    We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations—as well as their extensive fluctuations—suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin. PMID:22280374

  2. A Systematic Framework for Molecular Dynamics Simulations of Protein Post-Translational Modifications

    PubMed Central

    Grandits, Melanie; Oostenbrink, Chris; Zagrovic, Bojan

    2013-01-01

    By directly affecting structure, dynamics and interaction networks of their targets, post-translational modifications (PTMs) of proteins play a key role in different cellular processes ranging from enzymatic activation to regulation of signal transduction to cell-cycle control. Despite the great importance of understanding how PTMs affect proteins at the atomistic level, a systematic framework for treating post-translationally modified amino acids by molecular dynamics (MD) simulations, a premier high-resolution computational biology tool, has never been developed. Here, we report and validate force field parameters (GROMOS 45a3 and 54a7) required to run and analyze MD simulations of more than 250 different types of enzymatic and non-enzymatic PTMs. The newly developed GROMOS 54a7 parameters in particular exhibit near chemical accuracy in matching experimentally measured hydration free energies (RMSE = 4.2 kJ/mol over the validation set). Using this tool, we quantitatively show that the majority of PTMs greatly alter the hydrophobicity and other physico-chemical properties of target amino acids, with the extent of change in many cases being comparable to the complete range spanned by native amino acids. PMID:23874192

  3. A systematic framework for molecular dynamics simulations of protein post-translational modifications.

    PubMed

    Petrov, Drazen; Margreitter, Christian; Grandits, Melanie; Oostenbrink, Chris; Zagrovic, Bojan

    2013-01-01

    By directly affecting structure, dynamics and interaction networks of their targets, post-translational modifications (PTMs) of proteins play a key role in different cellular processes ranging from enzymatic activation to regulation of signal transduction to cell-cycle control. Despite the great importance of understanding how PTMs affect proteins at the atomistic level, a systematic framework for treating post-translationally modified amino acids by molecular dynamics (MD) simulations, a premier high-resolution computational biology tool, has never been developed. Here, we report and validate force field parameters (GROMOS 45a3 and 54a7) required to run and analyze MD simulations of more than 250 different types of enzymatic and non-enzymatic PTMs. The newly developed GROMOS 54a7 parameters in particular exhibit near chemical accuracy in matching experimentally measured hydration free energies (RMSE=4.2 kJ/mol over the validation set). Using this tool, we quantitatively show that the majority of PTMs greatly alter the hydrophobicity and other physico-chemical properties of target amino acids, with the extent of change in many cases being comparable to the complete range spanned by native amino acids. PMID:23874192

  4. Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

    PubMed

    Quan, Xiao-Jiang; Yuan, Liqun; Tiberi, Luca; Claeys, Annelies; De Geest, Natalie; Yan, Jiekun; van der Kant, Rob; Xie, Wei R; Klisch, Tiemo J; Shymkowitz, Joost; Rousseau, Frederic; Bollen, Mathieu; Beullens, Monique; Zoghbi, Huda Y; Vanderhaeghen, Pierre; Hassan, Bassem A

    2016-01-28

    Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects. PMID:26824657

  5. eIF2 interactions with initiator tRNA and eIF2B are regulated by post-translational modifications and conformational dynamics

    PubMed Central

    Beilsten-Edmands, Victoria; Gordiyenko, Yuliya; Kung, Jocky CK; Mohammed, Shabaz; Schmidt, Carla; Robinson, Carol V

    2015-01-01

    Translation of messenger RNA (mRNA) into proteins is key to eukaryotic gene expression and begins when initiation factor-2 (eIF2) delivers methionyl initiator tRNA (Met-tRNAi Met) to ribosomes. This first step is controlled by eIF2B mediating guanine nucleotide exchange on eIF2. We isolated eIF2 from yeast and used mass spectrometry to study the intact complex, and found that eIF2β is the most labile of the three subunits (eIF2α/β/γ). We then compared conformational dynamics of the ternary complex eIF2:GTP:Met-tRNAi Met with apo eIF2 using comparative chemical cross-linking. Results revealed high conformational dynamics for eIF2α in apo eIF2 while in the ternary complex all three subunits are constrained. Novel post-translational modifications identified here in both eIF2 and eIF2B were combined with established sites, and located within protein sequences and homology models. We found clustering at subunit interfaces and highly phosphorylated unstructured regions, at the N-terminus of eIF2β, and also between the eIF2Bε core and catalytic domains. We propose that modifications of these unstructured regions have a key role in regulating interactions between eIF2 and eIF2B, as well as other eIFs.

  6. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

    PubMed Central

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D.

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  7. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression.

    PubMed

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81-106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  8. 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

  9. Post-Translational Modifications of TRP Channels

    PubMed Central

    Voolstra, Olaf; Huber, Armin

    2014-01-01

    Transient receptor potential (TRP) channels constitute an ancient family of cation channels that have been found in many eukaryotic organisms from yeast to human. TRP channels exert a multitude of physiological functions ranging from Ca2+ homeostasis in the kidney to pain reception and vision. These channels are activated by a wide range of stimuli and undergo covalent post-translational modifications that affect and modulate their subcellular targeting, their biophysical properties, or channel gating. These modifications include N-linked glycosylation, protein phosphorylation, and covalent attachment of chemicals that reversibly bind to specific cysteine residues. The latter modification represents an unusual activation mechanism of ligand-gated ion channels that is in contrast to the lock-and-key paradigm of receptor activation by its agonists. In this review, we summarize the post-translational modifications identified on TRP channels and, when available, explain their physiological role. PMID:24717323

  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

    2015-01-01

    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. PMID:26518898

  11. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  12. Post-translational Modification and Quality Control

    PubMed Central

    Wang, Xuejun; Pattison, J. Scott; Su, Huabo

    2013-01-01

    Protein quality control (PQC) functions to minimize the level and toxicity of misfolded proteins in the cell. PQC is performed by intricate collaboration among chaperones and target protein degradation. The latter is carried out primarily by the ubiquitin-proteasome system and perhaps autophagy. Terminally misfolded proteins that are not timely removed tend to form aggregates. Their clearance requires macroautophagy. Macroautophagy serves in intracellular quality control also by selectively segregating defective organelles (e.g., mitochondria) and targeting them for degradation by the lysosome. Inadequate PQC is observed in a large subset of failing human hearts with a variety of etiologies and its pathogenic role has been experimentally demonstrated. Multiple post-translational modifications (PTMs) can occur to substrate proteins and/or PQC machineries, promoting or hindering the removal of the misfolded proteins. This article highlights recent advances in PTMs-mediated regulation of intracellular quality control mechanisms and its known involvement in cardiac pathology. PMID:23329792

  13. 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

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

    PubMed

    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

  15. Lysine post-translational modifications of collagen

    PubMed Central

    Yamauchi, Mitsuo; Sricholpech, Marnisa

    2012-01-01

    Type I collagen is the most abundant structural protein in vertebrates. It is a heterotrimeric molecule composed of two α1 chains and one α2 chain, forming a long uninterrupted triple helical structure with short non-triple helical telopeptides at both the N- and C-termini. During biosynthesis, collagen acquires a number of post-translational modifications, including lysine modifications, that are critical to the structure and biological functions of this protein. Lysine modifications of collagen are highly complicated sequential processes catalysed by several groups of enzymes leading to the final step of biosynthesis, covalent intermolecular cross-linking. In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then specific hydroxylysine residues located in the helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose. Outside the cell, lysine and hydroxylysine residues in the N- and C-telopeptides can be oxidatively deaminated to produce reactive aldehydes that undergo a series of non-enzymatic condensation reactions to form covalent intra- and inter-molecular cross-links. Owing to the recent advances in molecular and cellular biology, and analytical technologies, the biological significance and molecular mechanisms of these modifications have been gradually elucidated. This chapter provides an overview on these enzymatic lysine modifications and subsequent cross-linking. PMID:22708567

  16. 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

    2014-12-01

    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. PMID:25252820

  17. Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications

    PubMed Central

    Chesarino, Nicholas M; McMichael, Temet M; Yount, Jacob S

    2014-01-01

    IFITM3 restricts cellular infection by multiple important viral pathogens, and is particularly critical for the innate immune response against influenza virus. Expression of IFITM3 expands acidic endolysosomal compartments and prevents fusion of endocytosed viruses, leading to their degradation. This small, 133 amino acid, antiviral protein is controlled by at least four distinct post-translational modifications. Positive regulation of IFITM3 antiviral activity is provided by S-palmitoylation, while negative regulatory mechanisms include lysine ubiquitination, lysine methylation and tyrosine phosphorylation. Herein, we describe specific insights into IFITM3 trafficking and activity that were provided by studies of IFITM3 post-translational modifications, and discuss evidence suggesting that IFITM3 adopts multiple membrane topologies involving at least one intramembrane domain in its antivirally active conformation. PMID:25405885

  18. 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

  19. 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

  20. Rosuvastatin Modulates the Post-Translational Acetylome in Endothelial Cells

    PubMed Central

    Lin, Ming Chung; Hsing, Chung Hsi; Li, Fu An; Wu, Chien Hsing; Fu, Yaw Syan; Cheng, Jen Kun; Huang, Bin

    2014-01-01

    Background Statins are lipid-lowering drugs that can simultaneously evoke pleiotropic effects on cardioprotection, vasodilation, and diabetes prevention. Recently, statins have been reported to be able to activate the AMP-activated protein kinase, thereby up-regulating sirtuin (SIRT) that functions as non-histone deacetylases. Therefore, it is essential to investigate the post-translational acetylome that might explain the mechanism of statin-modulated pleiotropic effects. Methods Endothelial cells EAhy 926 treated with rosuvastatin were used to monitor the expression of SIRTs proteins. The protein lysates of both mock- and rosuvastatin-treated cells were further separated by two- dimensional gel electrophoresis coupled with western blotting analysis. The significantly changed acetyl- containing proteins detected by using an anti-acetyl lysine antibody were collected from another preparative gel for mass spectrometric assay to identify the acetylated site in the proteins. Results Rosuvastatin treatment was shown to increase the SIRT1 expression when compared with SIRT2. Among 100 detected proteins with acetylated signal, 12 showed an increased level of acetylation, whereas 6 showed a decreased level of acetylation (deacetylation). The acetylated lysine (K) sites of 3 heat shock proteins, i.e., HSP47/K165, HSP70/K380, and heat shock-inducible protein/K417, were determined. We also found that beta-filamin, elongation factor, galectin and hCG22067 have 2 acetylated lysine sites in their peptide sequences. These dynamic acetylations might alter the protein’s function and are thought to be important in regulating statin-mediated pleiotropic effect. Conclusions Our study provided a feasible methodology for detecting acetylated proteins. This acetylome information may be utilized to explain, at least partially, the mechanisms of statin-derived pleiotropic effects. PMID:27122770

  1. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels.

    PubMed

    Shipston, Michael J

    2014-01-01

    Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

  2. S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

    PubMed Central

    Shipston, Michael J.

    2014-01-01

    Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

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

    PubMed Central

    Ravikumar, Vaishnavi; Jers, Carsten; Mijakovic, Ivan

    2015-01-01

    Microbes with the capability to survive in the host tissue and efficiently subvert its innate immune responses can cause various health hazards. There is an inherent need to understand microbial infection patterns and mechanisms in order to develop efficient therapeutics. Microbial pathogens display host specificity through a complex network of molecular interactions that aid their survival and propagation. Co-infection states further lead to complications by increasing the microbial burden and risk factors. Quantitative proteomics based approaches and post-translational modification analysis can be efficiently applied to gain an insight into the molecular mechanisms involved. The measurement of the proteome and post-translationally modified proteome dynamics using mass spectrometry, results in a wide array of information, such as significant changes in protein expression, protein abundance, the modification status, the site occupancy level, interactors, functional significance of key players, potential drug targets, etc. This mini review discusses the potential of proteomics to investigate the involvement of post-translational modifications in bacterial pathogenesis and host–pathogen interactions. PMID:26635773

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

    PubMed Central

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

    2016-01-01

    Post-translationally modified proteins make up the majority of the proteome and establish, to a large part, the impressive level of functional diversity in higher, multi-cellular organisms. Most eukaryotic post-translational protein modifications (PTMs) denote reversible, covalent additions of small chemical entities such as phosphate-, acyl-, alkyl- and glycosyl-groups onto selected subsets of modifiable amino acids. In turn, these modifications induce highly specific changes in the chemical environments of individual protein residues, which are readily detected by high-resolution NMR spectroscopy. In the following, we provide a concise compendium of NMR characteristics of the main types of eukaryotic PTMs: serine, threonine, tyrosine and histidine phosphorylation, lysine acetylation, lysine and arginine methylation, and serine, threonine O-glycosylation. We further delineate the previously uncharacterized NMR properties of lysine propionylation, butyrylation, succinylation, malonylation and crotonylation, which, altogether, define an initial reference frame for comprehensive PTM studies by high-resolution NMR spectroscopy. PMID:23011410

  5. Post-translational modification of PII signal transduction proteins

    PubMed Central

    Merrick, Mike

    2015-01-01

    The PII proteins constitute one of the most widely distributed families of signal transduction proteins in nature. They are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. Quite remarkably PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and in all known cases they achieve their regulatory effect by direct interaction with their target. PII proteins in the Proteobacteria and the Actinobacteria are subject to post-translational modification by uridylylation or adenylylation respectively, whilst in some Cyanobacteria they can be modified by phosphorylation. In all these cases the protein’s modification state is influenced by the cellular nitrogen status and is thought to regulate its activity. However, in many organisms there is no evidence for modification of PII proteins and indeed the ability of these proteins to respond to the cellular nitrogen status is fundamentally independent of post-translational modification. In this review we explore the role of post-translational modification in PII proteins in the light of recent studies. PMID:25610437

  6. 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.

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

    SciTech Connect

    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.

  8. 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.

  9. Top-down characterization of the post-translationally modified intact periplasmic proteome of the bacterium Novosphingobium aromaticivorans

    SciTech Connect

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

    2013-03-10

    In this study, the intact periplasmic proteome of Novosphingobium aromaticivorans was analyzed. We identified 55 proteins in the periplasm, and characterized their post translational modifications. Proteins were first categorized based on their N-terminal processing: 17 proteins were identified with removal of signal peptides containing the canonical A-X-A motif, 8 proteins were identified with removal of signal peptides containing non A-X-A motif, 24 proteins were identified with N-terminal methione excision (NME), and 4 proteins were identified with other N-terminal processing (e.g. complex proteolysis). Only 2 proteins were identified with no N-terminal modifications. Other observed protein post-translational modifications included acetylation, glutathiolynation, pyroglutamate modification, disulfide bond formation, etc. In summary, we analyzed the intact periplasmic proteins of N. aromaticivorans in a high throughput fashion, and provided a catalogue of information on post-translational modifications observed in this dynamic subcellular fraction. This study provides the first experimental evidence for the expression and periplasmic localization of hypothetical and uncharacterized proteins, and the first unrestrictive, large-scale data on post-translational modifications in the bacterial periplasm.

  10. 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

  11. Protein Interactions, Post-translational Modifications and Topologies in Human Cells*

    PubMed Central

    Chavez, Juan D.; Weisbrod, Chad R.; Zheng, Chunxiang; Eng, Jimmy K.; Bruce, James E.

    2013-01-01

    The unique and remarkable physicochemical properties of protein surface topologies give rise to highly specific biomolecular interactions, which form the framework through which living systems are able to carry out their vast array of functions. Technological limitations undermine efforts to probe protein structures and interactions within unperturbed living systems on a large scale. Rapid chemical stabilization of proteins and protein complexes through chemical cross-linking offers the alluring possibility to study details of the protein structure to function relationships as they exist within living cells. Here we apply the latest technological advances in chemical cross-linking combined with mass spectrometry to study protein topologies and interactions from living human cells identifying a total of 368 cross-links. These include cross-links from all major cellular compartments including membrane, cytosolic and nuclear proteins. Intraprotein and interprotein cross-links were also observed for core histone proteins, including several cross-links containing post-translational modifications which are known histone marks conferring distinct epigenetic functions. Excitingly, these results demonstrate the applicability of cross-linking to make direct topological measurements on post-translationally modified proteins. The results presented here provide new details on the structures of known multi-protein complexes as well as evidence for new protein-protein interactions. PMID:23354917

  12. Linking post-translational modifications and variation of phenotypic traits.

    PubMed

    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

    2013-03-01

    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

  13. 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

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

    PubMed

    Margreitter, Christian; Petrov, Drazen; Zagrovic, Bojan

    2013-07-01

    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

  15. 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

  16. Regulating the Regulator: Post-Translational Modification of Ras

    PubMed Central

    Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

    2013-01-01

    Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

  17. Post-Translational Modification Control of Innate Immunity.

    PubMed

    Liu, Juan; Qian, Cheng; Cao, Xuetao

    2016-07-19

    A coordinated balance between the positive and negative regulation of pattern-recognition receptor (PRR)-initiated innate inflammatory responses is required to ensure the most favorable outcome for the host. Post-translational modifications (PTMs) of innate sensors and downstream signaling molecules influence their activity and function by inducing their covalent linkage to new functional groups. PTMs including phosphorylation and polyubiquitination have been shown to potently regulate innate inflammatory responses through the activation, cellular translocation, and interaction of innate receptors, adaptors, and downstream signaling molecules in response to infectious and dangerous signals. Other PTMs such as methylation, acetylation, SUMOylation, and succinylation are increasingly implicated in the regulation of innate immunity and inflammation. In this review, we focus on the roles of PTMs in controlling PRR-triggered innate immunity and inflammatory responses. The emerging roles of PTMs in the pathogenesis and potential treatment of infectious and inflammatory immune diseases are also discussed. PMID:27438764

  18. Nine Post-translational Modifications during the Biosynthesis of Cinnamycin

    PubMed Central

    2011-01-01

    Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides that are characterized by the thioether cross-linked amino acids lanthionine (Lan) and methyllanthionine (MeLan). Cinnamycin is a 19 amino acid lantibiotic that contains one Lan and two MeLan. Cinnamycin also contains an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-l-aspartic acid resulting from the hydroxylation of l-aspartate at position 15. These modifications are critical in mediating the interactions of cinnamycin with its target, phosphatidylethanolamine. Recently, the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005 was reported. Herein, we investigated the biosynthetic machinery using both in vitro studies and heterologous expression in Escherichia coli. CinX is an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide CinA. In addition, CinM catalyzes dehydration of four Ser and Thr residues and subsequent cyclization of Cys residues to form the three (Me)Lan bridges. The order of the post-translational modifications catalyzed by CinM and CinX is interchangeable in vitro. CinX did not require the leader sequence at the N-terminus of CinA for activity, but the leader peptide was necessary for CinM function. Although CinM dehydrated serine 6, it did not catalyze the formation of Lal. A small protein encoded by cinorf7 is critical for the formation of the cross-link between Lys19 and dehydroalanine 6 as shown by coexpression studies of CinA, CinM, CinX, and Cinorf7 in E. coli. PMID:21770392

  19. 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. PMID:25808548

  20. Post-translational regulation and modifications of flavivirus structural proteins.

    PubMed

    Roby, Justin A; Setoh, Yin Xiang; Hall, Roy A; Khromykh, Alexander A

    2015-07-01

    Flaviviruses are a group of single-stranded, positive-sense RNA viruses that generally circulate between arthropod vectors and susceptible vertebrate hosts, producing significant human and veterinary disease burdens. Intensive research efforts have broadened our scientific understanding of the replication cycles of these viruses and have revealed several elegant and tightly co-ordinated post-translational modifications that regulate the activity of viral proteins. The three structural proteins in particular - capsid (C), pre-membrane (prM) and envelope (E) - are subjected to strict regulatory modifications as they progress from translation through virus particle assembly and egress. The timing of proteolytic cleavage events at the C-prM junction directly influences the degree of genomic RNA packaging into nascent virions. Proteolytic maturation of prM by host furin during Golgi transit facilitates rearrangement of the E proteins at the virion surface, exposing the fusion loop and thus increasing particle infectivity. Specific interactions between the prM and E proteins are also important for particle assembly, as prM acts as a chaperone, facilitating correct conformational folding of E. It is only once prM/E heterodimers form that these proteins can be secreted efficiently. The addition of branched glycans to the prM and E proteins during virion transit also plays a key role in modulating the rate of secretion, pH sensitivity and infectivity of flavivirus particles. The insights gained from research into post-translational regulation of structural proteins are beginning to be applied in the rational design of improved flavivirus vaccine candidates and make attractive targets for the development of novel therapeutics. PMID:25711963

  1. Formylglycine, a post-translationally generated residue with unique catalytic capabilities and biotechnology applications.

    PubMed

    Appel, Mason J; Bertozzi, Carolyn R

    2015-01-16

    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

  2. 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

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

    PubMed

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

    2015-11-27

    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

  4. 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

  5. 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.

  6. 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. PMID:26254007

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

    PubMed Central

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

    2014-01-01

    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. PMID:24419378

  8. 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.

  9. Multiple Post-translational Modifications Affect Heterologous Protein Synthesis*

    PubMed Central

    Tokmakov, Alexander A.; Kurotani, Atsushi; Takagi, Tetsuo; Toyama, Mitsutoshi; Shirouzu, Mikako; Fukami, Yasuo; Yokoyama, Shigeyuki

    2012-01-01

    Post-translational modifications (PTMs) are required for proper folding of many proteins. The low capacity for PTMs hinders the production of heterologous proteins in the widely used prokaryotic systems of protein synthesis. Until now, a systematic and comprehensive study concerning the specific effects of individual PTMs on heterologous protein synthesis has not been presented. To address this issue, we expressed 1488 human proteins and their domains in a bacterial cell-free system, and we examined the correlation of the expression yields with the presence of multiple PTM sites bioinformatically predicted in these proteins. This approach revealed a number of previously unknown statistically significant correlations. Prediction of some PTMs, such as myristoylation, glycosylation, palmitoylation, and disulfide bond formation, was found to significantly worsen protein amenability to soluble expression. The presence of other PTMs, such as aspartyl hydroxylation, C-terminal amidation, and Tyr sulfation, did not correlate with the yield of heterologous protein expression. Surprisingly, the predicted presence of several PTMs, such as phosphorylation, ubiquitination, SUMOylation, and prenylation, was associated with the increased production of properly folded soluble proteins. The plausible rationales for the existence of the observed correlations are presented. Our findings suggest that identification of potential PTMs in polypeptide sequences can be of practical use for predicting expression success and optimizing heterologous protein synthesis. In sum, this study provides the most compelling evidence so far for the role of multiple PTMs in the stability and solubility of heterologously expressed recombinant proteins. PMID:22674579

  10. Co- and Post-Translational Protein Folding in the ER.

    PubMed

    Ellgaard, Lars; McCaul, Nicholas; Chatsisvili, Anna; Braakman, Ineke

    2016-06-01

    The biophysical rules that govern folding of small, single-domain proteins in dilute solutions are now quite well understood. The mechanisms underlying co-translational folding of multidomain and membrane-spanning proteins in complex cellular environments are often less clear. The endoplasmic reticulum (ER) produces a plethora of membrane and secretory proteins, which must fold and assemble correctly before ER exit - if these processes fail, misfolded species accumulate in the ER or are degraded. The ER differs from other cellular organelles in terms of the physicochemical environment and the variety of ER-specific protein modifications. Here, we review chaperone-assisted co- and post-translational folding and assembly in the ER and underline the influence of protein modifications on these processes. We emphasize how method development has helped advance the field by allowing researchers to monitor the progression of folding as it occurs inside living cells, while at the same time probing the intricate relationship between protein modifications during folding. PMID:26947578

  11. Post-translational modifications of nuclear receptors and human disease

    PubMed Central

    Anbalagan, Muralidharan; Huderson, Brandy; Murphy, Leigh; Rowan, Brian G.

    2012-01-01

    Nuclear receptors (NR) impact a myriad of physiological processes including homeostasis, reproduction, development, and metabolism. NRs are regulated by post-translational modifications (PTM) that markedly impact receptor function. Recent studies have identified NR PTMs that are involved in the onset and progression of human diseases, including cancer. The majority of evidence linking NR PTMs with disease has been demonstrated for phosphorylation, acetylation and sumoylation of androgen receptor (AR), estrogen receptor α (ERα), glucocorticoid receptor (GR) and peroxisome proliferator activated receptor γ (PPARγ). Phosphorylation of AR has been associated with hormone refractory prostate cancer and decreased disease-specific survival. AR acetylation and sumoylation increased growth of prostate cancer tumor models. AR phosphorylation reduced the toxicity of the expanded polyglutamine AR in Kennedy’s Disease as a consequence of reduced ligand binding. A comprehensive evaluation of ERα phosphorylation in breast cancer revealed several sites associated with better clinical outcome to tamoxifen therapy, whereas other phosphorylation sites were associated with poorer clinical outcome. ERα acetylation and sumoylation may also have predictive value for breast cancer. GR phosphorylation and acetylation impact GR responsiveness to glucocorticoids that are used as anti-inflammatory drugs. PPARγ phosphorylation can regulate the balance between growth and differentiation in adipose tissue that is linked to obesity and insulin resistance. Sumoylation of PPARγ is linked to repression of inflammatory genes important in patients with inflammatory diseases. NR PTMs provide an additional measure of NR function that can be used as both biomarkers of disease progression, and predictive markers for patient response to NR-directed treatments. PMID:22438791

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

    PubMed Central

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

    2014-01-01

    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

  13. 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

  14. Fluorescent Polymer-Based Post-Translational Differentiation and Subtyping of Breast Cancer Cells

    PubMed Central

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

    2012-01-01

    Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were differently modulated in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions. PMID:23061092

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

    PubMed Central

    2010-01-01

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

  16. DAPPLE 2: a Tool for the Homology-Based Prediction of Post-Translational Modification Sites.

    PubMed

    Trost, Brett; Maleki, Farhad; Kusalik, Anthony; Napper, Scott

    2016-08-01

    The post-translational modification of proteins is critical for regulating their function. Although many post-translational modification sites have been experimentally determined, particularly in certain model organisms, experimental knowledge of these sites is severely lacking for many species. Thus, it is important to be able to predict sites of post-translational modification in such species. Previously, we described DAPPLE, a tool that facilitates the homology-based prediction of one particular post-translational modification, phosphorylation, in an organism of interest using known phosphorylation sites from other organisms. Here, we describe DAPPLE 2, which expands and improves upon DAPPLE in three major ways. First, it predicts sites for many post-translational modifications (20 different types) using data from several sources (15 online databases). Second, it has the ability to make predictions approximately 2-7 times faster than DAPPLE depending on the database size and the organism of interest. Third, it simplifies and accelerates the process of selecting predicted sites of interest by categorizing them based on gene ontology terms, keywords, and signaling pathways. We show that DAPPLE 2 can successfully predict known human post-translational modification sites using, as input, known sites from species that are either closely (e.g., mouse) or distantly (e.g., yeast) related to humans. DAPPLE 2 can be accessed at http://saphire.usask.ca/saphire/dapple2 . PMID:27367363

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

    PubMed Central

    Wani, Revati; Nagata, Asako; Murray, Brion W.

    2014-01-01

    The perception of reactive oxygen species has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g., cancer). New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically distinct alterations to the protein (e.g., sulfenic/sulfinic/sulfonic acid, disulfides). These post-translational modifications (PTM) are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology. PMID:25339904

  18. Novel post-translational incorporation of tyrosine in PMA-activated polymorphonuclear leukocytes (PMN)

    SciTech Connect

    Nath, J.; Oliver, C.; Ohno, Y.; Gallin, J.I.

    1986-03-05

    During studies undertaken to determine whether stimulation of tubulin tyrosinolation occurs in PMA-activated PMN, a distinctly different and novel post-translational incorporation of tyrosine into multiple PMN proteins was observed. The reaction also occurred in organelle-depleted neutrophil cytoplasts and was highly exaggerated in organelle-enriched karyogranuloplasts. The incorporation was specific for tyrosine, did not require extracellular Ca/sup 2 +/ and was inhibited in the presence of a variety of reducing agents, intracellular scavengers of oxygen radicals and inhibitors of peroxidase-mediated reactions. The PMA-induced incorporation of tyrosine was completely absent in PMN from patients with chronic granulomatous disease, but occurred normally in PMN of a patient with myeloperoxidase deficiency. Moreover, the incorporation of tyrosine was blocked by N-acetyl-L-tyrosine but not by phenylalanine suggesting a requirement for the phenolic group. A two-fold increase in stable protein carbonyl derivatives was demonstrated suggesting an increased oxidative modification of the proteins. SDS urea PAGE and reversed phase HPLC did not reveal any detectable changes in the extent of protein cross-linking. The PMN tyrosine pool was approximately 900 ..mu..M and yet only 1 ..mu..M tyrosine was added in these experiments. The functional significance of this reaction is not yet clear.

  19. Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms.

    PubMed

    Weeks, Kate L; Avkiran, Metin

    2015-04-15

    Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms. PMID:25362149

  20. Markov chain Monte Carlo based analysis of post-translationally modified VDAC gating kinetics

    PubMed Central

    Tewari, Shivendra G.; Zhou, Yifan; Otto, Bradley J.; Dash, Ranjan K.; Kwok, Wai-Meng; Beard, Daniel A.

    2015-01-01

    The voltage-dependent anion channel (VDAC) is the main conduit for permeation of solutes (including nucleotides and metabolites) of up to 5 kDa across the mitochondrial outer membrane (MOM). Recent studies suggest that VDAC activity is regulated via post-translational modifications (PTMs). Yet the nature and effect of these modifications is not understood. Herein, single channel currents of wild-type, nitrosated, and phosphorylated VDAC are analyzed using a generalized continuous-time Markov chain Monte Carlo (MCMC) method. This developed method describes three distinct conducting states (open, half-open, and closed) of VDAC activity. Lipid bilayer experiments are also performed to record single VDAC activity under un-phosphorylated and phosphorylated conditions, and are analyzed using the developed stochastic search method. Experimental data show significant alteration in VDAC gating kinetics and conductance as a result of PTMs. The effect of PTMs on VDAC kinetics is captured in the parameters associated with the identified Markov model. Stationary distributions of the Markov model suggest that nitrosation of VDAC not only decreased its conductance but also significantly locked VDAC in a closed state. On the other hand, stationary distributions of the model associated with un-phosphorylated and phosphorylated VDAC suggest a reversal in channel conformation from relatively closed state to an open state. Model analyses of the nitrosated data suggest that faster reaction of nitric oxide with Cys-127 thiol group might be responsible for the biphasic effect of nitric oxide on basal VDAC conductance. PMID:25628567

  1. Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms

    PubMed Central

    Weeks, Kate L; Avkiran, Metin

    2015-01-01

    Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms. PMID:25362149

  2. 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

  3. Pdx1 Is Post-Translationally Modified In vivo and Serine 61 Is the Principal Site of Phosphorylation

    PubMed Central

    Frogne, Thomas; Sylvestersen, Kathrine Beck; Kubicek, Stefan; Nielsen, Michael Lund; Hecksher-Sørensen, Jacob

    2012-01-01

    Maintaining sufficient levels of Pdx1 activity is a prerequisite for proper regulation of blood glucose homeostasis and beta cell function. Mice that are haploinsufficient for Pdx1 display impaired glucose tolerance and lack the ability to increase beta cell mass in response to decreased insulin signaling. Several studies have shown that post-translational modifications are regulating Pdx1 activity through intracellular localization and binding to co-factors. Understanding the signaling cues converging on Pdx1 and modulating its activity is therefore an attractive approach in diabetes treatment. We employed a novel technique called Nanofluidic Proteomic Immunoassay to characterize the post-translational profile of Pdx1. Following isoelectric focusing in nano-capillaries, this technology relies on a pan specific antibody for detection and it therefore allows the relative abundance of differently charged protein species to be examined simultaneously. In all eukaryotic cells tested we find that the Pdx1 protein separates into four distinct peaks whereas Pdx1 protein from bacteria only produces one peak. Of the four peaks in eukaryotic cells we correlate one of them to a phosphorylation Using alanine scanning and mass spectrometry we map this phosphorylation to serine 61 in both Min6 cells and in exogenous Pdx1 over-expressed in HEK293 cells. A single phosphorylation is also present in cultured islets but it remains unaffected by changes in glucose levels. It is present during embryogenesis but is not required for pancreas development. PMID:22509401

  4. Post-Translational Modification Biology of Glutamate Receptors and Drug Addiction

    PubMed Central

    Mao, Li-Min; Guo, Ming-Lei; Jin, Dao-Zhong; Fibuch, Eugene E.; Choe, Eun Sang; Wang, John Q.

    2011-01-01

    Post-translational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues in their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling, and protein–protein interactions), subcellular redistribution (trafficking, endocytosis, synaptic delivery, and clustering), and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamine). Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction. PMID:21441996

  5. Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding*

    PubMed Central

    Houde, Damian; Peng, Yucai; Berkowitz, Steven A.; Engen, John R.

    2010-01-01

    Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications. PMID:20103567

  6. Post-translational modification of plant-made foreign proteins; glycosylation and beyond.

    PubMed

    Webster, Diane E; Thomas, Merlin C

    2012-01-01

    The complex and diverse nature of the post-translational modification (PTM) of proteins represents an efficient and cost-effective mechanism for the exponential diversification of the genome. PTMs have been shown to affect almost every aspect of protein activity, including function, localisation, stability, and dynamic interactions with other molecules. Although many PTMs are evolutionarily conserved there are also important kingdom-specific modifications which should be considered when expressing recombinant proteins. Plants are gaining increasing acceptance as an expression system for recombinant proteins, particularly where eukaryotic-like PTMs are required. Glycosylation is the most extensively studied PTM of plant-made recombinant proteins. However, other types of protein processing and modification also occur which are important for the production of high quality recombinant protein, such as hydroxylation and lipidation. Plant and/or protein engineering approaches offer many opportunities to exploit PTM pathways allowing the molecular farmer to produce a humanised product with modifications functionally similar or identical to the native protein. Indeed, plants have demonstrated a high degree of tolerance to changes in PTM pathways allowing recombinant proteins to be modified in a specific and controlled manner, frequently resulting in a homogeneity of product which is currently unrivalled by alternative expression platforms. Whether a recombinant protein is intended for use as a scientific reagent, a cosmetic additive or as a pharmaceutical, PTMs through their presence and complexity, offer an extensive range of options for the rational design of humanised (biosimilar), enhanced (biobetter) or novel products. PMID:21839159

  7. Protein post-translational modifications and regulation of pluripotency in human stem cells

    PubMed Central

    Wang, Yu-Chieh; Peterson, Suzanne E; Loring, Jeanne F

    2014-01-01

    Post-translational modifications (PTMs) are known to be essential mechanisms used by eukaryotic cells to diversify their protein functions and dynamically coordinate their signaling networks. Defects in PTMs have been linked to numerous developmental disorders and human diseases, highlighting the importance of PTMs in maintaining normal cellular states. Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), are capable of self-renewal and differentiation into a variety of functional somatic cells; these cells hold a great promise for the advancement of biomedical research and clinical therapy. The mechanisms underlying cellular pluripotency in human cells have been extensively explored in the past decade. In addition to the vast amount of knowledge obtained from the genetic and transcriptional research in hPSCs, there is a rapidly growing interest in the stem cell biology field to examine pluripotency at the protein and PTM level. This review addresses recent progress toward understanding the role of PTMs (glycosylation, phosphorylation, acetylation and methylation) in the regulation of cellular pluripotency. PMID:24217768

  8. Molecular Determinants of Co- and Post-translational N-glycosylation of Type I Transmembrane Peptides

    PubMed Central

    Malaby, Heidi L. H.; Kobertz, William R.

    2013-01-01

    Type I transmembrane peptides acquire N-linked glycans during and after protein synthesis to facilitate anterograde trafficking through the secretory pathway. Mutations in N-glycosylation consensus sites (NXT and NXS, where X ≠ P) that alter the kinetics of the initial N-glycan attachment have been associated with cardiac arrhythmias; however, the molecular determinants that define co- and post-translational consensus sites in proteins are not known. Here, we identified co- and post-translational consensus sites in the KCNE family of K+ channel regulatory subunits to uncover three determinants that favor co-translational N-glycosylation kinetics of type I transmembrane peptides that lack a cleavable signal sequence: threonine containing-consensus sites (NXT), multiple N-terminal consensus sites, and long C-termini. The identification of these three molecular determinants now makes it possible to predict co- and post-translational consensus sites in type I transmembrane peptides. PMID:23718681

  9. 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

  10. Haloferax mediterranei GlnK proteins are post-translationally modified by uridylylation.

    PubMed

    Pedro-Roig, Laia; Camacho, Mónica; Bonete, María José

    2013-04-01

    In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2 ), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family. PMID:23420616

  11. Detection of post-translational modifications in single peptides using electron tunnelling currents

    NASA Astrophysics Data System (ADS)

    Ohshiro, Takahito; Tsutsui, Makusu; Yokota, Kazumichi; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2014-10-01

    Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids. These modifications allow proteins to perform their primary biological functions, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes. We identify 12 different amino acids and the post-translational modification phosphotyrosine, which is involved in the process that switches enzymes on and off. Furthermore, we show that the conductance measurements can be used to partially sequence peptides of an epidermal growth factor receptor substrate, and can discriminate a peptide from its phosphorylated variant.

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

    PubMed

    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

  13. 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

  14. Structural Mapping of Post-translational Modifications in Human Interleukin-24

    PubMed Central

    Fuson, Kerry L.; Zheng, Mingzhong; Craxton, Molly; Pataer, Abujiang; Ramesh, Rajagopal; Chada, Sunil; Sutton, R. Bryan

    2009-01-01

    Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications. PMID:19734147

  15. Post-translational modification profiling - A novel tool for mapping the protein modification landscape in cancer.

    PubMed

    Eisenberg-Lerner, Avital; Ciechanover, Aaron; Merbl, Yifat

    2016-08-01

    The ubiquitin system plays an important role in essentially every cellular process, regulating numerous pathways ranging from development, transcription, DNA damage response, cell cycle, and signal transduction. Its best studied role involves removal of faulty proteins or those that are not necessary anymore. Aberrations in the ubiquitin system have been implicated in various pathologies including cancer, where specific mutations in E3 ligases such as Mdm2, pVHL, and BRCA1 have been linked to disease progression, prognosis, and resistance to drugs. Yet, there are hundreds of E3 ligases in the human genome and our knowledge of their target proteins and their dynamic regulation in the cellular environment is largely limited. In addition, fundamental questions related to recognition and specificity in ubiquitin conjugation remain unanswered. It is thus of major importance to characterize the ubiquitin landscape under various cellular conditions, and study how the regulatory network is altered in health and disease. To do so, analytical tools that allow identification of ubiquitin substrates, the conjugation and removal of ubiquitin, and the nature of specific ubiquitin linkages that are formed are needed. In this mini-review, we discuss common proteomic methodologies applied to studying the ubiquitome, and specifically focus on our recently developed post-translational modification (PTM) profiling approach. PTM profiling is a functional assay, amenable to biochemical manipulation, which allows the detection of protein modifications in a high-throughput manner. We discuss in detail the advantages and limitations of this system, focusing primarily on examples for analyzing the ubiquitin system in cancer. Uncovering the intricate signaling dynamics governed by and regulating ubiquitin modifications should clearly evolve into a new paradigm in understanding the molecular basis of malignant transformation and the development of novel therapeutic modalities. PMID:27229346

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

    PubMed

    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

    2016-07-21

    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

  17. 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

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

    PubMed

    Fernandez-Rodriguez, Jesus; Voigt, Christopher A

    2016-07-27

    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

  19. 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. PMID:26685169

  20. Characterization of neurohistone variants and post-translational modifications by electron capture dissociation mass spectrometry

    NASA Astrophysics Data System (ADS)

    Garcia, Benjamin A.; Siuti, Nertila; Thomas, C. Eric; Mizzen, Craig A.; Kelleher, Neil L.

    2007-01-01

    Post-translational modifications (PTMs) of histones are intimately involved in chromatin structure and thus have roles in cellular processes through their impact on gene activation or repression. At the forefront in histone PTM analysis are mass spectrometry-based techniques, which have capabilities to produce improved views of processes affected by chromatin remodeling via histone modifications. In this report, we take the first mass spectrometric look at histone variant expression and post-translational modifications from histones isolated from rat brain tissue. Analyses of whole rat brain identified specific histone H2A and H2B gene family members and several H4 and H3 post-translational modification sites by electron capture dissociation (ECD) mass spectrometry. We subsequently compared these results to selected rat brain regions. Major differences in the expression profiles of H2A and H2B gene family members or in the post-translational modifications on histone H4 were not observed from the different brain regions using a Top Down approach. However, "Middle Down" mass spectrometry facilitating improved characterization of the histone H3 tail (1-50 residues), revealed an enrichment of trimethylation on Lys9 from cerebellum tissue compared to H3 extracted from whole brain, cerebral cortex or hypothalamus tissue. We forward this study in honor of Professor Donald F. Hunt, whose pioneering efforts in protein and PTM analyses have spawned new eras and numerous careers, many exemplified in this special issue.

  1. The alpha subunit of nitrile hydratase is sufficient for catalytic activity and post-translational modification.

    PubMed

    Nelp, Micah T; Astashkin, Andrei V; Breci, Linda A; McCarty, Reid M; Bandarian, Vahe

    2014-06-24

    Nitrile hydratases (NHases) possess a mononuclear iron or cobalt cofactor whose coordination environment includes rare post-translationally oxidized cysteine sulfenic and sulfinic acid ligands. This cofactor is located in the α-subunit at the interfacial active site of the heterodimeric enzyme. Unlike canonical NHases, toyocamycin nitrile hydratase (TNHase) from Streptomyces rimosus is a unique three-subunit member of this family involved in the biosynthesis of pyrrolopyrimidine antibiotics. The subunits of TNHase are homologous to the α- and β-subunits of prototypical NHases. Herein we report the expression, purification, and characterization of the α-subunit of TNHase. The UV-visible, EPR, and mass spectra of the α-subunit TNHase provide evidence that this subunit alone is capable of synthesizing the active site complex with full post-translational modifications. Remarkably, the isolated post-translationally modified α-subunit is also catalytically active with the natural substrate, toyocamycin, as well as the niacin precursor 3-cyanopyridine. Comparisons of the steady state kinetic parameters of the single subunit variant to the heterotrimeric protein clearly show that the additional subunits impart substrate specificity and catalytic efficiency. We conclude that the α-subunit is the minimal sequence needed for nitrile hydration providing a simplified scaffold to study the mechanism and post-translational modification of this important class of catalysts. PMID:24914472

  2. Two Distinct Dynamic Modes Subtend the Detection of Unexpected Sounds

    PubMed Central

    King, Jean-Rémi; Gramfort, Alexandre; Schurger, Aaron; Naccache, Lionel; Dehaene, Stanislas

    2014-01-01

    The brain response to auditory novelty comprises two main EEG components: an early mismatch negativity and a late P300. Whereas the former has been proposed to reflect a prediction error, the latter is often associated with working memory updating. Interestingly, these two proposals predict fundamentally different dynamics: prediction errors are thought to propagate serially through several distinct brain areas, while working memory supposes that activity is sustained over time within a stable set of brain areas. Here we test this temporal dissociation by showing how the generalization of brain activity patterns across time can characterize the dynamics of the underlying neural processes. This method is applied to magnetoencephalography (MEG) recordings acquired from healthy participants who were presented with two types of auditory novelty. Following our predictions, the results show that the mismatch evoked by a local novelty leads to the sequential recruitment of distinct and short-lived patterns of brain activity. In sharp contrast, the global novelty evoked by an unexpected sequence of five sounds elicits a sustained state of brain activity that lasts for several hundreds of milliseconds. The present results highlight how MEG combined with multivariate pattern analyses can characterize the dynamics of human cortical processes. PMID:24475052

  3. How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy.

    PubMed

    Feng, Yuchen; Yao, Zhiyuan; Klionsky, Daniel J

    2015-06-01

    Macroautophagy (hereafter autophagy), literally defined as a type of self-eating, is a dynamic cellular process in which cytoplasm is sequestered within a unique compartment termed the phagophore. Upon completion, the phagophore matures into a double-membrane autophagosome that fuses with the lysosome or vacuole, allowing degradation of the cargo. Nonselective autophagy is primarily a cytoprotective response to various types of stress; however, the process can also be highly selective. Autophagy is involved in various aspects of cell physiology, and its dysregulation is associated with a range of diseases. The regulation of autophagy is complex, and the process must be properly modulated to maintain cellular homeostasis. In this review, we focus on the current state of knowledge concerning transcriptional, post-transcriptional, and post-translational regulation of autophagy in yeast and mammals. PMID:25759175

  4. 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.

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

    DOE PAGESBeta

    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

  6. The exploration of network motifs as potential drug targets from post-translational regulatory networks

    PubMed Central

    Zhang, Xiao-Dong; Song, Jiangning; Bork, Peer; Zhao, Xing-Ming

    2016-01-01

    Phosphorylation and proteolysis are among the most common post-translational modifications (PTMs), and play critical roles in various biological processes. More recent discoveries imply that the crosstalks between these two PTMs are involved in many diseases. In this work, we construct a post-translational regulatory network (PTRN) consists of phosphorylation and proteolysis processes, which enables us to investigate the regulatory interplays between these two PTMs. With the PTRN, we identify some functional network motifs that are significantly enriched with drug targets, some of which are further found to contain multiple proteins targeted by combinatorial drugs. These findings imply that the network motifs may be used to predict targets when designing new drugs. Inspired by this, we propose a novel computational approach called NetTar for predicting drug targets using the identified network motifs. Benchmarking results on real data indicate that our approach can be used for accurate prediction of novel proteins targeted by known drugs. PMID:26853265

  7. Post-translational modifications as key regulators of TNF-induced necroptosis.

    PubMed

    Liu, X; Shi, F; Li, Y; Yu, X; Peng, S; Li, W; Luo, X; Cao, Y

    2016-01-01

    Necroptosis is a novel form of programmed cell death that is independent of caspase activity. Different stimuli can trigger necroptosis. At present, the most informative studies about necroptosis derive from the tumor necrosis factor (TNF)-triggered system. The initiation of TNF-induced necroptosis requires the kinase activity of receptor-interacting protein 1 and 3 (RIP1 and RIP3). Evidence now reveals that the ability of RIP1 and RIP3 to modulate this key cellular event is tightly controlled by post-translational modifications, including ubiquitination, phosphorylation, caspase 8-mediated cleavage and GlcNAcylation. These regulatory events coordinately determine whether a cell will survive or die by apoptosis or necroptosis. In this review, we highlight recent advances in the study of post-translational modifications during TNF-induced necroptosis and discuss how these modifications regulate the complex and delicate control of programmed necrosis. PMID:27383048

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

    PubMed

    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

  9. 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

  10. The exploration of network motifs as potential drug targets from post-translational regulatory networks.

    PubMed

    Zhang, Xiao-Dong; Song, Jiangning; Bork, Peer; Zhao, Xing-Ming

    2016-01-01

    Phosphorylation and proteolysis are among the most common post-translational modifications (PTMs), and play critical roles in various biological processes. More recent discoveries imply that the crosstalks between these two PTMs are involved in many diseases. In this work, we construct a post-translational regulatory network (PTRN) consists of phosphorylation and proteolysis processes, which enables us to investigate the regulatory interplays between these two PTMs. With the PTRN, we identify some functional network motifs that are significantly enriched with drug targets, some of which are further found to contain multiple proteins targeted by combinatorial drugs. These findings imply that the network motifs may be used to predict targets when designing new drugs. Inspired by this, we propose a novel computational approach called NetTar for predicting drug targets using the identified network motifs. Benchmarking results on real data indicate that our approach can be used for accurate prediction of novel proteins targeted by known drugs. PMID:26853265

  11. Post-Translational Modifications of Nucleosomal Histones in Oligodendrocyte Lineage Cells in Development and Disease

    PubMed Central

    Shen, Siming; Casaccia-Bonnefil, Patrizia

    2008-01-01

    The role of epigenetics in modulating gene expression in the development of organs and tissues and in disease states is becoming increasingly evident. Epigenetics refers to the several mechanisms modulating inheritable changes in gene expression that are independent of modifications of the primary DNA sequence and include post-translational modifications of nucleosomal histones, changes in DNA methylation, and the role of microRNA. This review focuses on the epigenetic regulation of gene expression in oligodendroglial lineage cells. The biological effects that post-translational modifications of critical residues in the N-terminal tails of nucleosomal histones have on oligodendroglial cells are reviewed, and the implications for disease and repair are critically discussed. PMID:17999198

  12. 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

  13. Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

    PubMed Central

    Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

    2012-01-01

    Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

  14. Post-translational control of protein function with light using a LOV-intein fusion protein.

    PubMed

    Jones, D C; Mistry, I N; Tavassoli, A

    2016-04-01

    Methods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells. PMID:26940144

  15. High-throughput mass spectrometric discovery of protein post-translational modifications.

    PubMed

    Wilkins, M R; Gasteiger, E; Gooley, A A; Herbert, B R; Molloy, M P; Binz, P A; Ou, K; Sanchez, J C; Bairoch, A; Williams, K L; Hochstrasser, D F

    1999-06-11

    The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a

  16. Collagen prolyl3-hydroxylation: a major role for a minor post-translational modification?

    PubMed Central

    Hudson, David M.; Eyre, David R.

    2014-01-01

    Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap−/− mouse was missing 3-hydroxyproline in type I collagen α-chains. This finding led to the discovery mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzymes have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent. PMID:23772978

  17. Post-translationally-modified structures in the autophagy machinery: an integrative perspective.

    PubMed

    Popelka, Hana; Klionsky, Daniel J

    2015-09-01

    Autophagy is a self-cleaning process that occurs at a constitutive basal level, and is upregulated in response to stress. Macroautophagy (hereafter autophagy) is the most robust type of autophagy, where cargo (specific or nonspecific) is engulfed within a double-membrane structure termed an autophagosome. This process needs to be tightly regulated to maintain normal cellular homeostasis and prevent dysfunction; therefore, a fuller knowledge of the mechanisms of autophagy regulation is crucial for understanding the entire pathway. The autophagy-related proteins are the primary components that carry out autophagy. Many of these proteins are conserved from yeast to humans. A number of significant discoveries with regard to protein functional domains, protein-protein interactions or post-translational modifications of proteins involved in autophagy have been reported in parallel with, or followed by, solving the NMR or crystal structures of autophagy proteins or their protein domains. In the present review, we summarize structural insights gathered to date on the proteins of the autophagy machinery that are modulated by a post-translational modification, specifically phosphorylation, acetylation, ubiquitination and/or SUMOylation. For each protein, we link the reported results with information on the propensity of the corresponding amino acid sequence toward order/disorder. This integrative approach yields a comprehensive overview for each post-translationally modified protein, and also reveals areas for further investigation. PMID:26108642

  18. 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

  19. T Cell Epitopes and Post-Translationally Modified Epitopes in Type 1 Diabetes

    PubMed Central

    McGinty, John W.; Marré, Meghan L.; Bajzik, Veronique; Piganelli, Jon D.; James, Eddie A.

    2016-01-01

    Type 1 diabetes (T1D) is an autoimmune disease in which progressive loss of self-tolerance, evidenced by accumulation of auto-antibodies and auto-reactive T cells that recognize diverse self-proteins, leads to immune-mediated destruction of pancreatic beta cells and loss of insulin secretion. In this review, we discuss antigens and epitopes in T1D and the role that post-translational modifications play in circumventing tolerance mechanisms and increasing antigenic diversity. Emerging data suggest that, analogous to other autoimmune diseases such as rheumatoid arthritis and celiac disease, enzymatically modified epitopes are preferentially recognized in T1D. Modifying enzymes such as peptidyl deiminases and tissue transglutaminase are activated in response to beta cell stress, providing a mechanistic link between post-translational modification and interactions with the environment. Although studies of such responses in the at-risk population have been limited, current data suggests that breakdown in tolerance through post-translational modification represents an important checkpoint in the development of T1D. PMID:26370701

  20. Distinct representations and theta dynamics in dorsal and ventral hippocampus

    PubMed Central

    Royer, Sébastien; Sirota, Anton; Patel, Jagdish; Buzsáki, György

    2010-01-01

    Although anatomical, lesion and imaging studies of the hippocampus indicate qualitatively different information processing along its septo-temporal axis, physiological mechanisms supporting such distinction are missing. We found fundamental differences between the dorsal (dCA3) and the ventral-most parts (vCA3) of the hippocampus in both environmental representation and temporal dynamics. Discrete place fields of dCA3 neurons evenly covered all parts of the testing environments. In contrast, vCA3 neurons i) rarely showed continuous two-dimensional place fields, ii) differentiated open and closed arms of a radial maze, and iii) discharged similar firing patterns with respect to the goals, both on multiple arms of a radial maze and during opposite journeys in a zig-zag maze. In addition, theta power and the fraction of theta-rhythmic neurons were substantially reduced in the ventral as compared to dorsal hippocampus. We hypothesize that the spatial representation in the septo-temporal axis of the hippocampus is progressively decreased. This change is paralleled with a reduction of theta rhythm and an increased representation of non-spatial information. PMID:20130187

  1. Structural Studies and the Assembly of the Heptameric Post-translational Translocon Complex

    SciTech Connect

    Harada, Y.; Li, H.; Li, H.; Wall, J. S.; Lennarz, W. J.

    2011-01-28

    In Saccharomyces cerevisiae, some of the nascent chains can be post-translationally translocated into the endoplasmic reticulum through the heptameric post-translational translocon complex (post-translocon). This membrane-protein complex is composed of the protein-conducting channel and the tetrameric Sec62/63 complex. The Sec62/63 complex plays crucial roles in targeting of the signal recognition particle-independent protein substrate to the protein-conducting channel and in assembly of the post-translocon. Although the molecular mechanism of the post-translational translocation process has been well established, the structure of the post-translocon and how the channel and the Sec62/63 complex form the heptameric complex are largely uncharacterized. Here, we report a 20-{angstrom} resolution cryo-electron microscopy structure of the post-translocon. The purified post-translocon was found to have a mass of 287 kDa, which is consistent with the unit stoichiometry of the seven subunits as determined by a cysteine labeling experiment. We demonstrated that Triton X-100 dissociated the heptameric complex into three subcomplexes identified as the trimeric translocon Sec61-Sbh1-Sss1, the Sec63-Sec71-Sec72 trimer, and the heterotetramer Sec62-Sec63-Sec71-Sec72, respectively. Additionally, a role of the sixth cytosolic loop of Sec61 in assembly of the post-translocon was demonstrated. Mutations of conserved, positively charged amino acid residues in the loop caused decreased formation of the post-translocon. These studies provide the first architectural description of the yeast post-translocon.

  2. 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

  3. 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

  4. 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. PMID:26952777

  5. Vimentin and post-translational modifications in cell motility during cancer - a review.

    PubMed

    Shi, A-M; Tao, Z-Q; Li, R; Wang, Y-Q; Wang, X; Zhao, J

    2016-07-01

    The post-translational modifications (PTMs) are defined as the covalent modification or enzymatic modification of proteins during or after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling. Moreover, it is a known fact that PTM regulation offers an immense array and depth of regulatory possibilities. The present review article will focus on their possible role in cancer cell motility with special reference to vimentin, an intermediate filament (IF), as the later is an important process responsible for life-threatening state viz. cancer metastasis. PMID:27383311

  6. Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease*

    PubMed Central

    Mathias, Rommel A.; Guise, Amanda J.; Cristea, Ileana M.

    2015-01-01

    Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous, and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal histone deacetylase functions by regulating subcellular localization, transcriptional functions, and cell cycle-dependent activity, ultimately impacting on human disease. We discuss the contribution of these modifications to cardiac and vascular hypertrophy, myoblast differentiation, neuronal cell survival, and neurodegenerative disorders. PMID:25616866

  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. Modulation of Intrinsically Disordered Protein Function by Post-translational Modifications.

    PubMed

    Bah, Alaji; Forman-Kay, Julie D

    2016-03-25

    Post-translational modifications (PTMs) produce significant changes in the structural properties of intrinsically disordered proteins (IDPs) by affecting their energy landscapes. PTMs can induce a range of effects, from local stabilization or destabilization of transient secondary structure to global disorder-to-order transitions, potentially driving complete state changes between intrinsically disordered and folded states or dispersed monomeric and phase-separated states. Here, we discuss diverse biological processes that are dependent on PTM regulation of IDPs. We also present recent tools for generating homogenously modified IDPs for studies of PTM-mediated IDP regulatory mechanisms. PMID:26851279

  9. Dynamic melody recognition: distinctiveness and the role of musical expertise.

    PubMed

    Bailes, Freya

    2010-07-01

    The hypothesis that melodies are recognized at moments when they exhibit a distinctive musical pattern was tested. In a melody recognition experiment, point-of-recognition (POR) data were gathered from 32 listeners (16 musicians and 16 nonmusicians) judging 120 melodies. A series of models of melody recognition were developed, resulting from a stepwise multiple regression of two classes of information relating to melodic familiarity and melodic distinctiveness. Melodic distinctiveness measures were assembled through statistical analyses of over 15,000 Western themes and melodies. A significant model, explaining 85% of the variance, entered measures primarily of timing distinctiveness and pitch distinctiveness, but excluding familiarity, as predictors of POR. Differences between nonmusician and musician models suggest a processing shift from momentary to accumulated information with increased exposure to music. Supplemental materials for this article may be downloaded from http://mc.psychonomic-journals.org/content/supplemental. PMID:20551343

  10. Protein Phosphatase 1 (PP1) Is a Post-Translational Regulator of the Mammalian Circadian Clock

    PubMed Central

    Schmutz, Isabelle; Wendt, Sabrina; Schnell, Anna; Kramer, Achim; Mansuy, Isabelle M.; Albrecht, Urs

    2011-01-01

    Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock. PMID:21712997

  11. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications.

    PubMed

    Serrano-Gomez, Silvia Juliana; Maziveyi, Mazvita; Alahari, Suresh K

    2016-01-01

    The epithelial to mesenchymal transition (EMT) is a biological process in which a non-motile epithelial cell changes to a mesenchymal phenotype with invasive capacities. This phenomenon has been well documented in multiple biological processes including embryogenesis, fibrosis, tumor progression and metastasis. The hallmark of EMT is the loss of epithelial surface markers, most notably E-cadherin, and the acquisition of mesenchymal markers including vimentin and N-cadherin. The downregulation of E-cadherin during EMT can be mediated by its transcriptional repression through the binding of EMT transcription factors (EMT-TFs) such as SNAIL, SLUG and TWIST to E-boxes present in the E-cadherin promoter. Additionally, EMT-TFs can also cooperate with several enzymes to repress the expression of E-cadherin and regulate EMT at the epigenetic and post- translational level. In this review, we will focus on epigenetic and post- translational modifications that are important in EMT. In addition, we will provide an overview of the various therapeutic approaches currently being investigated to undermine EMT and hence, the metastatic progression of cancer as well. PMID:26905733

  12. Post-translational modification of the pyruvate phosphate dikinase from Trypanosoma cruzi.

    PubMed

    González-Marcano, Eglys; Mijares, Alfredo; Quiñones, Wilfredo; Cáceres, Ana; Concepción, Juan Luis

    2014-02-01

    In kinetoplastids such as Trypanosoma cruzi, glycolysis is compartmentalized in peroxisome-like organelles called glycosomes. Pyruvate phosphate dikinase (PPDK), an auxiliary enzyme of glycolysis, is also located in the glycosomes. We have detected that this protein is post-translationally modified by phosphorylation and proteolytic cleavage. On western blots of T. cruzi epimastigotes, two PPDK forms were found with apparent MW of 100 kDa and 75 kDa, the latter one being phosphorylated at Thr481, a residue present in a highly conserved region. In subcellular localization assays the 75 kDa PPDK was located peripherally at the glycosomal membrane. Both PPDK forms were found in all life-cycle stages of the parasite. When probing for both PPDK forms during a growth of epimastigotes in batch culture, an increase in the level of the 75 kDa form and a decrease of the 100 kDa one were observed by western blot analysis, signifying that glucose starvation and the concomitant switch of the metabolism to amino acid catabolism may play a role in the post-translational processing of the PPDK. Either one or both of the processes, phosphorylation and proteolytic cleavage of PPDK, result in inactivation of the enzyme. It remains to be established whether the phenomenon exerts a regulatory function. PMID:24060543

  13. Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties

    PubMed Central

    Cancellotti, Enrico; Mahal, Sukhvir P; Somerville, Robert; Diack, Abigail; Brown, Deborah; Piccardo, Pedro; Weissmann, Charles; Manson, Jean C

    2013-01-01

    The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrPSc, an abnormal conformer of the host glycoprotein PrPC. TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrPSc with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrPSc was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains. PMID:23395905

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

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.08697.001 PMID:26408961

  15. Sonic hedgehog multimerization: a self-organizing event driven by post-translational modifications?

    PubMed

    Koleva, Mirella V; Rothery, Stephen; Spitaler, Martin; Neil, Mark A A; Magee, Anthony I

    2015-01-01

    Sonic hedgehog (Shh) is a morphogen active during vertebrate development and tissue homeostasis in adulthood. Dysregulation of the Shh signalling pathway is known to incite carcinogenesis. Due to the highly lipophilic nature of this protein imparted by two post-translational modifications, Shh's method of transit through the aqueous extracellular milieu has been a long-standing conundrum, prompting the proposition of numerous hypotheses to explain the manner of its displacement from the surface of the producing cell. Detection of high molecular-weight complexes of Shh in the intercellular environment has indicated that the protein achieves this by accumulating into multimeric structures prior to release from producing cells. The mechanism of assembly of the multimers, however, has hitherto remained mysterious and contentious. Here, with the aid of high-resolution optical imaging and post-translational modification mutants of Shh, we show that the C-terminal cholesterol and the N-terminal palmitate adducts contribute to the assembly of large multimers and regulate their shape. Moreover, we show that small Shh multimers are produced in the absence of any lipid modifications. Based on an assessment of the distribution of various dimensional characteristics of individual Shh clusters, in parallel with deductions about the kinetics of release of the protein from the producing cells, we conclude that multimerization is driven by self-assembly underpinned by the law of mass action. We speculate that the lipid modifications augment the size of the multimolecular complexes through prolonging their association with the exoplasmic membrane. PMID:26312641

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

    PubMed

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

    2016-05-01

    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

  17. 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.

  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. S-Nitrosylation: Specificity, Occupancy, and Interaction with Other Post-Translational Modifications

    PubMed Central

    Kohr, Mark J.; Murphy, Elizabeth

    2013-01-01

    Abstract Significance: S-nitrosylation (SNO) has been identified throughout the body as an important signaling modification both in physiology and a variety of diseases. SNO is a multifaceted post-translational modification, in that it can either act as a signaling molecule itself or as an intermediate to other modifications. Recent Advances and Critical Issues: Through extensive SNO research, we have made progress toward understanding the importance of single cysteine-SNO sites; however, we are just beginning to explore the importance of specific SNO within the context of other SNO sites and post-translational modifications. Additionally, compartmentalization and SNO occupancy may play an important role in the consequences of the SNO modification. Future Directions: In this review, we will consider the context of SNO signaling and discuss how the transient nature of SNO, its role as an oxidative intermediate, and the pattern of SNO, should be considered when determining the impact of SNO signaling. Antioxid. Redox Signal. 19, 1209–1219. PMID:23157187

  20. 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.

  1. Post-translationally modified tubulins in Artemia: prelarval development in the absence of detyrosinated tubulin.

    PubMed

    Langdon, C M; Freeman, J A; MacRae, T H

    1991-11-01

    The synthesis of post-translationally modified tubulins was examined during Artemia development. Tubulin, either purified to homogeneity or in cell-free extracts, was blotted to nitrocellulose and probed with a panel of antibodies. When purified tubulin was examined, tyrosinated tubulin underwent a large decrease as development progressed and this was accompanied by the appearance of detyrosinated tubulin in samples from organisms developed 24 hr. The inclusion of carboxypeptidase inhibitors had a small effect on the relative amounts of tyrosinated and detyrosinated tubulins in 24-hr preparations. The amount of alpha- and beta-tubulin in cell-free extracts of Artemia either remained relatively constant during development or increased slightly. The same result was obtained for acetylated and tyrosinated tubulin. Detyrosinated tubulin first appeared in 24-hr cell-free extracts and was only post-translationally modified tubulin to increase, relative to the total amount of tubulin, as the brine shrimp developed. As revealed by immunofluorescence staining, detyrosinated tubulin occurred in many cell types of developing nauplii and was prominently displayed in mitotic figures. Artemia, a complex metazoan animal, is thus able to grow for an extended period of time in the absence of detyrosinated tubulin. This isoform is however, synthesized in early larvae and may be required for the development of elongated cells including those which encircle the gut. Detyrosination remains as the only developmentally related change observed for brine shrimp tubulin. PMID:1936554

  2. Non-enzymatic post-translational protein modifications and proteostasis network deregulation in carcinogenesis.

    PubMed

    Trougakos, Ioannis P; Sesti, Fabiola; Tsakiri, Eleni; Gorgoulis, Vassilis G

    2013-10-30

    Organisms are constantly challenged by stressors and thus the maintenance of biomolecules functionality is essential for the assurance of cellular homeostasis. Proteins carry out the vast majority of cellular functions by mostly participating in multimeric protein assemblies that operate as protein machines. Cells have evolved a complex proteome quality control network for the rescue, when possible, or the degradation of damaged polypeptides. Nevertheless, despite these proteostasis ensuring mechanisms, new protein synthesis, and the replication-mediated dilution of proteome damage in mitotic cells, the gradual accumulation of stressors during aging (or due to lifestyle) results in increasingly damaged proteome. Non-enzymatic post-translational protein modifications mostly arise by unbalanced redox homeostasis and/or high glucose levels and may cause disruption of proteostasis as they can alter protein function. This outcome may then increase genomic instability due to reduced fidelity in processes like DNA replication or repair. Herein, we present a synopsis of the major non-enzymatic post-translation protein modifications and of the proteostasis network deregulation in carcinogenesis. We propose that activation of the proteostasis ensuring mechanisms in premalignant cells has tumor-preventive effects, whereas considering that over-activation of these mechanisms represents a hallmark of advanced tumors, their inhibition provides a strategy for the development of anti-tumor therapies. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. PMID:23500136

  3. Investigation of splicing changes and post-translational processing of LMNA in sporadic inclusion body myositis.

    PubMed

    Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell; Fabian, Vicki; Needham, Merrilee; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-01-01

    Some features of sporadic inclusion body myositis (s-IBM) suggest that there is acceleration of the normal ageing process in muscle tissue. LMNA encodes the nuclear lamina proteins lamin A/C through alternative splicing, and aberrant splicing of exon 11 leads to the premature ageing disease, Hutchinson-Gilford progeria syndrome. Progerin, the pathogenic isoform expressed in HGPS tissues, has also been detected at low levels in tissues of normal individuals with aging. We therefore investigated the alternative splicing of LMNA gene transcripts, and the post-translational processing of prelamin A, in s-IBM and control muscle samples. Age-related low level expression of the progerin transcript was detected in both s-IBM and control muscles, but was not increased in s-IBM and there was no increase in progerin protein or demonstrable accumulation of intermediate prelamin isoforms in the s-IBM muscles. However, an age-related shift in the balance of splicing towards lamin A-related transcripts, which was present in normal muscles, was not found in s-IBM. Our findings indicate that while there are changes in the patterns of LMNA splicing in s-IBM muscle which are probably secondary to the underlying pathological process, it is unlikely that aberrant splicing of exon 11 or defective post-translational processing of prelamin A are involved in the pathogenesis of the disease. PMID:24040437

  4. 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. PMID:27181949

  5. Phycobiliprotein biosynthesis in cyanobacteria: structure and function of enzymes involved in post-translational modification.

    PubMed

    Schluchter, Wendy M; Shen, Gaozhong; Alvey, Richard M; Biswas, Avijit; Saunée, Nicolle A; Williams, Shervonda R; Mille, Crystal A; Bryant, Donald A

    2010-01-01

    Cyanobacterial phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at beta-72, which occurs on the beta-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to phycobiliproteins as well as the Asn methyl transferase for beta-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies. PMID:20532743

  6. Reversible Post-Translational Carboxylation Modulates The Enzymatic Activity Of N-Acetyl-L-Ornithine Transcarbamylase†

    PubMed Central

    Li, Yongdong; Yu, Xiaolin; Ho, Jeremy; Fushman, David; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang

    2010-01-01

    N-acetyl-L-ornithine transcarbamylase (AOTCase), rather than ornithine transcarbamylase (OTCase), is the essential carbamylase enzyme in the arginine biosynthesis of several plant and human pathogens. The specificity of this unique enzyme provides a potential target for controlling the spread of these pathogens. Recently, several crystal structures of AOTCase from Xanthomonas campestris (xc) have been determined. In these structures, an unexplained electron density at the tip of Lys302 side-chain was observed. Using 13C NMR spectroscopy, we show herein that Lys302 is post-translationally carboxylated. The structure of wild-type AOTCase complexed with the bisubstrate analogue, Nδ-(phosphonoacetyl)-Nα-acetyl-L-ornithine (PALAO), indicates that the carboxyl group on Lys302 forms a strong hydrogen bonding network with surrounding active site residues, Lys252, Ser253, His293, and Glu92 from the adjacent subunit either directly or via a water molecule. Furthermore, the carboxyl group is involved in binding N-acetyl-L-ornithine via a water molecule. Activity assays with the wild-type enzyme and several mutants demonstrate that the post translational modification of lysine 302 has an important role in catalysis. PMID:20695527

  7. 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

  8. 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

  9. 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, Weijun; 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.

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

    DOE PAGESBeta

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; Meng, Da; Zhao, Rui; Tolić, Nikola; Cao, Li; Shukla, Anil; Monroe, Matthew E.; Moore, Ronald J.; et al

    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

  11. 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

  12. Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly.

    PubMed

    Sun, Qing; Chen, Qi; Blackstock, Daniel; Chen, Wilfred

    2015-08-25

    Context driven biosensor assembly with modular targeting and detection moieties is gaining significant attentions. Although protein-based nanoparticles have emerged as an excellent platform for biosensor assembly, current strategies of decorating bionanoparticles with targeting and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. Herein, we report a highly modular post-translational modification approach for biosensor assembly based on sortase A-mediated ligation. This approach enables the simultaneous modifications of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA aptamer, and dye decoration. The resulting easy-purification platform offers a high degree of targeting and detection modularity with signal amplification. This flexibility is demonstrated for the detection of both immobilized antigens and cancer cells. PMID:26235232

  13. Post-translational control of RIPK3 and MLKL mediated necroptotic cell death

    PubMed Central

    2015-01-01

    Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis. PMID:27158445

  14. Protein deacetylation by SIRT1: an emerging key post-translational modification in metabolic regulation

    PubMed Central

    Yu, Jiujiu; Auwerx, Johan

    2013-01-01

    The biological function of most proteins relies on reversible post-translational modifications, among which phosphorylation is most prominently studied and well recognized. Recently, a growing amount of evidence indicates that acetylation-deacetylation reactions, when applied to crucial mediators, can also robustly affect the function of target proteins and thereby have wide-ranging physiological impacts. Sirtuin 1 (SIRT1), which functions as a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, deacetylates a wide variety of metabolic molecules in response to the cellular energy and redox status and as such causes significant changes in metabolic homeostasis. This review surveys the evidence for the emerging role of SIRT1-mediated deacetylation in the control of metabolic homeostasis. PMID:20026274

  15. The role of post-translational modifications in hearing and deafness.

    PubMed

    Mateo Sánchez, Susana; Freeman, Stephen D; Delacroix, Laurence; Malgrange, Brigitte

    2016-09-01

    Post-translational modifications (PTMs) are key molecular events that modify proteins after their synthesis and modulate their ultimate functional properties by affecting their stability, localisation, interaction potential or activity. These chemical changes expand the size of the proteome adding diversity to the molecular pathways governing the biological outcome of cells. PTMs are, thus, crucial in regulating a variety of cellular processes such as apoptosis, proliferation and differentiation and have been shown to be instrumental during embryonic development. In addition, alterations in protein PTMs have been implicated in the pathogenesis of many human diseases, including deafness. In this review, we summarize the recent progress made in understanding the roles of PTMs during cochlear development, with particular emphasis on the enzymes driving protein phosphorylation, acetylation, methylation, glycosylation, ubiquitination and SUMOylation. We also discuss how these enzymes may contribute to hearing impairment and deafness. PMID:27147466

  16. 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

  17. Post-translational oxidative modification of fibrinogen is associated with coagulopathy after traumatic injury.

    PubMed

    White, Nathan J; Wang, Yi; Fu, Xiaoyun; Cardenas, Jessica C; Martin, Erika J; Brophy, Donald F; Wade, Charles E; Wang, Xu; St John, Alexander E; Lim, Esther B; Stern, Susan A; Ward, Kevin R; López, José A; Chung, Dominic

    2016-07-01

    Victims of trauma often develop impaired blood clot formation (coagulopathy) that contributes to bleeding and mortality. Fibrin polymerization is one critical component of clot formation that can be impacted by post-translational oxidative modifications of fibrinogen after exposure to oxidants. In vitro evidence suggests that Aα-C domain methionine sulfoxide formation, in particular, can induce conformational changes that prevent lateral aggregation of fibrin protofibrils during polymerization. We used mass spectrometry of plasma from trauma patients to find that fibrinogen Aα-C domain methionine sulfoxide content was selectively-increased in patients with coagulopathy vs. those without coagulopathy. This evidence supports a novel linkage between oxidative stress, coagulopathy, and bleeding after injury. PMID:27105953

  18. 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

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

    PubMed

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

    2015-11-01

    The investigation of post-translational modifications (PTMs) represents one of the main research focuses for the study of protein function and cell signaling. Mass spectrometry instrumentation with increasing sensitivity improved protocols for PTM enrichment and recently established pipelines for high-throughput experiments allow large-scale identification and quantification of several PTM types. This review addresses the concurrently emerging challenges for the computational analysis of the resulting data and presents PTM-centered approaches for spectra identification, statistical analysis, multivariate analysis and data interpretation. We furthermore discuss the potential of future developments that will help to gain deep insight into the PTM-ome and its biological role in cells. This article is part of a Special Issue entitled: Computational Proteomics. PMID:26216596

  20. [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. PMID:26351162

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

    PubMed

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

    2014-01-31

    The post-translational modification (PTM) of proteins plays a critical role in the regulation of a broad range of cellular processes in eukaryotes. Yet their role in governing similar systems in the conventionally presumed 'simpler' forms of life has been largely neglected and, until recently, was thought to occur only rarely, with some modifications assumed to be limited to higher organisms alone. Recent developments in mass spectrometry-based proteomics have provided an unparalleled power to enrich, identify and quantify peptides with PTMs. Additional modifications to biological molecules such as lipids and carbohydrates that are essential for bacterial pathophysiology have only recently been detected on proteins. Here we review bacterial protein PTMs, focusing on phosphorylation, acetylation, proteolytic degradation, methylation and lipidation and the roles they play in bacterial adaptation - thus highlighting the importance of proteomic techniques in a field that is only just in its infancy. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. PMID:23994099

  2. 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.

  3. The Emerging Immunological Role of Post-Translational Modifications by Reactive Nitrogen Species in Cancer Microenvironment

    PubMed Central

    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. PMID:24605112

  4. Quantitation of protein post-translational modifications using isobaric tandem mass tags.

    PubMed

    Liang, Hui-Chung; Lahert, Emma; Pike, Ian; Ward, Malcolm

    2015-01-01

    Post-translational modifications (PTMs) of proteins are known to modulate many cellular processes and their qualitative and quantitative evaluation is fundamental for understanding the mechanisms of biological events. Over the past decade, improvements in sample preparation techniques and enrichment strategies, the development of quantitative labeling strategies, the launch of a new generation of mass spectrometers and the creation of bioinformatics tools for the interrogation of ever larger datasets has established MS-based quantitative proteomics as a powerful workflow for global proteomics, PTM analysis and the elucidation of key biological mechanisms. With the advantage of their multiplexing capacity and the flexibility of an ever-growing family of different peptide-reactive groups, isobaric tandem mass tags facilitate quantitative proteomics and PTM experiments and enable higher sample throughput. In this review, we focus on the technical concept and utility of the isobaric tandem mass tag labeling approach to PTM analysis, including phosphorylation, glycosylation and S-nitrosylation. PMID:25697195

  5. Post-Translational Decrease in Respiratory Chain Proteins in the Polg Mutator Mouse Brain

    PubMed Central

    Hauser, David N.; Dillman, Allissa A.; Ding, Jinhui; Li, Yan; Cookson, Mark R.

    2014-01-01

    Mitochondrial DNA damage is thought to be a causal contributor to aging as mice with inactivating mutations in polymerase gamma (Polg) develop a progeroid phenotype. To further understand the molecular mechanisms underlying this phenotype, we used iTRAQ and RNA-Seq to determine differences in protein and mRNA abundance respectively in the brains of one year old Polg mutator mice compared to control animals. We found that mitochondrial respiratory chain proteins are specifically decreased in abundance in the brains of the mutator mice, including several nuclear encoded mitochondrial components. However, we found no evidence that the changes we observed in protein levels were the result of decreases in mRNA expression. These results show that there are post-translational effects associated with mutations in Polg. PMID:24722488

  6. Post-translational cleavage and self-interaction of the phytoplasma effector SAP11.

    PubMed

    Lu, Yen-Ting; Cheng, Kai-Tan; Jiang, Shin-Ying; Yang, Jun-Yi

    2014-04-28

    Phytoplasmas are insect-transmitted intracellular plant bacterial pathogens that secrete effector molecules into host cells that interfere with the host's developmental or metabolic processes. Recently, the secreted Aster Yellows phytoplasma strain Witches' Broom protein11 (SAP11) has been shown to act as a virulence factor that alters the development, hormone biosynthesis, phosphate (Pi) homeostasis, and defense responses in the affected plants. We found that SAP11 undergoes proteolytic processing in planta and self-interaction in vitro. These biochemical studies provide foundational insights necessary for the functional characterization of SAP11; however, the biological relevance of post-translational cleavage and self-interaction of SAP11 to its role as a virulence factor warrants further investigation. PMID:24776784

  7. Regulation of post-translational modifications of muskelin by protein kinase C.

    PubMed

    Prag, Soren; De Arcangelis, Adèle; Georges-Labouesse, Elisabeth; Adams, Josephine C

    2007-01-01

    Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface. PMID:17049906

  8. 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

  9. 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

  10. Host-Mediated Post-Translational Prenylation of Novel Dot/Icm-Translocated Effectors of Legionella Pneumophila

    PubMed Central

    Price, Christopher T. D.; Jones, Snake C.; Amundson, Karen E.; Kwaik, Yousef Abu

    2010-01-01

    The Dot/Icm type IV translocated Ankyrin B (AnkB) effector of Legionella pneumophila is modified by the host prenylation machinery that anchors it into the outer leaflet of the Legionella-containing vacuole (LCV), which is essential for biological function of the effector in vitro and in vivo. Prenylation involves the covalent linkage of an isoprenoid lipid moiety to a C-terminal CaaX motif in eukaryotic proteins enabling their anchoring into membranes. We show here that the LCV harboring an ankB null mutant is decorated with prenylated proteins in a Dot/Icm-dependent manner, indicating that other LCV membrane-anchored proteins are prenylated. In silico analyses of four sequenced L. pneumophila genomes revealed the presence of eleven other genes that encode proteins with a C-terminal eukaryotic CaaX prenylation motif. Of these eleven designated Prenylated effectors of Legionella (Pel), seven are also found in L. pneumophila AA100. We show that six L. pneumophila AA100 Pel proteins exhibit distinct cellular localization when ectopically expressed in mammalian cells and this is dependent on action of the host prenylation machinery and the conserved cysteine residue of the CaaX motif. Although inhibition of the host prenylation machinery completely blocks intra-vacuolar proliferation of L. pneumophila, it only had a modest effect on intracellular trafficking of the LCV. Five of the Pel proteins are injected into human macrophages by the Dot/Icm type IV translocation system of L. pneumophila. Taken together, the Pel proteins are novel Dot/Icm-translocated effectors of L. pneumophila that are post-translationally modified by the host prenylation machinery, which enables their anchoring into cellular membranes, and the prenylated effectors contribute to evasion of lysosomal fusion by the LCV. PMID:21687755

  11. Dynamic regulation of macroautophagy by distinctive, ubiquitin-like proteins

    PubMed Central

    Klionsky, Daniel J.; Schulman, Brenda A.

    2014-01-01

    Autophagy complements the ubiquitin-proteasome system in mediating protein turnover. Whereas the proteasome degrades individual proteins modified with ubiquitin chains, autophagy degrades many proteins and organelles en masse. Macromolecules destined for autophagic degradation are “selected” through sequestration within a specialized double-membrane compartment termed the “phagophore”, the precursor to an “autophagosome”, and then hydrolyzed in a lysosome/vacuole-dependent manner. Notably, a pair of distinctive ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate degradation by autophagy in unique ways, by controlling autophagosome biogenesis and recruitment of specific cargos during selective autophagy. Here we review structural mechanisms underlying functions and conjugation of these UBLs that are specialized to provide interaction platforms linked to phagophore membranes. PMID:24699082

  12. Oral cavity contains distinct niches with dynamic microbial communities.

    PubMed

    Xu, Xin; He, Jinzhi; Xue, Jing; Wang, Yan; Li, Kun; Zhang, Keke; Guo, Qiang; Liu, Xianghong; Zhou, Yuan; Cheng, Lei; Li, Mingyun; Li, Yuqing; Li, Yan; Shi, Wenyuan; Zhou, Xuedong

    2015-03-01

    Microbes colonize human oral surfaces within hours after delivery. During postnatal development, physiological changes, such as the eruption of primary teeth and replacement of the primary dentition with permanent dentition, greatly alter the microbial habitats, which, in return, may lead to community composition shifts at different phases in people's lives. By profiling saliva, supragingival and mucosal plaque samples from healthy volunteers at different ages and dentition stages, we observed that the oral cavity is a highly heterogeneous ecological system containing distinct niches with significantly different microbial communities. More importantly, the phylogenetic microbial structure varies with ageing. In addition, only a few taxa were present across the whole populations, indicating a core oral microbiome should be defined based on age and oral niches. PMID:24800728

  13. Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation.

    PubMed

    Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole N

    2016-08-01

    Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12(-/-)), PRC1 (Ring1A/B(-/-)) and (Dnmt1/3a/3b(-/-)) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http

  14. Dynamic functional integration of distinct neural empathy systems

    PubMed Central

    2014-01-01

    Recent evidence points to two separate systems for empathy: a vicarious sharing emotional system that supports our ability to share emotions and mental states and a cognitive system that involves cognitive understanding of the perspective of others. Several recent models offer new evidence regarding the brain regions involved in these systems, but no study till date has examined how regions within each system dynamically interact. The study by Raz et al. in this issue of Social, Cognitive, & Affective Neuroscience is among the first to use a novel approach of functional magnetic resonance imaging analysis of fluctuations in network cohesion while an individual is experiencing empathy. Their results substantiate the approach positing two empathy mechanisms and, more broadly, demonstrate how dynamic analysis of emotions can further our understanding of social behavior. PMID:23956080

  15. A Detailed Map of Oxidative Post-translational Modifications of Human p21ras sing Fourier Transform Mass Spectrometry

    PubMed Central

    Zhao, Cheng; Sethuraman, Mahadevan; Clavreul, Nicolas; Kaur, Parminder; Cohen, Richard A.

    2010-01-01

    P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high resolution and high mass accuracy Fourier Transform Mass Spectrometry (FTMS) was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by “bottom-up” analysis and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, “top-down” analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications especially using top-down analysis. PMID:16841939

  16. Cell specific post-translational processing of pikachurin, a protein involved in retinal synaptogenesis.

    PubMed

    Han, Jianzhong; Townes-Anderson, Ellen

    2012-01-01

    Pikachurin is a recently identified, highly conserved, extracellular matrix-like protein. Murine pikachurin has 1,017 amino acids (~110 kDa), can bind to α-dystroglycan, and has been found to localize mainly in the synaptic cleft of photoreceptor ribbon synapses. Its knockout selectively disrupts synaptogenesis between photoreceptor and bipolar cells. To further characterize this synaptic protein, we used an antibody raised against the N-terminal of murine pikachurin on Western blots of mammalian and amphibian retinas and found, unexpectedly, that a low weight ~60-kDa band was the predominant signal for endogenous pikachurin. This band was predicted to be an N-terminal product of post-translational cleavage of pikachurin. A similar sized protein was also detected in human Y79 retinoblastoma cells, a cell line with characteristics of photoreceptor cells. In Y79 cells, endogenous pikachurin immunofluorescence was found on the cell surface of living cells. The expression of the N-fragment was not significantly affected by dystroglycan overexpression in spite of the biochemical evidence for pikachurin-α-dystroglycan binding. The presence of a corresponding endogenous C-fragment was not determined because of the lack of a suitable antibody. However, a protein of ~65 kDa was detected in Y79 cells expressing recombinant pikachurin with a C-terminal tag. In contrast, in QBI-HEK 293A cells, whose endogenous pikachurin protein level is negligible, recombinant pikachurin did not appear to be cleaved. Instead pikachurin was found either intact or as dimers. Finally, whole and N- and C-fragments of recombinant pikachurin were present in the conditioned media of Y79 cells indicating the secretion of pikachurin. The site of cleavage, however, was not conclusively determined. Our data suggest the existence of post-translational cleavage of pikachurin protein as well as the extracellular localization of cleaved protein specifically by retinal cells. The functions of the

  17. 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

  18. Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo*S

    PubMed Central

    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.

    2007-01-01

    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 β 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 IC50 of ∼3 μm for both nitroalkenes, an IC50 equivalent to the potent thiol oxidant peroxynitrite (ONOO−) and an IC50 30-fold less than H2O2, 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 occurrence of these

  19. A novel post-translational modification of nucleolin, SUMOylation at Lys-294, mediates arsenite-induced cell death by regulating gadd45α mRNA stability.

    PubMed

    Zhang, Dongyun; Liang, Yuguang; Xie, Qipeng; Gao, Guangxun; Wei, Jinlong; Huang, Haishan; Li, Jingxia; Gao, Jimin; Huang, Chuanshu

    2015-02-20

    Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation. PMID:25561743

  20. A Novel Post-translational Modification of Nucleolin, SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability*

    PubMed Central

    Zhang, Dongyun; Liang, Yuguang; Xie, Qipeng; Gao, Guangxun; Wei, Jinlong; Huang, Haishan; Li, Jingxia; Gao, Jimin; Huang, Chuanshu

    2015-01-01

    Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation. PMID:25561743

  1. Dynamic Remodeling of Microbial Biofilms by Functionally Distinct Exopolysaccharides

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. PMID:25096883

  2. Multiple post-translational modifications in hepatocyte nuclear factor 4{alpha}

    SciTech Connect

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

    2011-07-15

    Highlights: {yields} We performed comprehensive PTM analysis for HNF4{alpha} protein. {yields} We identified 8 PTMs in HNF4{alpha} protein including newly identified PTMs. {yields} Among them, we found acetylation at lysine 458 was one of the prime PTMs for HNF4{alpha} function. {yields} Acetylation at lysine 458 was inhibitory for HNF4{alpha} transcription function. {yields} This modification fluctuated in response to extracellular condition. -- Abstract: To investigate the role of post-translational modifications (PTMs) in the hepatocyte nuclear factor 4{alpha} (HNF4{alpha})-mediated transcription, we took a comprehensive survey of PTMs in HNF4{alpha} protein by massspectrometry and identified totally 8 PTM sites including newly identified ubiquitilation and acetylation sites. To assess the impact of identified PTMs in HNF4{alpha}-function, we introduced point mutations at the identified PTM sites and, tested transcriptional activity of the HNF4{alpha}. Among the point-mutations, an acetylation site at lysine 458 was found significant in the HNF4{alpha}-mediated transcriptional control. An acetylation negative mutant at lysine 458 showed an increased transcriptional activity by about 2-fold, while an acetylation mimic mutant had a lowered transcriptional activation. Furthermore, this acetylation appeared to be fluctuated in response to extracellular nutrient conditions. Thus, by applying an comprehensive analysis of PTMs, multiple PTMs were newly identified in HNF4{alpha} and unexpected role of an HNF4{alpha} acetylation could be uncovered.

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

    PubMed Central

    Michard, Céline; Doublet, Patricia

    2015-01-01

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

  4. 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

  5. Protein post-translational modifications and misfolding: new concepts in heart failure.

    PubMed

    Del Monte, Federica; Agnetti, Giulio

    2014-08-01

    A new concept in the field of heart-failure (HF) research points to a role of misfolded proteins, forming preamyloid oligomers (PAOs), in cardiac toxicity. This is largely based on few studies reporting the presence of PAOs, similar to those observed in neurodegenerative diseases, in experimental and human HF. As the majority of proteinopathies are sporadic in nature, protein post-translational modifications (PTMs) likely play a major role in this growing class of diseases. In fact, PTMs are known regulators of protein folding and of the formation of amyloid species in well-established proteinopathies. Proteomics has been instrumental in identifying both chemical and enzymatic PTMs, with a potential impact on protein mis-/folding. Here we provide the basics on how proteins fold along with a few examples of PTMs known to modulate protein misfolding and aggregation, with particular focus on the heart. Due to its innovative content and the growing awareness of the toxicity of misfolded proteins, an "Alzheimer's theory of HF" is timely. Moreover, the continuous innovations in proteomic technologies will help pinpoint PTMs that could contribute to the process. This nuptial between biology and technology could greatly assist in identifying biomarkers with increased specificity as well as more effective therapies. PMID:24946239

  6. Post-Translational Regulation via Clp Protease Is Critical for Survival of Mycobacterium tuberculosis

    PubMed Central

    Raju, Ravikiran M.; Jedrychowski, Mark P.; Wei, Jun-Rong; Pinkham, Jessica T.; Park, Annie S.; O'Brien, Kathryn; Rehren, German; Schnappinger, Dirk; Gygi, Steven P.; Rubin, Eric J.

    2014-01-01

    Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology. PMID:24603869

  7. Post-translational activation introduces a free radical into pyruvate formate-lyase.

    PubMed Central

    Knappe, J; Neugebauer, F A; Blaschkowski, H P; Gänzler, M

    1984-01-01

    Pyruvate formate-lyase (formate acetyltransferase; EC 2.3.1.54) of Escherichia coli cells is post-translationally interconverted between inactive and active forms. Conversion of the inactive to the active form is catalyzed by an Fe2+-dependent activating enzyme and requires adenosylmethionine and dihydroflavodoxin. This process is shown here to introduce a paramagnetic moiety into the structure of pyruvate formate-lyase. It displays an EPR signal at g = 2 with a doublet splitting of 1.5 mT and could comprise an organic free radical located on an amino acid residue of the polypeptide chain. Hypophosphite was discovered as a specific reagent that destroys both the enzyme radical and the enzyme activity; it becomes covalently bound to the protein. The enzymatic generation of the radical, which is linked to adenosylmethionine cleavage into 5'-deoxyadenosine and methionine, possibly occurs through an Fe-adenosyl complex. These results suggest a radical mechanism for the catalytic cycle of pyruvate formate-lyase. PMID:6369325

  8. A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes.

    PubMed

    Karthikeyan, Kailash; Barker, Kristi; Tang, Yanyang; Kahn, Peter; Wiktor, Peter; Brunner, Al; Knabben, Vinicius; Takulapalli, Bharath; Buckner, Jane; Nepom, Gerald; LaBaer, Joshua; Qiu, Ji

    2016-07-01

    Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity. PMID:27141097

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

    PubMed

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

    2016-02-01

    Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC-MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

  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. ProteomeScout: a repository and analysis resource for post-translational modifications and proteins.

    PubMed

    Matlock, Matthew K; Holehouse, Alex S; Naegle, Kristen M

    2015-01-01

    ProteomeScout (https://proteomescout.wustl.edu) is a resource for the study of proteins and their post-translational modifications (PTMs) consisting of a database of PTMs, a repository for experimental data, an analysis suite for PTM experiments, and a tool for visualizing the relationships between complex protein annotations. The PTM database is a compendium of public PTM data, coupled with user-uploaded experimental data. ProteomeScout provides analysis tools for experimental datasets, including summary views and subset selection, which can identify relationships within subsets of data by testing for statistically significant enrichment of protein annotations. Protein annotations are incorporated in the ProteomeScout database from external resources and include terms such as Gene Ontology annotations, domains, secondary structure and non-synonymous polymorphisms. These annotations are available in the database download, in the analysis tools and in the protein viewer. The protein viewer allows for the simultaneous visualization of annotations in an interactive web graphic, which can be exported in Scalable Vector Graphics (SVG) format. Finally, quantitative data measurements associated with public experiments are also easily viewable within protein records, allowing researchers to see how PTMs change across different contexts. ProteomeScout should prove useful for protein researchers and should benefit the proteomics community by providing a stable repository for PTM experiments. PMID:25414335

  12. 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

  13. 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

  14. 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. PMID:24140149

  15. 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

  16. 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

  17. ProteomeScout: a repository and analysis resource for post-translational modifications and proteins

    PubMed Central

    Matlock, Matthew K.; Holehouse, Alex S.; Naegle, Kristen M.

    2015-01-01

    ProteomeScout (https://proteomescout.wustl.edu) is a resource for the study of proteins and their post-translational modifications (PTMs) consisting of a database of PTMs, a repository for experimental data, an analysis suite for PTM experiments, and a tool for visualizing the relationships between complex protein annotations. The PTM database is a compendium of public PTM data, coupled with user-uploaded experimental data. ProteomeScout provides analysis tools for experimental datasets, including summary views and subset selection, which can identify relationships within subsets of data by testing for statistically significant enrichment of protein annotations. Protein annotations are incorporated in the ProteomeScout database from external resources and include terms such as Gene Ontology annotations, domains, secondary structure and non-synonymous polymorphisms. These annotations are available in the database download, in the analysis tools and in the protein viewer. The protein viewer allows for the simultaneous visualization of annotations in an interactive web graphic, which can be exported in Scalable Vector Graphics (SVG) format. Finally, quantitative data measurements associated with public experiments are also easily viewable within protein records, allowing researchers to see how PTMs change across different contexts. ProteomeScout should prove useful for protein researchers and should benefit the proteomics community by providing a stable repository for PTM experiments. PMID:25414335

  18. 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

  19. Post-Translational Modification of Constitutive Nitric Oxide Synthase in the Penis

    PubMed Central

    Musicki, Biljana; Ross, Ashley E.; Champion, Hunter C.; Burnett, Arthur L.; Bivalacqua, Trinity J.

    2009-01-01

    Erectile dysfunction (ED) is a common men's health problem characterized by the consistent inability to sustain an erection sufficient for sexual intercourse. Basic science research on erectile physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin and/or neurogenic dysfunction. The constitutive forms of nitric oxide synthase [NOS; endothelial NOS (eNOS) and neuronal NOS (nNOS)] are important enzymes involved in the production of nitric oxide (NO) and thus regulate penile vascular homeostasis. Given the impact of endothelial- and neuronal-derived NO in penile vascular biology, a great deal of research over the past decade has focused on the role of NO synthesis from the endothelium and nitrergic nerve terminal in normal erectile physiology as well as in disease states. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED. Therefore, molecular mechanisms involved in dysregulation of these NOS isoforms in the development of ED are essential to discovering the pathogenesis of ED in various disease states. This communication reviews the role of eNOS and nNOS in erectile physiology and discusses the alterations in eNOS and nNOS via post-translation modification in various vascular diseases of the penis. PMID:19342700

  20. 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 .

  1. PTMcode: a database of known and predicted functional associations between post-translational modifications in proteins

    PubMed Central

    Minguez, Pablo; Letunic, Ivica; Parca, Luca; Bork, Peer

    2013-01-01

    Post-translational modifications (PTMs) are involved in the regulation and structural stabilization of eukaryotic proteins. The combination of individual PTM states is a key to modulate cellular functions as became evident in a few well-studied proteins. This combinatorial setting, dubbed the PTM code, has been proposed to be extended to whole proteomes in eukaryotes. Although we are still far from deciphering such a complex language, thousands of protein PTM sites are being mapped by high-throughput technologies, thus providing sufficient data for comparative analysis. PTMcode (http://ptmcode.embl.de) aims to compile known and predicted PTM associations to provide a framework that would enable hypothesis-driven experimental or computational analysis of various scales. In its first release, PTMcode provides PTM functional associations of 13 different PTM types within proteins in 8 eukaryotes. They are based on five evidence channels: a literature survey, residue co-evolution, structural proximity, PTMs at the same residue and location within PTM highly enriched protein regions (hotspots). PTMcode is presented as a protein-based searchable database with an interactive web interface providing the context of the co-regulation of nearly 75 000 residues in >10 000 proteins. PMID:23193284

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

    PubMed

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

    2016-08-01

    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. PMID:27291504

  3. 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.

  4. The Multiplicity of Post-Translational Modifications in Pro-Opiomelanocortin-Derived Peptides

    PubMed Central

    Yasuda, Akikazu; Jones, Leslie Sargent; Shigeri, Yasushi

    2013-01-01

    The precursor protein, pro-opiomelanocortin (POMC) undergoes extensive post-translational processing in a tissue-specific manner to yield various biologically active peptides involved in diverse cellular functions. The recently developed method of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for direct tissue analysis has proved to be a powerful tool for investigating the distribution of peptides and proteins. In particular, topological mass spectrometry analysis using MALDI-MS can selectively provide a mass profile of the hormones included in cell secretory granules. An advantage of this technology is that it is possible to analyze a frozen thin slice section, avoiding an extraction procedure. Subsequently, tandem mass spectrometry (MS/MS) has a profound impact on addressing the modified residues in the hormone molecules. Based on these strategies with mass spectrometry, several interesting molecular forms of POMC-derived peptides have been found in the fish pituitary, such as novel sites of acetylation in α-melanocyte-stimulating hormone (MSH), hydroxylation of a proline residue in β-MSH, and the phosphorylated form of corticotropin-like intermediate lobe peptide. PMID:24348461

  5. 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

    2016-01-01

    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

  6. PPARG Post-translational Modifications Regulate Bone Formation and Bone Resorption.

    PubMed

    Stechschulte, L A; Czernik, P J; Rotter, Z C; Tausif, F N; Corzo, C A; Marciano, D P; Asteian, A; Zheng, J; Bruning, J B; Kamenecka, T M; Rosen, C J; Griffin, P R; Lecka-Czernik, B

    2016-08-01

    The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease. PMID:27422345

  7. 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

  8. 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

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

    PubMed

    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

  10. 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

  11. 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

  12. Annotation of post-translational modifications in the Swiss-Prot knowledge base.

    PubMed

    Farriol-Mathis, Nathalie; Garavelli, John S; Boeckmann, Brigitte; Duvaud, Séverine; Gasteiger, Elisabeth; Gateau, Alain; Veuthey, Anne-Lise; Bairoch, Amos

    2004-06-01

    High-throughput proteomic studies produce a wealth of new information regarding post-translational modifications (PTMs). The Swiss-Prot knowledge base is faced with the challenge of including this information in a consistent and structured way, in order to facilitate easy retrieval and promote understanding by biologist expert users as well as computer programs. We are therefore standardizing the annotation of PTM features represented in Swiss-Prot. Indeed, a controlled vocabulary has been associated with every described PTM. In this paper, we present the major update of the feature annotation, and, by showing a few examples, explain how the annotation is implemented and what it means. Mod-Prot, a future companion database of Swiss-Prot, devoted to the biological aspects of PTMs (i.e., general description of the process, identity of the modification enzyme(s), taxonomic range, mass modification) is briefly described. Finally we encourage once again the scientific community (i.e., both individual researchers and database maintainers) to interact with us, so that we can continuously enhance the quality and swiftness of our services. PMID:15174124

  13. Human islets and dendritic cells generate post-translationally modified islet autoantigens.

    PubMed

    McLaughlin, R J; de Haan, A; Zaldumbide, A; de Koning, E J; de Ru, A H; van Veelen, P A; van Lummel, M; Roep, B O

    2016-08-01

    The initiation of type 1 diabetes (T1D) requires a break in peripheral tolerance. New insights into neoepitope formation indicate that post-translational modification of islet autoantigens, for example via deamidation, may be an important component of disease initiation or exacerbation. Indeed, deamidation of islet autoantigens increases their binding affinity to the T1D highest-risk human leucocyte antigen (HLA) haplotypes HLA-DR3/DQ2 and -DR4/DQ8, increasing the chance that T cells reactive to deamidated autoantigens can be activated upon T cell receptor ligation. Here we investigated human pancreatic islets and inflammatory and tolerogenic human dendritic cells (DC and tolDC) as potential sources of deamidated islet autoantigens and examined whether deamidation is altered in an inflammatory environment. Islets, DC and tolDC contained tissue transglutaminase, the key enzyme responsible for peptide deamidation, and enzyme activity increased following an inflammatory insult. Islets treated with inflammatory cytokines were found to contain deamidated insulin C-peptide. DC, heterozygous for the T1D highest-risk DQ2/8, pulsed with native islet autoantigens could present naturally processed deamidated neoepitopes. HLA-DQ2 or -DQ8 homozygous DC did not present deamidated islet peptides. This study identifies both human islets and DC as sources of deamidated islet autoantigens and implicates inflammatory activation of tissue transglutaminase as a potential mechanism for islet and DC deamidation. PMID:26861694

  14. Human Oncogenic Herpesvirus and Post-translational Modifications – Phosphorylation and SUMOylation

    PubMed Central

    Chang, Pei-Ching; Campbell, Mel; Robertson, Erle S.

    2016-01-01

    Pathogens, especially viruses, evolve abilities to utilize cellular machineries to facilitate their survival and propagation. Post-translational modifications (PTMs), especially phosphorylation and SUMOylation, that reversibly modulate the function and interactions of target proteins are among the most important features in cell signaling pathways. PTM-dependent events also serve as one of the favorite targets for viruses. Among the seven unambiguous human oncogenic viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), human papillomavirus (HPV), Human T lymphotrophic virus-1 (HTLV-1), and Merkel cell polyomavirus (MCPyV), two are herpesviruses. The life cycle of herpesviruses consists of latent and lytic phases and the rapid switch between these states includes global remodeling of the viral genome from heterochromatin-to-euchromatin. The balance between lytic replication and latency is essential for herpesvirus to maintain a persistent infection through a combination of viral propagation and evasion of the host immune response, which consequently may contribute to tumorigenesis. It is no surprise that the swift reversibility of PTMs, especially SUMOylation, a modification that epigenetically regulates chromatin structure, is a major hijack target of the host for oncogenic herpesviruses. In this brief review, we summarize the varied ways in which herpesviruses engage the host immune components through PTMs, focusing on phosphorylation and SUMOylation. PMID:27379086

  15. 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

  16. Regulation of molecular chaperones through post-translational modifications: Decrypting the chaperone code

    PubMed Central

    Cloutier, Philippe; Coulombe, Benoit

    2015-01-01

    Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point toward the more ambitious endeavor of deciphering the “chaperone code”. PMID:23459247

  17. Status of Large-scale Analysis of Post-translational Modifications by Mass Spectrometry*

    PubMed Central

    Olsen, Jesper V.; Mann, Matthias

    2013-01-01

    Cellular function can be controlled through the gene expression program, but often protein post-translational modifications (PTMs) provide a more precise and elegant mechanism. Key functional roles of specific modification events—for instance, during the cell cycle—have been known for decades, but only in the past 10 years has mass-spectrometry-(MS)-based proteomics begun to reveal the true extent of the PTM universe. In this overview for the special PTM issue of Molecular and Cellular Proteomics, we take stock of where MS-based proteomics stands in the large-scale analysis of protein modifications. For many PTMs, including phosphorylation, ubiquitination, glycosylation, and acetylation, tens of thousands of sites can now be confidently identified and localized in the sequence of the protein. The quantification of PTM levels between different cellular states is likewise established, with label-free methods showing particular promise. It is also becoming possible to determine the absolute occupancy or stoichiometry of PTM sites on a large scale. Powerful software for the bioinformatic analysis of thousands of PTM sites has been developed. However, a complete inventory of sites has not been established for any PTM, and this situation will persist into the foreseeable future. Furthermore, although PTM coverage by MS-based methods is impressive, it still needs to be improved, especially in tissues and in clinically relevant systems. The central challenge for the field is to develop streamlined methods for determining biological functions for the myriad of modifications now known to exist. PMID:24187339

  18. Functional decorations: post-translational modifications and heart disease delineated by targeted proteomics

    PubMed Central

    2013-01-01

    The more than 300 currently identified post-translational modifications (PTMs) provides great scope for subtle or dramatic alteration of protein structure and function. Furthermore, the rapid and transient nature of many PTMs allows efficient signal transmission in response to internal and environmental stimuli. PTMs are predominantly added by enzymes, and the enzymes responsible (such as kinases) are thus attractive targets for therapeutic interventions. Modifications can be grouped according to their stability or transience (reversible versus irreversible): irreversible types (such as irreversible redox modifications or protein deamidation) are often associated with aging or tissue injury, whereas transient modifications are associated with signal propagation and regulation. This is particularly important in the setting of heart disease, which comprises a diverse range of acute (such as ischemia/reperfusion), chronic (such as heart failure, dilated cardiomyopathy) and genetic (such as hypertrophic cardiomyopathy) disease states, all of which have been associated with protein PTM. Recently the interplay between diverse PTMs has been suggested to also influence cellular function, with cooperation or competition for sites of modification possible. Here we discuss the utility of proteomics for examining PTMs in the context of the molecular mechanisms of heart disease. PMID:23445784

  19. 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.

  20. Post-translational processing of cholecystokinin in pig brain and gut.

    PubMed Central

    Eng, J; Shiina, Y; Straus, E; Yalow, R S

    1982-01-01

    A sequential extraction method employing methanol extraction of the COOH-terminal fragments of cholecystokinin (CCK) from pig tissues followed by HCl extraction of intact CCK and its NH2-terminal fragments is described. Radioimmunoassay of extracts and their fractionation by Sephadex chromatography and HPLC demonstrate that the distributions of COOH-terminal and NH2-terminal immunoreactivities among various regions of brain are similar and independent of the concentrations in individual regions. The distribution in gut differs from that in brain. Greatest concentrations of CCK immunoreactivity are located in cortical tissue in the brain and in duodenal mucosa in gut. Both brain and gut contain CCK octapeptide (CCK8) and an NH2-terminal fragment that is likely to be desoctapeptide-CCK33. Intact CCK33 is extractable from gut but not from brain. Brain contains another NH2-terminal immunoreactive molecule lacking COOH-terminal immunoreactivity that may be a peptide with a COOH-terminal extension, as has been described for gastrin, or one that may not be derived from a CCK33-like precursor. This peptide is much less prominent in gut, or may be nonexistent there. The failure to find CCK33 in the brain and the presence in the brain of this as-yet-uncharacterized NH2-terminal peptide raises the question as to whether the differences between neuronal and mucosal tissues are a consequence of differences in post-translational processing or in the DNA templates. PMID:6964399

  1. 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. PMID:26265041

  2. novPTMenzy: a database for enzymes involved in novel post-translational modifications

    PubMed Central

    Khater, Shradha; Mohanty, Debasisa

    2015-01-01

    With the recent discoveries of novel post-translational modifications (PTMs) which play important roles in signaling and biosynthetic pathways, identification of such PTM catalyzing enzymes by genome mining has been an area of major interest. Unlike well-known PTMs like phosphorylation, glycosylation, SUMOylation, no bioinformatics resources are available for enzymes associated with novel and unusual PTMs. Therefore, we have developed the novPTMenzy database which catalogs information on the sequence, structure, active site and genomic neighborhood of experimentally characterized enzymes involved in five novel PTMs, namely AMPylation, Eliminylation, Sulfation, Hydroxylation and Deamidation. Based on a comprehensive analysis of the sequence and structural features of these known PTM catalyzing enzymes, we have created Hidden Markov Model profiles for the identification of similar PTM catalyzing enzymatic domains in genomic sequences. We have also created predictive rules for grouping them into functional subfamilies and deciphering their mechanistic details by structure-based analysis of their active site pockets. These analytical modules have been made available as user friendly search interfaces of novPTMenzy database. It also has a specialized analysis interface for some PTMs like AMPylation and Eliminylation. The novPTMenzy database is a unique resource that can aid in discovery of unusual PTM catalyzing enzymes in newly sequenced genomes. Database URL: http://www.nii.ac.in/novptmenzy.html PMID:25931459

  3. Human Oncogenic Herpesvirus and Post-translational Modifications - Phosphorylation and SUMOylation.

    PubMed

    Chang, Pei-Ching; Campbell, Mel; Robertson, Erle S

    2016-01-01

    Pathogens, especially viruses, evolve abilities to utilize cellular machineries to facilitate their survival and propagation. Post-translational modifications (PTMs), especially phosphorylation and SUMOylation, that reversibly modulate the function and interactions of target proteins are among the most important features in cell signaling pathways. PTM-dependent events also serve as one of the favorite targets for viruses. Among the seven unambiguous human oncogenic viruses, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), human papillomavirus (HPV), Human T lymphotrophic virus-1 (HTLV-1), and Merkel cell polyomavirus (MCPyV), two are herpesviruses. The life cycle of herpesviruses consists of latent and lytic phases and the rapid switch between these states includes global remodeling of the viral genome from heterochromatin-to-euchromatin. The balance between lytic replication and latency is essential for herpesvirus to maintain a persistent infection through a combination of viral propagation and evasion of the host immune response, which consequently may contribute to tumorigenesis. It is no surprise that the swift reversibility of PTMs, especially SUMOylation, a modification that epigenetically regulates chromatin structure, is a major hijack target of the host for oncogenic herpesviruses. In this brief review, we summarize the varied ways in which herpesviruses engage the host immune components through PTMs, focusing on phosphorylation and SUMOylation. PMID:27379086

  4. 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

    2014-01-01

    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

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

    PubMed

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

    2008-10-15

    De novo sequencing is a spectrum analysis approach for mass spectrometry data to discover post-translational modifications in proteins; however, such an approach is still in its infancy and is still not widely applied to proteomic practices due to its limited reliability. In this work, we describe a de novo sequencing approach for the discovery of protein modifications based on identification of the proteome UStags (Shen, Y.; Tolić, N.; Hixson, K. K.; Purvine, S. O.; Pasa-Tolić, L.; Qian, W. J.; Adkins, J. N.; Moore, R. J.; Smith, R. D. Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry data for peptides and polypeptides from a yeast lysate, and the de novo sequences obtained were selected based on filter levels designed to provide a limited yet high quality subset 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 possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances within several yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. To determine false discovery rates, two random (false) databases were independently used for sequence matching, 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 of the approach were investigated and described. The combined de novo-UStag approach complements the UStag method previously reported by enabling the discovery of new protein modifications. PMID:18783246

  6. Targeted mass spectrometry methods for detecting oxidative post-translational modifications.

    PubMed

    Tveen-Jensen, Karina; Reis, Ana; Spickett, Corinne M; Pitt, Andrew R

    2014-10-01

    Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases. PMID:26461406

  7. 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

  8. Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

    PubMed Central

    Oren, M; Maltzman, W; Levine, A J

    1981-01-01

    The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen. Images PMID:6100960

  9. 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

  10. Transcriptional and post-translational regulation of mouse cation transport regulator homolog 1.

    PubMed

    Oh-Hashi, Kentaro; Nomura, Yuki; Shimada, Kiyo; Koga, Hisashi; Hirata, Yoko; Kiuchi, Kazutoshi

    2013-08-01

    Recently, cation transport regulator homolog 1 (Chac1) has been identified as a novel pro-apoptotic factor in cells under endoplasmic reticulum (ER) stress. Of the three major ER stress sensors, it is suggested that ATF4 participates in the transcriptional regulation of Chac1 gene expression. The precise characterization of the Chac1 promoter, however, has not yet been elucidated. In this study, we detected the induction of Chac1 mRNA expression using DNA array analysis and RT-PCR of thapsigargin (Tg)-inducible genes in Neuro2a cells. Chac1 mRNA expression was also induced immediately following treatment with tunicamycin (Tm) and brefeldin A. Characterization of the mouse Chac1 promoter activity using a luciferase reporter assay revealed that the CREB/ATF element and amino acid response element in the mouse Chac1 promoter are functional and respond to Tm stimulation and ATF4 overexpression. Mutations in either element in the Chac1 promoter did not inhibit the responsiveness of this promoter to Tm and ATF4; however, mutations in both of these elements dramatically decreased the basal activity and response to ER stress stimuli. In addition to the transcriptional regulation, we found that Chac1 protein expression was only detected in the presence of MG132, a proteasome inhibitor, even though mouse Chac1 gene was transiently overexpressed in Neuro2a cells. Taken together, we are the first to demonstrate the transcriptional and post-translational regulation of Chac1 expression in a neuronal cell line. PMID:23615711

  11. A new role for α-ketoglutarate dehydrogenase complex: regulating metabolism through post-translational modification of other enzymes.

    PubMed

    McKenna, Mary C; Rae, Caroline D

    2015-07-01

    This Editorial highlights a study by Gibson et al. published in this issue of JNeurochem, in which the authors reveal a novel role for the α-ketoglutarate dehydrogenase complex (KGDHC) in post-translational modification of proteins. KGDHC may catalyze post-translational modification of itself as well as several other proteins by succinylation of lysine residues. The authors' report of an enzyme responsible for succinylation of key mitochondrial enzymes represents a major step toward our understanding of the complex functional metabolome. TCA, tricarboxylic acid; KG, α-ketoglutarate; KGDHC, α-ketoglutarate dehydrogenase complex; FUM, fumarase; MDH, malate dehydrogenase; ME, malic enzyme; GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; GS, glutamine synthetase; PAG, phosphate-activated glutaminase; SIRT3, silent information regulator 3; SIRT5, silent information regulator 5. PMID:26052752

  12. Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo

    PubMed Central

    Mohamed, Bashir M; Verma, Navin K; Davies, Anthony M; McGowan, Aoife; Crosbie-Staunton, Kieran; Prina-Mello, Adriele; Kelleher, Dermot; Botting, Catherine H; Causey, Corey P; Thompson, Paul R; Pruijn, Ger JM; Kisin, Elena R; Tkach, Alexey V; Shvedova, Anna A; Volkov, Yuri

    2012-01-01

    Aim Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca2+-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination. Materials & methods Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated. Results The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets. Conclusion Nanoparticle exposure facilitated post-translational citrullination of proteins. PMID:22625207

  13. 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. PMID:25981296

  14. Identification of a post-translationally myristoylated autophagy-inducing domain released by caspase cleavage of huntingtin.

    PubMed

    Martin, Dale D O; Heit, Ryan J; Yap, Megan C; Davidson, Michael W; Hayden, Michael R; Berthiaume, Luc G

    2014-06-15

    Huntington disease (HD) is a debilitating neurodegenerative disease characterized by the loss of motor control and cognitive ability that ultimately leads to death. It is caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein, which leads to aggregation of the protein and eventually cellular death. Both the wild-type and mutant form of the protein are highly regulated by post-translational modifications including proteolysis, palmitoylation and phosphorylation. We now demonstrate the existence of a new post-translational modification of HTT: the addition of the 14 carbon fatty acid myristate to a glycine residue exposed on a caspase-3-cleaved fragment (post-translational myristoylation) and that myristoylation of this fragment is altered in a physiologically relevant model of mutant HTT. Myristoylated HTT553-585-EGFP, but not its non-myristoylated variant, initially localized to the ER, induced the formation of autophagosomes and accumulated in abnormally large autophagolysosomal/lysosomal structures in a variety of cell types, including neuronal cell lines under nutrient-rich conditions. Our results suggest that accumulation of myristoylated HTT553-586 in cells may alter the rate of production of autophagosomes and/or their clearance through the heterotypic autophagosomal/lysosomal fusion process. Overall, our novel observations establish a role for the post-translational myristoylation of a caspase-3-cleaved fragment of HTT, highly similar to the Barkor/ATG14L autophagosome-targeting sequence domain thought to sense, maintain and/or promote membrane curvature in the regulation of autophagy. Abnormal processing or production of this myristoylated HTT fragment might be involved in the pathophysiology of HD. PMID:24459296

  15. Identification of a post-translationally myristoylated autophagy-inducing domain released by caspase cleavage of Huntingtin

    PubMed Central

    Martin, Dale D.O.; Heit, Ryan J.; Yap, Megan C.; Davidson, Michael W.; Hayden, Michael R.; Berthiaume, Luc G.

    2014-01-01

    Huntington disease (HD) is a debilitating neurodegenerative disease characterized by the loss of motor control and cognitive ability that ultimately leads to death. It is caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein, which leads to aggregation of the protein and eventually cellular death. Both the wild-type and mutant form of the protein are highly regulated by post-translational modifications including proteolysis, palmitoylation and phosphorylation. We now demonstrate the existence of a new post-translational modification of HTT: the addition of the 14 carbon fatty acid myristate to a glycine residue exposed on a caspase-3-cleaved fragment (post-translational myristoylation) and that myristoylation of this fragment is altered in a physiologically relevant model of mutant HTT. Myristoylated HTT553–585–EGFP, but not its non-myristoylated variant, initially localized to the ER, induced the formation of autophagosomes and accumulated in abnormally large autophagolysosomal/lysosomal structures in a variety of cell types, including neuronal cell lines under nutrient-rich conditions. Our results suggest that accumulation of myristoylated HTT553–586 in cells may alter the rate of production of autophagosomes and/or their clearance through the heterotypic autophagosomal/lysosomal fusion process. Overall, our novel observations establish a role for the post-translational myristoylation of a caspase-3-cleaved fragment of HTT, highly similar to the Barkor/ATG14L autophagosome-targeting sequence domain thought to sense, maintain and/or promote membrane curvature in the regulation of autophagy. Abnormal processing or production of this myristoylated HTT fragment might be involved in the pathophysiology of HD. PMID:24459296

  16. 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. PMID:23395536

  17. Complete Post-Translational Modification Mapping of Pathogenic N. meningitidis Pilins Requires Top-Down Mass Spectrometry

    PubMed Central

    Gault, Joseph; Malosse, Christian; Machata, Silke; Millien, Corinne; Podglajen, Isabelle; Ploy, Marie-Cécile; Costello, Catherine E.; Duménil, Guillaume; Chamot-Rooke, Julia

    2014-01-01

    In pathogenic bacteria post-translationally modified proteins have been found to promote bacterial survival, replication and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15–18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harbouring its own set of post-translational modifications (PTMs) and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down mass spectrometry therefore appears to be a promising tool for the analysis of highly post-translationally modified proteins involved in bacterial virulence. PMID:24459079

  18. PTMcode v2: a resource for functional associations of post-translational modifications within and between proteins

    PubMed Central

    Minguez, Pablo; Letunic, Ivica; Parca, Luca; Garcia-Alonso, Luz; Dopazo, Joaquin; Huerta-Cepas, Jaime; Bork, Peer

    2015-01-01

    The post-translational regulation of proteins is mainly driven by two molecular events, their modification by several types of moieties and their interaction with other proteins. These two processes are interdependent and together are responsible for the function of the protein in a particular cell state. Several databases focus on the prediction and compilation of protein–protein interactions (PPIs) and no less on the collection and analysis of protein post-translational modifications (PTMs), however, there are no resources that concentrate on describing the regulatory role of PTMs in PPIs. We developed several methods based on residue co-evolution and proximity to predict the functional associations of pairs of PTMs that we apply to modifications in the same protein and between two interacting proteins. In order to make data available for understudied organisms, PTMcode v2 (http://ptmcode.embl.de) includes a new strategy to propagate PTMs from validated modified sites through orthologous proteins. The second release of PTMcode covers 19 eukaryotic species from which we collected more than 300 000 experimentally verified PTMs (>1 300 000 propagated) of 69 types extracting the post-translational regulation of >100 000 proteins and >100 000 interactions. In total, we report 8 million associations of PTMs regulating single proteins and over 9.4 million interplays tuning PPIs. PMID:25361965

  19. Profiles of epigenetic histone post-translational modifications at type 1 diabetes susceptible genes.

    PubMed

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

    2012-05-11

    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

  20. Prediction and Analysis of Post-Translational Pyruvoyl Residue Modification Sites from Internal Serines in Proteins

    PubMed Central

    Jiang, Yang; Li, Bi-Qing; Zhang, Yuchao; Feng, Yuan-Ming; Gao, Yu-Fei; Zhang, Ning; Cai, Yu-Dong

    2013-01-01

    Most of pyruvoyl-dependent proteins observed in prokaryotes and eukaryotes are critical regulatory enzymes, which are primary targets of inhibitors for anti-cancer and anti-parasitic therapy. These proteins undergo an autocatalytic, intramolecular self-cleavage reaction in which a covalently bound pyruvoyl group is generated on a conserved serine residue. Traditional detections of the modified serine sites are performed by experimental approaches, which are often labor-intensive and time-consuming. In this study, we initiated in an attempt for the computational predictions of such serine sites with Feature Selection based on a Random Forest. Since only a small number of experimentally verified pyruvoyl-modified proteins are collected in the protein database at its current version, we only used a small dataset in this study. After removing proteins with sequence identities >60%, a non-redundant dataset was generated and was used, which contained only 46 proteins, with one pyruvoyl serine site for each protein. Several types of features were considered in our method including PSSM conservation scores, disorders, secondary structures, solvent accessibilities, amino acid factors and amino acid occurrence frequencies. As a result, a pretty good performance was achieved in our dataset. The best 100.00% accuracy and 1.0000 MCC value were obtained from the training dataset, and 93.75% accuracy and 0.8441 MCC value from the testing dataset. The optimal feature set contained 9 features. Analysis of the optimal feature set indicated the important roles of some specific features in determining the pyruvoyl-group-serine sites, which were consistent with several results of earlier experimental studies. These selected features may shed some light on the in-depth understanding of the mechanism of the post-translational self-maturation process, providing guidelines for experimental validation. Future work should be made as more pyruvoyl-modified proteins are found and the method

  1. 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

  2. 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

  3. Complexity analyses show two distinct types of nonlinear dynamics in short heart period variability recordings

    PubMed Central

    Porta, Alberto; Bari, Vlasta; Marchi, Andrea; De Maria, Beatrice; Cysarz, Dirk; Van Leeuwen, Peter; Takahashi, Anielle C. M.; Catai, Aparecida M.; Gnecchi-Ruscone, Tomaso

    2015-01-01

    Two diverse complexity metrics quantifying time irreversibility and local prediction, in connection with a surrogate data approach, were utilized to detect nonlinear dynamics in short heart period (HP) variability series recorded in fetuses, as a function of the gestational period, and in healthy humans, as a function of the magnitude of the orthostatic challenge. The metrics indicated the presence of two distinct types of nonlinear HP dynamics characterized by diverse ranges of time scales. These findings stress the need to render more specific the analysis of nonlinear components of HP dynamics by accounting for different temporal scales. PMID:25806002

  4. Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

    PubMed Central

    Burén, Stefan; Ortega-Villasante, Cristina; Blanco-Rivero, Amaya; Martínez-Bernardini, Andrea; Shutova, Tatiana; Shevela, Dmitriy; Messinger, Johannes; Bako, Laszlo; Villarejo, Arsenio; Samuelsson, Göran

    2011-01-01

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

  5. 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. PMID:25561502

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

    PubMed

    Søgaard, M; Andersen, J S; Roepstorff, P; Svensson, B

    1993-10-01

    We have used electrospray mass spectrometry (ESMS) in combination with protein chemistry and genetics to delineate post-translational modifications in yeast of barley alpha-amylase 1 (AMY1), a 45 kD enzyme crucial for production of malt, an important starting material in the manufacture of beer and whisky. In addition to signal peptide processing these modifications are: (1) removal of C-terminal Arg-Ser by Kex1p, (2) glutathionylation of Cys95, (3) O-glycosylation, and (4) additional degradation of the C-terminus. PMID:7764097

  7. Distinct prion strains are defined by amyloid core structure and chaperone binding site dynamics.

    PubMed

    Frederick, Kendra K; Debelouchina, Galia T; Kayatekin, Can; Dorminy, Tea; Jacavone, Angela C; Griffin, Robert G; Lindquist, Susan

    2014-02-20

    Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance. PMID:24485763

  8. 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.

  9. Protein deacetylation by sirtuins: delineating a post-translational regulatory program responsive to nutrient and redox stressors

    PubMed Central

    Bao, Jianjun

    2012-01-01

    Lysine acetylation/deacetylation is increasingly being recognized as common post-translational modification that appears to be broadly operational throughout the cell. The functional roles of these modifications, outside of the nucleus, have not been extensively studied. Moreover, as acetyl-CoA donates the acetyl group for acetylation, nutrient availability and energetic status may be pivotal in this modification. Similarly, nutrient limitation is associated with the deacetylation reaction. This modification is orchestrated by a novel family of sirtuin deacetylases that function in a nutrient and redox dependent manner and targets non-histone protein deacetylation. In compartment-specific locations, candidate target proteins undergoing lysine-residue deacetylation are being identified. Through these investigations, the functional role of this post-translational modification is being delineated. We review the sirtuin family proteins, discuss their functional effects on target proteins, and postulate on potential biological programs and disease processes that may be modified by sirtuin-mediated deacetylation of target proteins. PMID:20680393

  10. Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

    PubMed Central

    Arur, Swathi; Schedl, Tim

    2014-01-01

    Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

  11. Post-Translational Modifications of Kaposi’s Sarcoma-Associated Herpesvirus Regulatory Proteins – SUMO and KSHV

    PubMed Central

    Campbell, Mel; Izumiya, Yoshihiro

    2011-01-01

    KSHV latency can be envisioned as an outcome that is balanced between factors that promote viral gene expression and lytic replication against those that facilitate gene silencing and establish or maintain latency. A large body of work has focused on the activities of the key viral regulatory proteins involved in KSHV latent or lytic states. Moreover, recent studies have also begun to document the importance of epigenetic landscape evolution of the KSHV viral genome during latency and reactivation. However, one area of KSHV molecular virology that remains largely unanswered is the precise role of post-translational modifications on the activities of viral factors that function during latency and reactivation. In this review, we will summarize the post-translational modifications associated with three viral factors whose activities contribute to the viral state. The viral proteins discussed are the two major KSHV encoded transcription factors, K-Rta (KSHV replication and transcriptional activator) and K-bZIP (KSHV basic leucine zipper) and the viral latency-associated nuclear antigen (LANA). A special emphasis will be placed on the role of the sumoylation pathway in the modulation of the KSHV lifecycle. Newly uncovered small ubiquitin-like modifier (SUMO)-associated properties of LANA and K-Rta will also be presented, namely LANA histone targeting SUMO E3 ligase activity and K-Rta SUMO-targeted ubiquitin ligase function. PMID:22347876

  12. 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.

  13. A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

    PubMed Central

    Almeida, Karen H.; Sobol, Robert W.

    2007-01-01

    Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

  14. Dual Pili Post-translational Modifications Synergize to Mediate Meningococcal Adherence to Platelet Activating Factor Receptor on Human Airway Cells

    PubMed Central

    Schulz, Benjamin L.; Power, Peter M.; Swords, W. Edward; Weiser, Jeffery N.; Apicella, Michael A.; Edwards, Jennifer L.; Jennings, Michael P.

    2013-01-01

    Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells. PMID:23696740

  15. Mechanistic insights into the inhibition of Sec61-dependent co- and post-translational translocation by mycolactone

    PubMed Central

    McKenna, Michael; Simmonds, Rachel E.; High, Stephen

    2016-01-01

    ABSTRACT The virulence factor mycolactone is responsible for the immunosuppression and tissue necrosis that characterise Buruli ulcer, a disease caused by infection with Mycobacterium ulcerans. In this study, we confirm that Sec61, the protein-conducting channel that coordinates entry of secretory proteins into the endoplasmic reticulum, is a primary target of mycolactone, and characterise the nature of its inhibitory effect. We conclude that mycolactone constrains the ribosome–nascent-chain–Sec61 complex, consistent with its broad-ranging perturbation of the co-translational translocation of classical secretory proteins. In contrast, the effect of mycolactone on the post-translational ribosome-independent translocation of short secretory proteins through the Sec61 complex is dependent on both signal sequence hydrophobicity and the translocation competence of the mature domain. Changes to protease sensitivity strongly suggest that mycolactone acts by inducing a conformational change in the pore-forming Sec61α subunit. These findings establish that mycolactone inhibits Sec61-mediated protein translocation and highlight differences between the co- and post-translational routes that the Sec61 complex mediates. We propose that mycolactone also provides a useful tool for further delineating the molecular mechanisms of Sec61-dependent protein translocation. PMID:26869228

  16. 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

  17. Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database.

    PubMed

    Khoury, George A; Baliban, Richard C; Floudas, Christodoulos A

    2011-09-13

    Post-translational modifications (PTMs) broadly contribute to the recent explosion of proteomic data and possess a complexity surpassing that of protein design. PTMs are the chemical modification of a protein after its translation, and have wide effects broadening its range of functionality. Based on previous estimates, it is widely believed that more than half of proteins are glycoproteins. Whereas mutations can only occur once per position, different forms of post-translational modifications may occur in tandem. With the number and abundances of modifications constantly being discovered, there is no method to readily assess their relative levels. Here we report the relative abundances of each PTM found experimentally and putatively, from high-quality, manually curated, proteome-wide data, and show that at best, less than one-fifth of proteins are glycosylated. We make available to the academic community a continuously updated resource (http://selene.princeton.edu/PTMCuration) containing the statistics so scientists can assess "how many" of each PTM exists. PMID:22034591

  18. Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana.

    PubMed

    Bergmüller, Eveline; Gehrig, Peter M; Gruissem, Wilhelm

    2007-09-01

    Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants. PMID:17691833

  19. On the road to nowhere: cross-talk between post-translational protein targeting and cytosolic quality control.

    PubMed

    Casson, Joseph; McKenna, Michael; High, Stephen

    2016-06-15

    A well-defined co-translational pathway couples the synthesis and translocation of nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), thereby minimizing the possibility of the hydrophobic signals and transmembrane domains that such proteins contain from being exposed to the cytosol. Nevertheless, a proportion of these co-translational substrates may fail to reach the ER, and therefore mislocalize to the cytosol where their intrinsic hydrophobicity makes them aggregation-prone. A range of hydrophobic precursor proteins that employ alternative, post-translational, routes for ER translocation also contribute to the cytosolic pool of mislocalized proteins (MLPs). In this review, we detail how mammalian cells can efficiently deal with these MLPs by selectively targeting them for proteasomal degradation. Strikingly, this pathway for MLP degradation is regulated by cytosolic components that also facilitate the TRC40-dependent, post-translational, delivery of tail-anchored membrane proteins (TA proteins) to the ER. Among these components are small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and Bcl-2-associated athanogene 6 (BAG6), which appear to play a decisive role in enforcing quality control over hydrophobic precursor proteins that have mislocalized to the cytosol, directing them to either productive membrane insertion or selective ubiquitination and proteasomal degradation. PMID:27284044

  20. 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

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

    PubMed

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

    2016-06-01

    Aberrant histone post-translational modifications (hPTMs) have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification patterns in patient samples (Noberini, 2015) [1]), are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD002669. PMID:27408908

  2. 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. PMID:26468507

  3. 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. PMID:26177826

  4. 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

  5. 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

  6. SiteSeek: Post-translational modification analysis using adaptive locality-effective kernel methods and new profiles

    PubMed Central

    Yoo, Paul D; Ho, Yung Shwen; Zhou, Bing Bing; Zomaya, Albert Y

    2008-01-01

    Background Post-translational modifications have a substantial influence on the structure and functions of protein. Post-translational phosphorylation is one of the most common modification that occur in intracellular proteins. Accurate prediction of protein phosphorylation sites is of great importance for the understanding of diverse cellular signalling processes in both the human body and in animals. In this study, we propose a new machine learning based protein phosphorylation site predictor, SiteSeek. SiteSeek is trained using a novel compact evolutionary and hydrophobicity profile to detect possible protein phosphorylation sites for a target sequence. The newly proposed method proves to be more accurate and exhibits a much stable predictive performance than currently existing phosphorylation site predictors. Results The performance of the proposed model was compared to nine existing different machine learning models and four widely known phosphorylation site predictors with the newly proposed PS-Benchmark_1 dataset to contrast their accuracy, sensitivity, specificity and correlation coefficient. SiteSeek showed better predictive performance with 86.6% accuracy, 83.8% sensitivity, 92.5% specificity and 0.77 correlation-coefficient on the four main kinase families (CDK, CK2, PKA, and PKC). Conclusion Our newly proposed methods used in SiteSeek were shown to be useful for the identification of protein phosphorylation sites as it performed much better than widely known predictors on the newly built PS-Benchmark_1 dataset. PMID:18541042

  7. Force-Induced Dynamical Properties of Multiple Cytoskeletal Filaments Are Distinct from that of Single Filaments

    PubMed Central

    Das, Dipjyoti; Das, Dibyendu; Padinhateeri, Ranjith

    2014-01-01

    How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis have not been studied extensively earlier within simple theoretical frameworks. In this paper, we study the collective dynamical properties of multiple filaments under force, and demonstrate the distinct properties of a multi-filament system in comparison to a single filament. Comparing stochastic simulation results with recent experimental data, we show that multi-filament collective catastrophes are slower than catastrophes of single filaments. Our study also shows further distinctions as follows: (i) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filaments, (ii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, and (iii) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a unified picture by establishing interconnections among all these collective phenomena. Additionally, we show that the collapse times during catastrophes can be sharp indicators of collective stall forces exceeding the additive contributions of single filaments. PMID:25531397

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

    PubMed

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

    2016-06-15

    Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs. PMID:26866564

  9. Prion protein localizes at the ciliary base during neural and cardiovascular development, and its depletion affects α-tubulin post-translational modifications

    PubMed Central

    Halliez, Sophie; Martin-Lannerée, Séverine; Passet, Bruno; Hernandez-Rapp, Julia; Castille, Johan; Urien, Céline; Chat, Sophie; Laude, Hubert; Vilotte, Jean-Luc; Mouillet-Richard, Sophie; Béringue, Vincent

    2015-01-01

    Although conversion of the cellular form of the prion protein (PrPC) into a misfolded isoform is the underlying cause of prion diseases, understanding PrPC physiological functions has remained challenging. PrPC depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrPC. Here, we report PrPC enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrPC depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and α-tubulin post-translational modifications. These processes were also affected over a limited time window in PrPC–ablated embryos. Thus, our study reveals PrPC as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions. PMID:26679898

  10. Proteome adaptations in Ethe1-deficient mice indicate a role in lipid catabolism and cytoskeleton organization via post-translational protein modifications

    PubMed Central

    Hildebrandt, Tatjana M.; Di Meo, Ivano; Zeviani, Massimo; Viscomi, Carlo; Braun, Hans-Peter

    2013-01-01

    Hydrogen sulfide is a physiologically relevant signalling molecule. However, circulating levels of this highly biologically active substance have to be maintained within tightly controlled limits in order to avoid toxic side effects. In patients suffering from EE (ethylmalonic encephalopathy), a block in sulfide oxidation at the level of the SDO (sulfur dioxygenase) ETHE1 leads to severe dysfunctions in microcirculation and cellular energy metabolism. We used an Ethe1-deficient mouse model to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Major disturbances in post-translational protein modifications indicate that the mitochondrial sulfide oxidation pathway could have a crucial function during sulfide signalling most probably via the regulation of cysteine S-modifications. Our results confirm the involvement of sulfide in redox regulation and cytoskeleton dynamics. In addition, they suggest that sulfide signalling specifically regulates mitochondrial catabolism of FAs (fatty acids) and BCAAs (branched-chain amino acids). These findings are particularly relevant in the context of EE since they may explain major symptoms of the disease. PMID:23800285