Mechanistic understanding of the cysteine capping modifications of antibodies enables selective chemical engineering in live mammalian cells.

PubMed

Zhong, Xiaotian; He, Tao; Prashad, Amar S; Wang, Wenge; Cohen, Justin; Ferguson, Darren; Tam, Amy S; Sousa, Eric; Lin, Laura; Tchistiakova, Lioudmila; Gatto, Scott; D'Antona, Aaron; Luan, Yen-Tung; Ma, Weijun; Zollner, Richard; Zhou, Jing; Arve, Bo; Somers, Will; Kriz, Ronald

2017-04-20

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein and DNA Modifications: Evolutionary Imprints of Bacterial Biochemical Diversification and Geochemistry on the Provenance of Eukaryotic Epigenetics

PubMed Central

Aravind, L.; Burroughs, A. Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M.

2014-01-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth’s history. PMID:24984775

Protein and DNA modifications: evolutionary imprints of bacterial biochemical diversification and geochemistry on the provenance of eukaryotic epigenetics.

PubMed

Aravind, L; Burroughs, A Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M

2014-07-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth's history. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Maillard-reaction-induced modification and aggregation of proteins and hardening of texture in protein bar model systems.

PubMed

Zhou, Peng; Guo, Mufan; Liu, Dasong; Liu, Xiaoming; Labuza, Teodore P

2013-03-01

The hardening of high-protein bars causes problems in their acceptability to consumers. The objective of this study was to determine the progress of the Maillard reaction in model systems of high-protein nutritional bars containing reducing sugars, and to illustrate the influences of the Maillard reaction on the modification and aggregation of proteins and the hardening of bar matrices during storage. The progress of the Maillard reaction, glycation, and aggregation of proteins, and textural changes in bar matrices were investigated during storage at 25, 35, and 45 °C. The initial development of the Maillard reaction caused little changes in hardness; however, further storage resulted in dramatic modification of protein with formation of high-molecular-weight polymers, resulting in the hardening in texture. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula minimized the changes in texture. The hardening of high-protein bars causes problems in their acceptability to consumers. Maillard reaction is one of the mechanisms contributing to the hardening of bar matrix, particularly for the late stage of storage. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula will minimize the changes in texture. © 2013 Institute of Food Technologists®

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95.

PubMed

Vallejo, Daniela; Codocedo, Juan F; Inestrosa, Nibaldo C

2017-04-01

The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

PubMed

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine.

PubMed

Ohno, Satoshi; Matsui, Megumi; Yokogawa, Takashi; Nakamura, Masashi; Hosoya, Takamitsu; Hiramatsu, Toshiyuki; Suzuki, Masaaki; Hayashi, Nobuhiro; Nishikawa, Kazuya

2007-03-01

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.

Novel roles for biogenic monoamines: from monoamines in transglutaminase-mediated post-translational protein modification to monoaminylation deregulation diseases.

PubMed

Walther, Diego J; Stahlberg, Silke; Vowinckel, Jakob

2011-12-01

Functional protein serotonylation is a newly recognized post-translational modification with the primary biogenic monoamine (PBMA) serotonin (5-HT). This covalent protein modification is catalyzed by transglutaminases (TGs) and, for example, acts in the constitutive activation of small GTPases. Multiple physiological roles have been identified since its description in 2003 and, importantly, deregulated serotonylation was shown in the etiology of bleeding disorders, primary pulmonary hypertension and diabetes. The PBMAs 5-HT, histamine, dopamine, and norepinephrine all act as neurotransmitters in the nervous system and as hormones in non-neuronal tissues, which points out their physiological importance. In analogy to serotonylation we have found that also the other PBMAs act through the TG-catalyzed mechanisms of 'histaminylation', 'dopaminylation' and 'norepinephrinylation'. Therefore, PBMAs deploy a considerable portion of their effects via protein monoaminylation in addition to their canonical receptor-mediated signaling. Here, the implications of these newly identified post-translational modifications are presented and discussed. Furthermore, the potential regulatory roles of protein monoaminylation in small GTPase, heterotrimeric G-protein and lipid signaling, as well as in modulating metabolic enzymes and nuclear processes, are critically assessed. © 2011 The Authors Journal compilation © 2011 FEBS.

m6A RNA Methylation Controls Neural Development and Is Involved in Human Diseases.

PubMed

Du, Kunzhao; Zhang, Longbin; Lee, Trevor; Sun, Tao

2018-06-16

RNA modifications are involved in many aspects of biological functions. N6-methyladenosine (m 6 A) is one of the most important forms of RNA methylation and plays a vital role in regulating gene expression, protein translation, cell behaviors, and physiological conditions in many species, including humans. The dynamic and reversible modification of m 6 A is conducted by three elements: methyltransferases ("writers"), such as methyltransferase-like protein 3 (METTL3) and METTL14; m 6 A-binding proteins ("readers"), such as the YTH domain family proteins (YTHDFs) and YTH domain-containing protein 1 (YTHDC1); and demethylases ("erasers"), such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). In this review, we summarize the current knowledge on mapping mRNA positions of m 6 A modification and revealing molecular processes of m 6 A. We further highlight the biological significance of m 6 A modification in neural cells during development of the nervous system and its association with human diseases. m 6 A RNA methylation is becoming a new frontier in neuroscience and should help us better understand neural development and neurological diseases from a novel point of view.

The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins.

PubMed

Akbari, Fariba; Eskandani, Morteza; Khosroushahi, Ahmad Yari

2014-11-01

Microalgae have been used in food, cosmetic, and biofuel industries as a natural source of lipids, vitamins, pigments and antioxidants for a long time. Green microalgae, as potent photobioreactors, can be considered as an economical expression system to produce recombinant therapeutical proteins at large-scale due to low cost of production and scaling-up capitalization owning to the inexpensive medium requirement, fast growth rate, and the ease of manipulation. These microalgae possess all benefit eukaryotic expression systems including the ability of post-translational modifications required for proper folding and stability of active proteins. Among the many items regarded as recombinant protein production, this review compares the different expression systems with green microalgae like Dunaliella by viewing the nuclear/chloroplast transformation challenges/benefits, related selection markers/reporter genes, and crucial factors/strategies affecting the increase of foreign protein expression in microalgae transformants. Some important factors were discussed regarding the increase of protein yielding in microalgae transformants including: transformation-associated genotypic modifications, endogenous regulatory factors, promoters, codon optimization, enhancer elements, and milking of recombinant protein.

Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

DOE PAGES

McMillan, Janet R.; Brodin, Jeffrey D.; Millan, Jaime A.; ...

2017-01-25

Here, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using Beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When assembled into crystalline superlattices with AuNPs, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB 2 packing. We probe the role of protein oligonucleotide number and conjugatemore » size on this observation, which revealed the importance of oligonucleotide distribution and number in this observed assembly behavior. These results indicate that proteins with defined DNA-modification patterns are powerful tools to control the nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.« less

Mass spectrometry based structural analysis and systems immunoproteomics strategies for deciphering the host response to endotoxin.

PubMed

Khan, Mohd M; Ernst, Orna; Sun, Jing; Fraser, Iain D C; Ernst, Robert K; Goodlett, David R; Nita-Lazar, Aleksandra

2018-06-24

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4 (TLR4)/myeloid differentiation factor-2 (MD2) complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress, and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry (MS)-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, MS-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications (PTMs). Copyright © 2018. Published by Elsevier Ltd.

Protein organic chemistry and applications for labeling and engineering in live-cell systems.

PubMed

Takaoka, Yousuke; Ojida, Akio; Hamachi, Itaru

2013-04-08

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry-based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test-tube settings; 2) the bioorthogonal reactions of proteins with non-natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag-probe pair for labeling natural amino acids; and 4) ligand-directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2-4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of methyllysine peptides binding to chromobox protein homolog 6 chromodomain in the human proteome.

PubMed

Li, Nan; Stein, Richard S L; He, Wei; Komives, Elizabeth; Wang, Wei

2013-10-01

Methylation is one of the important post-translational modifications that play critical roles in regulating protein functions. Proteomic identification of this post-translational modification and understanding how it affects protein activity remain great challenges. We tackled this problem from the aspect of methylation mediating protein-protein interaction. Using the chromodomain of human chromobox protein homolog 6 as a model system, we developed a systematic approach that integrates structure modeling, bioinformatics analysis, and peptide microarray experiments to identify lysine residues that are methylated and recognized by the chromodomain in the human proteome. Given the important role of chromobox protein homolog 6 as a reader of histone modifications, it was interesting to find that the majority of its interacting partners identified via this approach function in chromatin remodeling and transcriptional regulation. Our study not only illustrates a novel angle for identifying methyllysines on a proteome-wide scale and elucidating their potential roles in regulating protein function, but also suggests possible strategies for engineering the chromodomain-peptide interface to enhance the recognition of and manipulate the signal transduction mediated by such interactions.

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

PubMed

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

2011-12-01

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation. Copyright © 2011 Elsevier Inc. All rights reserved.

A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection.

PubMed

Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L; Chan, Leanne Jade G; Wei, Tong; Joe, Anna; Thomas, Nicholas; Pruitt, Rory; Adams, Paul D; Chern, Maw Sheng; Petzold, Christopher J; Liu, Chang C; Ronald, Pamela C

2016-04-18

Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

A second-generation expression system for tyrosine-sulfated proteins and its application in crop protection

DOE PAGES

Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L.; ...

2015-11-27

Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

PubMed

Gan, Jin; Qiao, Niu; Strahan, Roxanne; Zhu, Caixia; Liu, Lei; Verma, Subhash C; Wei, Fang; Cai, Qiliang

2016-11-01

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis. Copyright © 2016 John Wiley & Sons, Ltd.

Biochemical systems approaches for the analysis of histone modification readout.

PubMed

Soldi, Monica; Bremang, Michael; Bonaldi, Tiziana

2014-08-01

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

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

PubMed Central

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

2011-01-01

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

Posttranslational Modifications and Plant-Environment Interaction.

PubMed

Hashiguchi, A; Komatsu, S

2017-01-01

Posttranslational modifications (PTMs) of proteins such as phosphorylation and ubiquitination are crucial for controlling protein stability, localization, and conformation. Genetic information encoded in DNA is transcribed, translated, and increases its complexity by multiple PTMs. Conformational change introduced by PTMs affects interacting partners of each proteins and their downstream signaling; therefore, PTMs are the major level of modulations of total outcome of living cells. Plants are living in harsh environment that requires unremitting physiological modulation to survive, and the plant response to various environment stresses is regulated by PTMs of proteins. This review deals with the novel knowledge of PTM-focused proteomic studies on various life conditions. PTMs are focused that mediate plant-environment interaction such as stress perception, protein homeostasis, control of energy shift, and defense by immune system. Integration of diverse signals on a protein via multiple PTMs is discussed as well, considering current situation where signal integration became an emerging area approached by systems biology into account. © 2017 Elsevier Inc. All rights reserved.

Systemic Induction of NO-, Redox-, and cGMP Signaling in the Pumpkin Extrafascicular Phloem upon Local Leaf Wounding

PubMed Central

Gaupels, Frank; Furch, Alexandra C. U.; Zimmermann, Matthias R.; Chen, Faxing; Kaever, Volkhard; Buhtz, Anja; Kehr, Julia; Sarioglu, Hakan; Kogel, Karl-Heinz; Durner, Jörg

2016-01-01

Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima). Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint toward a wound-induced shift in the redox status of the EFP. Nitric oxide (NO) is another important player in stress-induced redox signaling in plants. Therefore, we analyzed NO-dependent protein modifications in the EFP. Six to forty eight hours after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1) and Cyclophilin 18 (CYP18) as well as the 26.5 kD isoform of Phloem Protein 2 (PP2) were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP) in the EFP and discuss the possible function of this second messenger in systemic NO and redox signaling within the EFP. PMID:26904092

Human Cells as Platform to Produce Gamma-Carboxylated Proteins.

PubMed

de Sousa Bomfim, Aline; de Freitas, Marcela Cristina Corrêa; Covas, Dimas Tadeu; de Sousa Russo, Elisa Maria

2018-01-01

The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.

Studies of protein oxidation as a product quality attribute on a scale-down model for cell culture process development.

PubMed

Lee, Nacole D; Kondragunta, Bhargavi; Uplekar, Shaunak; Vallejos, Jose; Moreira, Antonio; Rao, Govind

2015-01-01

Of importance to the biological properties of proteins produced in cell culture systems are the complex post-translational modifications that are affected by variations in process conditions. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by such process variations. Dissolved oxygen is a parameter of increasing interest since studies have shown that despite the necessity of oxygen for respiration, there may also be some detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components. Variation, or changes to cell culture products, can affect function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3-producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Relative protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. Protein oxidation, oxidative modification to intracellular proteins that involves cleavage of the polypeptide chain, and modifications of the amino acid side chains can be affected by variations in dissolved oxygen levels in cell culture systems. Studies have shown that despite the necessity of oxygen for respiration, there may be detrimental effects of oxygen to the cell. Production and accumulation of reactive oxygen species can cause damage to proteins as a result of oxidation of the cell and cellular components, affecting function, clearance rate, immunogenicity, and specific activity, which translates into clinical implications. The effect of increasing dissolved oxygen on protein oxidation in immunoglobulin G3-producing mouse hybridoma cells was studied using 50 mL high-throughput mini-bioreactors that employ non-invasive optical sensor technology for monitoring and closed feedback control of pH and dissolved oxygen. Protein carbonyl concentration of proteins produced under varying levels of dissolved oxygen was measured by enzyme-linked immunosorbent assay and used as an indicator of oxidative damage. A trend of increasing protein carbonyl content in response to increasing dissolved oxygen levels under controlled conditions was observed. © PDA, Inc. 2015.

Heritability of targeted gene modifications induced by plant-optimized CRISPR systems.

PubMed

Mao, Yanfei; Botella, Jose Ramon; Zhu, Jian-Kang

2017-03-01

The Streptococcus-derived CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system has emerged as a very powerful tool for targeted gene modifications in many living organisms including plants. Since the first application of this system for plant gene modification in 2013, this RNA-guided DNA endonuclease system has been extensively engineered to meet the requirements of functional genomics and crop trait improvement in a number of plant species. Given its short history, the emphasis of many studies has been the optimization of the technology to improve its reliability and efficiency to generate heritable gene modifications in plants. Here we review and analyze the features of customized CRISPR/Cas9 systems developed for plant genetic studies and crop breeding. We focus on two essential aspects: the heritability of gene modifications induced by CRISPR/Cas9 and the factors affecting its efficiency, and we provide strategies for future design of systems with improved activity and heritability in plants.

Strategies to improve plasma half life time of peptide and protein drugs.

PubMed

Werle, M; Bernkop-Schnürch, A

2006-06-01

Due to the obvious advantages of long-acting peptide and protein drugs, strategies to prolong plasma half life time of such compounds are highly on demand. Short plasma half life times are commonly due to fast renal clearance as well as to enzymatic degradation occurring during systemic circulation. Modifications of the peptide/protein can lead to prolonged plasma half life times. By shortening the overall amino acid amount of somatostatin and replacing L: -analogue amino acids with D: -amino acids, plasma half life time of the derivate octreotide was 1.5 hours in comparison to only few minutes of somatostatin. A PEG(2,40 K) conjugate of INF-alpha-2b exhibited a 330-fold prolonged plasma half life time compared to the native protein. It was the aim of this review to provide an overview of possible strategies to prolong plasma half life time such as modification of N- and C-terminus or PEGylation as well as methods to evaluate the effectiveness of drug modifications. Furthermore, fundamental data about most important proteolytic enzymes of human blood, liver and kidney as well as their cleavage specificity and inhibitors for them are provided in order to predict enzymatic cleavage of peptide and protein drugs during systemic circulation.

Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry

PubMed Central

Zhang, Han; Ge, Ying

2012-01-01

Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research. PMID:22187450

Effects of covalent modification by 4-hydroxy-2-nonenal on the noncovalent oligomerization of ubiquitin.

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

When lipid membranes containing ω-6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4-hydroxy-2-nonenal (HNE)-a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non-covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

PubMed

Krasitskaya, V V; Korneeva, S I; Kudryavtsev, A N; Markova, S V; Stepanyuk, G A; Frank, L A

2011-11-01

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

[Pichia pastoris as an expression system for recombinant protein production].

PubMed

Ciarkowska, Anna; Jakubowska, Anna

2013-01-01

Pichia pastoris has become increasingly popular as a host for recombinant protein production in recent years. P. pastoris is more cost effective and allows achieving higher expression levels than insect and mammalian cells. It also offers some significant advantages over E. coli expression systems, such as avoiding problems with proper protein folding. Also, P. pastoris as an eukaryotic organism can carry out posttranslational modifications of produced proteins. Additionally, P. pastoris can produce high levels of recombinant proteins in extracellular medium which simplifies protein purification. Having many advantages over other expression systems makes P. pastoris an organism of choice for industrial protein production.

S-sulfhydration: a cysteine posttranslational modification in plant systems.

PubMed

Aroca, Ángeles; Serna, Antonio; Gotor, Cecilia; Romero, Luis C

2015-05-01

Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems. © 2015 American Society of Plant Biologists. All Rights Reserved.

Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

PubMed

Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

2017-10-01

Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Adenovirus Type 5 Viral Particles Pseudotyped with Mutagenized Fiber Proteins Show Diminished Infectivity of Coxsackie B-Adenovirus Receptor-Bearing Cells

PubMed Central

Jakubczak, John L.; Rollence, Michele L.; Stewart, David A.; Jafari, Jonathon D.; Von Seggern, Dan J.; Nemerow, Glen R.; Stevenson, Susan C.; Hallenbeck, Paul L.

2001-01-01

A major limitation of adenovirus type 5 (Ad5)-based gene therapy, the inability to target therapeutic genes to selected cell types, is attributable to the natural tropism of the virus for the widely expressed coxsackievirus-adenovirus receptor (CAR) protein. Modifications of the Ad5 fiber knob domain have been shown to alter the tropism of the virus. We have developed a novel system to rapidly evaluate the function of modified fiber proteins in their most relevant context, the adenoviral capsid. This transient transfection/infection system combines transfection of cells with plasmids that express high levels of the modified fiber protein and infection with Ad5.βgal.ΔF, an E1-, E3-, and fiber-deleted adenoviral vector encoding β-galactosidase. We have used this system to test the adenoviral transduction efficiency mediated by a panel of fiber protein mutants that were proposed to influence CAR interaction. A series of amino acid modifications were incorporated via mutagenesis into the fiber expression plasmid, and the resulting fiber proteins were subsequently incorporated onto adenoviral particles. Mutations located in the fiber knob AB and CD loops demonstrated the greatest reduction in fiber-mediated gene transfer in HeLa cells. We also observed effects on transduction efficiency with mutations in the FG loop, indicating that the binding site may extend to the adjacent monomer in the fiber trimer and in the HI loop. These studies support the concept that modification of the fiber knob domain to diminish or ablate CAR interaction should result in a detargeted adenoviral vector that can be combined simultaneously with novel ligands for the development of a systemically administered, targeted adenoviral vector. PMID:11222722

Protein mass analysis of histones.

PubMed

Galasinski, Scott C; Resing, Katheryn A; Ahn, Natalie G

2003-09-01

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.

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

DOEpatents

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

2015-03-03

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, David N.; Reed, David W.; Thompson, Vicki S.

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.

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

[Methods of quantitative proteomics].

PubMed

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction.

PubMed

Jacobsen, Michael T; Fairhead, Michael; Fogelstrand, Per; Howarth, Mark

2017-08-17

Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping." Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore-friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(ii) catalyst† †Dedicated to Professor Stuart L. Schreiber on the occasion of his 60th birthday. ‡ ‡Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05313k Click here for additional data file.

PubMed Central

Kwan, Terence T.-L.; Boutureira, Omar; Frye, Elizabeth C.; Walsh, Stephen J.; Gupta, Moni K.; Wallace, Stephen; Wu, Yuteng; Zhang, Fengzhi; Sore, Hannah F.; Galloway, Warren R. J. D.; Chin, Jason W.; Welch, Martin; Bernardes, Gonçalo J. L.

2017-01-01

Transition metal catalysis has emerged as a powerful strategy to expand synthetic flexibility of protein modification. Herein, we report a cationic Ru(ii) system that enables the first example of alkyne hydrosilylation between dimethylarylsilanes and O-propargyl-functionalized proteins using a substoichiometric amount or low-loading of Ru(ii) catalyst to achieve the first C–Si bond formation on full-length substrates. The reaction proceeds under physiological conditions at a rate comparable to other widely used bioorthogonal reactions. Moreover, the resultant gem-disubstituted vinylsilane linkage can be further elaborated through thiol–ene coupling or fluoride-induced protodesilylation, demonstrating its utility in further rounds of targeted modifications. PMID:28966779

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

PubMed Central

Wang, Qi-En; Zhu, Qianzheng; Wani, Gulzar; El-Mahdy, Mohamed A.; Li, Jinyou; Wani, Altaf A.

2005-01-01

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein. PMID:16030353

General Characteristics of the Changes in the Thermal Stability of Proteins and Enzymes After the Chemical Modification of Their Functional Groups

NASA Astrophysics Data System (ADS)

Kutuzova, G. D.; Ugarova, N. N.; Berezin, Ilya V.

1984-11-01

The principal structural and physicochemical factors determining the stability of protein macromolecules in solution and the characteristics of the structure of the proteins from thermophilic microorganisms are examined. The mechanism of the changes in the thermal stability of proteins and enzymes after the chemical modification of their functional side groups and the experimental data concerning the influence of chemical modification on the thermal stability of proteins are analysed. The dependence of the stabilisation effect and of the changes in the structure of protein macromolecules on the degree of modification and on the nature of the modified groups and the groups introduced into proteins in the course of modification (their charge and hydrophobic properties) is demonstrated. The great practical value of the method of chemical modification for the preparation of stabilised forms of biocatalysts is shown in relation to specific examples. The bibliography includes 178 references.

Early events in copper-ion catalyzed oxidation of α-synuclein.

PubMed

Tiwari, Manish K; Leinisch, Fabian; Sahin, Cagla; Møller, Ian Max; Otzen, Daniel E; Davies, Michael J; Bjerrum, Morten J

2018-04-22

Previous studies on metal-ion catalyzed oxidation of α-synuclein oxidation have mostly used conditions that result in extensive modification precluding an understanding of the early events in this process. In this study, we have examined time-dependent oxidative events related to α-synuclein modification using six different molar ratios of Cu 2+ /H 2 O 2 /protein and Cu 2+ /H 2 O 2 /ascorbate/protein resulting in mild to moderate extents of oxidation. For a Cu 2+ /H 2 O 2 /protein molar ratio of 2.3:7.8:1 only low levels of carbonyls were detected (0.078 carbonyls per protein), whereas a molar ratio of 4.7:15.6:1 gave 0.22 carbonyls per α-synuclein within 15 min. With the latter conditions, rapid conversion of 3 out of 4 methionines (Met) to methionine sulfoxide, and 2 out of 4 tyrosines (Tyr) were converted to products including inter- and intra-molecular dityrosine cross-links and protein oligomers, as determined by SDS-PAGE and Western blot analysis. Limited histidine (His) modification was observed. The rapid formation of dityrosine cross-links was confirmed by fluorescence and mass-spectrometry. These data indicate that Met and Tyr oxidation are early events in Cu 2+ /H 2 O 2 -mediated damage, with carbonyl formation being a minor process. With the Cu 2+ /H 2 O 2 /ascorbate system, rapid protein carbonyl formation was detected with the first 5 min, but after this time point, little additional carbonyl formation was detected. With this system, lower levels of Met and Tyr oxidation were detected (2 Met and 1 Tyr modified with a Cu 2+ /H 2 O 2 /ascorbate/protein ratio of 2.3:7.8:7.8:1), but greater His oxidation. Only low levels of intra- dityrosine cross-links and no inter- dityrosine oligomers were detected under these conditions, suggesting that ascorbate limits Cu 2+ /H 2 O 2 -induced α-synuclein modification. Copyright © 2018. Published by Elsevier Inc.

Kinetic memory based on the enzyme-limited competition.

PubMed

Hatakeyama, Tetsuhiro S; Kaneko, Kunihiko

2014-08-01

Cellular memory, which allows cells to retain information from their environment, is important for a variety of cellular functions, such as adaptation to external stimuli, cell differentiation, and synaptic plasticity. Although posttranslational modifications have received much attention as a source of cellular memory, the mechanisms directing such alterations have not been fully uncovered. It may be possible to embed memory in multiple stable states in dynamical systems governing modifications. However, several experiments on modifications of proteins suggest long-term relaxation depending on experienced external conditions, without explicit switches over multi-stable states. As an alternative to a multistability memory scheme, we propose "kinetic memory" for epigenetic cellular memory, in which memory is stored as a slow-relaxation process far from a stable fixed state. Information from previous environmental exposure is retained as the long-term maintenance of a cellular state, rather than switches over fixed states. To demonstrate this kinetic memory, we study several models in which multimeric proteins undergo catalytic modifications (e.g., phosphorylation and methylation), and find that a slow relaxation process of the modification state, logarithmic in time, appears when the concentration of a catalyst (enzyme) involved in the modification reactions is lower than that of the substrates. Sharp transitions from a normal fast-relaxation phase into this slow-relaxation phase are revealed, and explained by enzyme-limited competition among modification reactions. The slow-relaxation process is confirmed by simulations of several models of catalytic reactions of protein modifications, and it enables the memorization of external stimuli, as its time course depends crucially on the history of the stimuli. This kinetic memory provides novel insight into a broad class of cellular memory and functions. In particular, applications for long-term potentiation are discussed, including dynamic modifications of calcium-calmodulin kinase II and cAMP-response element-binding protein essential for synaptic plasticity.

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

PubMed Central

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

2016-01-01

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

Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Hao; Wen, Jianzhong; Huang, Richard Y-C.

2012-02-01

Protein structure determines function in biology, and a variety of approaches have been employed to obtain structural information about proteins. Mass spectrometry-based protein footprinting is one fast-growing approach. One labeling-based footprinting approach is the use of a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) to modify solvent-accessible carboxyl groups on glutamate (E) and aspartate (D). This paper describes method development of carboxyl-group modification in protein footprinting. The modification protocol was evaluated by using the protein calmodulin as a model. Because carboxyl-group modification is a slow reaction relative to protein folding and unfolding, there is an issue that modificationsmore » at certain sites may induce protein unfolding and lead to additional modification at sites that are not solvent-accessible in the wild-type protein. We investigated this possibility by using hydrogen deuterium amide exchange (H/DX). The study demonstrated that application of carboxyl group modification in probing conformational changes in calmodulin induced by Ca{sup 2+} binding provides useful information that is not compromised by modification-induced protein unfolding.« less

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver.

PubMed

Krawczyńska, Agata; Herman, Andrzej P; Antushevich, Hanna; Bochenek, Joanna; Dziendzikowska, Katarzyna; Gajewska, Alina; Gromadzka-Ostrowska, Joanna

2017-01-01

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Progress in expression and molecular modification of microbial transglutaminase].

PubMed

Liu, Song; Zhang, Dongxu; Du, Guocheng; Chen, Jian

2011-12-01

Microbial transglutaminase, which could catalyze the cross-linking of many proteins or non-protein materials, has been widely used in food, pharmaceutical and textile industry. To enhance the yield of the enzyme and establish corresponding platform for molecular modification, the researchers of Japanese Ajinomoto began to construct the recombinant strain producing transglutaminase in the 1990s. So far, the enzyme has been successfully expressed in different expression systems. Some of the recombinant strains are more productive than wild strains. Recently, progress has been made in the molecular modification of microbial transglutaminase, and the activity, thermo-stability and specificity of the enzyme are improved. This review briefly summarized and analyzed the strategies involved in these studies, and noted its trends.

Synergistic Modification Induced Specific Recognition between Histone and TRIM24 via Fluctuation Correlation Network Analysis

NASA Astrophysics Data System (ADS)

Zhang, Jinmai; Luo, Huajie; Liu, Hao; Ye, Wei; Luo, Ray; Chen, Hai-Feng

2016-04-01

Histone modification plays a key role in gene regulation and gene expression. TRIM24 as a histone reader can recognize histone modification. However the specific recognition mechanism between TRIM24 and histone modification is unsolved. Here, systems biology method of dynamics correlation network based on molecular dynamics simulation was used to answer the question. Our network analysis shows that the dynamics correlation network of H3K23ac is distinctly different from that of wild type and other modifications. A hypothesis of “synergistic modification induced recognition” is then proposed to link histone modification and TRIM24 binding. These observations were further confirmed from community analysis of networks with mutation and network perturbation. Finally, a possible recognition pathway is also identified based on the shortest path search for H3K23ac. Significant difference of recognition pathway was found among different systems due to methylation and acetylation modifications. The analysis presented here and other studies show that the dynamic network-based analysis might be a useful general strategy to study the biology of protein post-translational modification and associated recognition.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-08-01

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthetic Proteins and Peptides for the Direct Interrogation of α-Synuclein Posttranslational Modifications

PubMed Central

Pratt, Matthew R.; Abeywardana, Tharindumala; Marotta, Nicholas P.

2015-01-01

α-Synuclein is the aggregation-prone protein associated with Parkinson’s disease (PD) and related neurodegenerative diseases. Complicating both its biological functions and toxic aggregation are a variety of posttranslational modifications. These modifications have the potential to either positively or negatively affect α-synuclein aggregation, raising the possibility that the enzymes that add or remove these modifications could be therapeutic targets in PD. Synthetic protein chemistry is uniquely positioned to generate site-specifically and homogeneously modified proteins for biochemical study. Here, we review the application of synthetic peptides and proteins towards understanding the effects of α-synuclein posttranslational modifications. PMID:26120904

Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

PubMed

Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-03-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

Roles of N-Terminal Fatty Acid Acylations in Membrane Compartment Partitioning: Arabidopsis h-Type Thioredoxins as a Case Study[C][W

PubMed Central

Traverso, José A.; Micalella, Chiara; Martinez, Aude; Brown, Spencer C.; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-01-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX–green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane. PMID:23543785

Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

PubMed Central

Thess, Andreas; Grund, Stefanie; Mui, Barbara L; Hope, Michael J; Baumhof, Patrick; Fotin-Mleczek, Mariola; Schlake, Thomas

2015-01-01

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies. PMID:26050989

Methyleneation of peptides by N,N,N,N-tetramethylethylenediamine (TEMED) under conditions used for free radical polymerization: a mechanistic study.

PubMed

Shirangi, Mehrnoosh; Sastre Toraño, Javier; Sellergren, Börje; Hennink, Wim E; Somsen, Govert W; van Nostrum, Cornelus F

2015-01-21

Free radical polymerization is often used to prepare protein and peptide-loaded hydrogels for the design of controlled release systems and molecular imprinting materials. Peroxodisulfates (ammonium peroxodisulfates (APS) or potassium peroxodisulfates (KPS)) with N,N,N,N-tetramethylethylenediamine (TEMED) are frequently used as initiator and catalyst. However, exposure to these free radical polymerization reagents may lead to modification of the protein and peptide. In this work, we show the modification of lysine residues by ammonium peroxodisulfate (APS)/TEMED of the immunostimulant thymopentin (TP5). Parallel studies on a decapeptide and a library of 15 dipeptides were performed to reveal the mechanism of modification. LC-MS of APS/TEMED-exposed TP5 revealed a major reaction product with an increased mass (+12 Da) with respect to TP5. LC-MS(2) and LC-MS(3) were performed to obtain structural information on the modified peptide and localize the actual modification site. Interpretation of the obtained data demonstrates the formation of a methylene bridge between the lysine and arginine residue in the presence of TEMED, while replacing TEMED with a sodium bisulfite catalyst did not show this modification. Studies with the other peptides showed that the TEMED radical can induce methyleneation on peptides when lysine is next to arginine, proline, cysteine, aspargine, glutamine, histidine, tyrosine, tryptophan, and aspartic acid residues. Stability of peptides and protein needs to be considered when using APS/TEMED in in situ polymerization systems. The use of an alternative catalyst such as sodium bisulfite may preserve the chemical integrity of peptides during in situ polymerization.

Novel approach using DNA-RNA hybrids in RNA nanotechnology | Center for Cancer Research

Cancer.gov

Developing simple approaches to detect interactions, modifications, and cellular locations of macromolecules is essential for understanding biochemical processes. The use of protein fragment complementation assays, also called split-protein systems, is a highly sensitive approach for studying protein interactions in biological systems. In this approach, functional proteins are split into non-functional fragments, and when attached to possible interacting partners, can reassemble and become functional again. Use of split-protein assays can establish differences between a healthy and a diseased state in the cell as well as determine the outcome of a therapeutic intervention.

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

PubMed Central

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

2015-01-01

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

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

PubMed

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

2016-02-15

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

New insights into metabolic signaling and cell survival: the role of beta-O-linkage of N-acetylglucosamine.

PubMed

Ngoh, Gladys A; Jones, Steven P

2008-12-01

The involvement of glucose in fundamental metabolic pathways represents a core element of biology. Late in the 20th century, a unique glucose-derived signal was discovered, which appeared to be involved in a variety of cellular processes, including mitosis, transcription, insulin signaling, stress responses, and potentially, Alzheimer's disease, and diabetes. By definition, this glucose-fed signaling system was a post-translational modification to proteins. However, unlike classical cotranslational N-glycosylation occurring in the endoplasmic reticulum and Golgi apparatus, this process occurs elsewhere throughout the cell in a highly dynamic fashion, similar to the quintessential post-translational modification, phosphorylation. This more recently described post-translational modification, the beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to nucleocytoplasmic proteins, represents an under-investigated area of biology. This signaling system operates in all of the tissues examined and seems to have persisted throughout all multicellular eukaryotes. Thus, it comes with little surprise that O-GlcNAc signaling is an integral system and viable target for biomedical investigation. This system may be a boundless source for insight into a variety of diseases and yield numerous opportunities for drug design. This Perspective will address recent insights into O-GlcNAc signaling in the cardiovascular system as a paradigm for its involvement in other biological systems.

Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches.

PubMed

Mast, Carole; Lyan, Bernard; Joly, Charlotte; Centeno, Delphine; Giacomoni, Franck; Martin, Jean-François; Mosoni, Laurent; Dardevet, Dominique; Pujos-Guillot, Estelle; Papet, Isabelle

2015-04-29

Liver protein can be altered under paracetamol (APAP) treatment. APAP-protein adducts and other protein modifications (oxidation/nitration, expression) play a role in hepatotoxicity induced by acute overdoses, but it is unknown whether liver protein modifications occur during long-term treatment with non-toxic doses of APAP. We quantified APAP-protein adducts and assessed other protein modifications in the liver from rats under chronic (17 days) treatment with two APAP doses (0.5% or 1% of APAP in the diet w/w). A targeted metabolomic method was validated and used to quantify APAP-protein adducts as APAP-cysteine adducts following proteolytic hydrolysis. The limit of detection was found to be 7ng APAP-cysteine/mL hydrolysate i.e. an APAP-Cys to tyrosine ratio of 0.016‰. Other protein modifications were assessed on the same protein hydrolysate by untargeted metabolomics including a new strategy to process the data and identify discriminant molecules. These two complementary mass spectrometry (MS)-based metabolic approaches enabled the assessment of a wide range of protein modifications induced by chronic treatment with APAP. APAP-protein adducts were detected even in the absence of glutathione depletion and hepatotoxicity, i.e. in the 0.5% APAP group, and increased by 218% in the 1% APAP group compared to the 0.5% APAP group. At the same time, the untargeted metabolomic method revealed a decrease in the binding of cysteine, cysteinyl-glycine and GSH to thiol groups of protein cysteine residues, an increase in the oxidation of tryptophan and proline residues and a modification in protein expression. This wide range of modifications in liver proteins occurred in rats under chronic treatment with APAP that did not induce hepatotoxicity. Copyright © 2015 Elsevier B.V. All rights reserved.

The different roles of selective autophagic protein degradation in mammalian cells.

PubMed

Wang, Da-wei; Peng, Zhen-ju; Ren, Guang-fang; Wang, Guang-xin

2015-11-10

Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases.

[Epigenome: what we learned from Rett syndrome, a neurological disease caused by mutation of a methyl-CpG binding protein].

PubMed

Kubota, Takeo

2013-01-01

Epigenome is defined as DNA and histone modification-dependent gene regulation system. Abnormalities in this system are known to cause various neuro-developmental diseases. We recently reported that neurological symptoms of Rett syndrome, which is an autistic disorder caused by mutations in methyl-CpG binding protein 2 (MeCP2), was associated with failure of epigenomic gene regulation in neuronal cells, and that clinical differences in the identical twins with Rett syndrome in the differences in DNA methylation in neuronal genes, but not caused by DNA sequence differences. Since central nervus system requires precise gene regulation, neurological diseases including Alzheimer and Parkinson diseases may be caused by acquired DNA modification (epigenomic) changes that results in aberrant gene regulation as well as DNA sequence changes congenitally occurred (mutation).

Dual Coordination of Post Translational Modifications in Human Protein Networks

PubMed Central

Woodsmith, Jonathan; Kamburov, Atanas; Stelzl, Ulrich

2013-01-01

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

Nanoliposome is a Promising Carrier of Protein and Peptide Biomolecule for the Treatment of Cancer.

PubMed

Kumar Giri, Tapan; Giri, Ayan; Kumar Barman, Tapan; Maity, Subhasis

2016-01-01

Nano-liposomes are the newly developed delivery systems for cancer therapy that are finding a position particularly suitable as peptide and protein carriers. These are three-layered self-assembled structures with nanoparticulate carrier systems. The overall pharmacological properties of commonly used protein and peptide in cancer therapy can be improved by the incorporation of protein and peptide into the nano-liposome. The surface modifications can be made liposomes to make compatible with targeting ligands has made these nanocarriers for targeted delivery. This review discusses the method of preparation and characterization of liposome based protein peptide delivery for the treatment of cancer. This review also explores latest work intended for targeted treatment of cancer by nano-liposomal protein and peptide delivery system. This type of delivery is targeting protein and peptide to tumor site by avoiding the reticuloendothelial system. Methods of nano-liposome delivery containing protein and peptide are also highlighted.

Signaling gateway molecule pages—a data model perspective

PubMed Central

Dinasarapu, Ashok Reddy; Saunders, Brian; Ozerlat, Iley; Azam, Kenan; Subramaniam, Shankar

2011-01-01

Summary: The Signaling Gateway Molecule Pages (SGMP) database provides highly structured data on proteins which exist in different functional states participating in signal transduction pathways. A molecule page starts with a state of a native protein, without any modification and/or interactions. New states are formed with every post-translational modification or interaction with one or more proteins, small molecules or class molecules and with each change in cellular location. State transitions are caused by a combination of one or more modifications, interactions and translocations which then might be associated with one or more biological processes. In a characterized biological state, a molecule can function as one of several entities or their combinations, including channel, receptor, enzyme, transcription factor and transporter. We have also exported SGMP data to the Biological Pathway Exchange (BioPAX) and Systems Biology Markup Language (SBML) as well as in our custom XML. Availability: SGMP is available at www.signaling-gateway.org/molecule. Contact: shankar@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21505029

The effect of reagents mimicking oxidative stress on fibrinogen function.

PubMed

Štikarová, Jana; Kotlín, Roman; Riedel, Tomáš; Suttnar, Jiří; Pimková, Kristýna; Chrastinová, Leona; Dyr, Jan E

2013-01-01

Fibrinogen is one of the plasma proteins most susceptible to oxidative modification. It has been suggested that modification of fibrinogen may cause thrombotic/bleeding complications associated with many pathophysiological states of organism. We exposed fibrinogen molecules to three different modification reagents-malondialdehyde, sodium hypochlorite, and peroxynitrite-that are presented to various degrees in different stages of oxidative stress. We studied the changes in fibrin network formation and platelet interactions with modified fibrinogens under flow conditions. The fastest modification of fibrinogen was caused by hypochlorite. Fibers from fibrinogen modified with either reagent were thinner in comparison with control fibers. We found that platelet dynamic adhesion was significantly lower on fibrinogen modified with malondialdehyde and significantly higher on fibrinogen modified either with hypochlorite or peroxynitrite reflecting different prothrombotic/antithrombotic properties of oxidatively modified fibrinogens. It seems that, in the complex reactions ongoing in living organisms at conditions of oxidation stress, hypochlorite modifies proteins (e.g., fibrinogen) faster and more preferentially than malondialdehyde. It suggests that the prothrombotic effects of prior fibrinogen modifications may outweigh the antithrombotic effect of malondialdehyde-modified fibrinogen in real living systems.

The Effect of Reagents Mimicking Oxidative Stress on Fibrinogen Function

PubMed Central

Štikarová, Jana; Kotlín, Roman; Riedel, Tomáš; Suttnar, Jiří; Pimková, Kristýna; Chrastinová, Leona; Dyr, Jan E.

2013-01-01

Fibrinogen is one of the plasma proteins most susceptible to oxidative modification. It has been suggested that modification of fibrinogen may cause thrombotic/bleeding complications associated with many pathophysiological states of organism. We exposed fibrinogen molecules to three different modification reagents—malondialdehyde, sodium hypochlorite, and peroxynitrite—that are presented to various degrees in different stages of oxidative stress. We studied the changes in fibrin network formation and platelet interactions with modified fibrinogens under flow conditions. The fastest modification of fibrinogen was caused by hypochlorite. Fibers from fibrinogen modified with either reagent were thinner in comparison with control fibers. We found that platelet dynamic adhesion was significantly lower on fibrinogen modified with malondialdehyde and significantly higher on fibrinogen modified either with hypochlorite or peroxynitrite reflecting different prothrombotic/antithrombotic properties of oxidatively modified fibrinogens. It seems that, in the complex reactions ongoing in living organisms at conditions of oxidation stress, hypochlorite modifies proteins (e.g., fibrinogen) faster and more preferentially than malondialdehyde. It suggests that the prothrombotic effects of prior fibrinogen modifications may outweigh the antithrombotic effect of malondialdehyde-modified fibrinogen in real living systems. PMID:24235886

Highly Predictive Reprogramming of tRNA Modifications Is Linked to Selective Expression of Codon-Biased Genes

PubMed Central

2016-01-01

Cells respond to stress by controlling gene expression at several levels, with little known about the role of translation. Here, we demonstrate a coordinated translational stress response system involving stress-specific reprogramming of tRNA wobble modifications that leads to selective translation of codon-biased mRNAs representing different classes of critical response proteins. In budding yeast exposed to four oxidants and five alkylating agents, tRNA modification patterns accurately distinguished among chemically similar stressors, with 14 modified ribonucleosides forming the basis for a data-driven model that predicts toxicant chemistry with >80% sensitivity and specificity. tRNA modification subpatterns also distinguish SN1 from SN2 alkylating agents, with SN2-induced increases in m3C in tRNA mechanistically linked to selective translation of threonine-rich membrane proteins from genes enriched with ACC and ACT degenerate codons for threonine. These results establish tRNA modifications as predictive biomarkers of exposure and illustrate a novel regulatory mechanism for translational control of cell stress response. PMID:25772370

tRNA wobble modifications and protein homeostasis

PubMed Central

Ranjan, Namit; Rodnina, Marina V.

2016-01-01

Abstract tRNA is a central component of the protein synthesis machinery in the cell. In living cells, tRNAs undergo numerous post-transcriptional modifications. In particular, modifications at the anticodon loop play an important role in ensuring efficient protein synthesis, maintaining protein homeostasis, and helping cell adaptation and survival. Hypo-modification of the wobble position of the tRNA anticodon loop is of particular relevance for translation regulation and is implicated in various human diseases. In this review we summarize recent evidence of how methyl and thiol modifications in eukaryotic tRNA at position 34 affect cellular fitness and modulate regulatory circuits at normal conditions and under stress. PMID:27335723

Redox modification of caveolar proteins in the cardiovascular system- role in cellular signalling and disease.

PubMed

Bubb, Kristen J; Birgisdottir, Asa Birna; Tang, Owen; Hansen, Thomas; Figtree, Gemma A

2017-08-01

Rapid and coordinated release of a variety of reactive oxygen species (ROS) such as superoxide (O 2 .- ), hydrogen peroxide (H 2 O 2 ) and peroxynitrite, in specific microdomains, play a crucial role in cell signalling in the cardiovascular system. These reactions are mediated by reversible and functional modifications of a wide variety of key proteins. Dysregulation of this oxidative signalling occurs in almost all forms of cardiovascular disease (CVD), including at the very early phases. Despite the heavily publicized failure of "antioxidants" to improve CVD progression, pharmacotherapies such as those targeting the renin-angiotensin system, or statins, exert at least part of their large clinical benefit via modulating cellular redox signalling. Over 250 proteins, including receptors, ion channels and pumps, and signalling proteins are found in the caveolae. An increasing proportion of these are being recognized as redox regulated-proteins, that reside in the immediate vicinity of the two major cellular sources of ROS, nicotinamide adenine dinucleotide phosphate oxidase (Nox) and uncoupled endothelial nitric oxide synthase (eNOS). This review focuses on what is known about redox signalling within the caveolae, as well as endogenous protective mechanisms utilized by the cell, and new approaches to targeting dysregulated redox signalling in the caveolae as a therapeutic strategy in CVD. Copyright © 2017. Published by Elsevier Inc.

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

NASA Astrophysics Data System (ADS)

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

2016-02-01

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

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

PubMed

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

2016-02-01

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

Chemical characteristics and enhanced hepatoprotective activities of Maillard reaction products derived from milk protein-sugar system.

PubMed

Oh, Nam Su; Young Lee, Ji; Lee, Hyun Ah; Joung, Jae Yeon; Shin, Yong Kook; Kim, Sae Hun; Kim, Younghoon; Lee, Kwang Won

2016-02-01

The objective of this study was to investigate the characteristics, antioxidative properties, and hepatoprotective effects of Maillard reaction products (MRP) from milk protein reacted with sugars. The MRP were obtained from milk protein, whey protein concentrates and sodium caseinate, using 2 types of sugars, lactose and glucose, by heating the mixture at 55°C for 7d in a sodium phosphate buffer (pH 7.4). Changes in the chemical modification of the milk protein were monitored by measuring the protein-bound carbonyls and PAGE protein profiles. The results showed that the amount of protein-bound carbonyls increased after Maillard reaction (MR). In addition, sodium dodecyl sulfate-PAGE analysis indicated a formation of high-molecular weight complexes through MR. The modification sites induced by MR of milk protein were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of tryptic-digested gel spots of MRP. As a result, modification and their localization in AA sequence of MRP was identified. Also, the MRP showed higher antioxidant activities than the intact milk protein, and they reduced intracellular reactive oxygen species production and inhibited the depletion of the reduced glutathione concentrations in the HepG2 cells. In particular, glucose-sodium caseinate MRP showed the highest biological activities among all MRP. Therefore, these results suggest that the MRP from milk protein reacting with sugars possess effective antioxidant activity and have a protective ability against oxidative damage. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Yeast synthetic biology for the production of recombinant therapeutic proteins.

PubMed

Kim, Hyunah; Yoo, Su Jin; Kang, Hyun Ah

2015-02-01

The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Chemoenzymatic Labeling of Proteins: Techniques and Approaches

PubMed Central

Rashidian, Mohammad; Dozier, Jonathan K.; Distefano, Mark D.

2013-01-01

Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally-occurring post-translational modifications, for creating antibody-drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics and protein-protein interactions and for the preparation of protein-polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups are not only inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase and N-myristoyl transferase. PMID:23837885

The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

USDA-ARS?s Scientific Manuscript database

Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

Main Strategies of Plant Expression System Glycoengineering for Producing Humanized Recombinant Pharmaceutical Proteins.

PubMed

Rozov, S M; Permyakova, N V; Deineko, E V

2018-03-01

Most the pharmaceutical proteins are derived not from their natural sources, rather their recombinant analogs are synthesized in various expression systems. Plant expression systems, unlike mammalian cell cultures, combine simplicity and low cost of procaryotic systems and the ability for posttranslational modifications inherent in eucaryotes. More than 50% of all human proteins and more than 40% of the currently used pharmaceutical proteins are glycosylated, that is, they are glycoproteins, and their biological activity, pharmacodynamics, and immunogenicity depend on the correct glycosylation pattern. This review examines in detail the similarities and differences between N- and O-glycosylation in plant and mammalian cells, as well as the effect of plant glycans on the activity, pharmacokinetics, immunity, and intensity of biosynthesis of pharmaceutical proteins. The main current strategies of glycoengineering of plant expression systems aimed at obtaining fully humanized proteins for pharmaceutical application are summarized.

Human cells: new platform for recombinant therapeutic protein production.

PubMed

Swiech, Kamilla; Picanço-Castro, Virgínia; Covas, Dimas Tadeu

2012-07-01

The demand for recombinant therapeutic proteins is significantly increasing. There is a constant need to improve the existing expression systems, and also developing novel approaches to face the therapeutic proteins demands. Human cell lines have emerged as a new and powerful alternative for the production of human therapeutic proteins because this expression system is expected to produce recombinant proteins with post translation modifications more similar to their natural counterpart and reduce the potential immunogenic reactions against nonhuman epitopes. Currently, little information about the cultivation of human cells for the production of biopharmaceuticals is available. These cells have shown efficient production in laboratory scale and represent an important tool for the pharmaceutical industry. This review presents the cell lines available for large-scale recombinant proteins production and evaluates critically the advantages of this expression system in comparison with other expression systems for recombinant therapeutic protein production. Copyright © 2012 Elsevier Inc. All rights reserved.

Proteome | Office of Cancer Clinical Proteomics Research

Cancer.gov

A proteome is the entire complement of proteins, including modifications made to a particular set of proteins, produced by an organism or a cellular system. This will vary with time and distinct requirements such as growth conditions and stresses, and thus is highly dynamic and spatial. Proteomics is the study of the proteome.

Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

PubMed Central

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-01-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

PubMed

Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

2014-02-01

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

Regulation of protein turnover by heat shock proteins.

PubMed

Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

2014-12-01

Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system. Copyright © 2014 Elsevier Inc. All rights reserved.

Surface modification of protein enhances encapsulation in chitosan nanoparticles

NASA Astrophysics Data System (ADS)

Koyani, Rina D.; Andrade, Mariana; Quester, Katrin; Gaytán, Paul; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

2018-04-01

Chitosan nanoparticles have a huge potential as nanocarriers for environmental and biomedical purposes. Protein encapsulation in nano-sized chitosan provides protection against inactivation, proteolysis, and other alterations due to environmental conditions, as well as the possibility to be targeted to specific tissues by ligand functionalization. In this work, we demonstrate that the chemical modification of the protein surface enhances the protein loading in chitosan nanocarriers. Encapsulation of green fluorescent protein and the cytochrome P450 was studied. The increase of electrostatic interactions between the free amino groups of chitosan and the increased number of free carboxylic groups in the protein surface enhance the protein loading, protein retention, and, thus, the enzymatic activity of chitosan nanoparticles. The chemical modification of protein surface with malonic acid moieties reduced drastically the protein isoelectric point increasing the protein interaction with the polycationic biomaterial and chitosan. The chemical modification of protein does not alter the morphology of chitosan nanoparticles that showed an average diameter of 18 nm, spheroidal in shape, and smooth surfaced. The strategy of chemical modification of protein surface, shown here, is a simple and efficient technique to enhance the protein loading in chitosan nanoparticles. This technique could be used for other nanoparticles based on polycationic or polyanionic materials. The increase of protein loading improves, doubtless, the performance of protein-loaded chitosan nanoparticles for biotechnological and biomedical applications.

In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

PubMed Central

Perrochia, Ludovic; Crozat, Estelle; Hecker, Arnaud; Zhang, Wenhua; Bareille, Joseph; Collinet, Bruno; van Tilbeurgh, Herman; Forterre, Patrick

2013-01-01

N6-threonylcarbamoyladenosine (t6A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t6A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t6A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t6A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t6A in nature. These findings shed light on the reaction mechanism of t6A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells. PMID:23258706

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

PubMed Central

Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

2015-01-01

From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. PMID:26478227

Redox regulation of cGMP-dependent protein kinase Iα in the cardiovascular system

PubMed Central

Prysyazhna, Oleksandra; Eaton, Philip

2015-01-01

Elevated levels of oxidants in biological systems have been historically referred to as “oxidative stress,” a choice of words that perhaps conveys an imbalanced view of reactive oxygen species in cells and tissues. The term stress suggests a harmful role, whereas a contemporary view is that oxidants are also crucial for the maintenance of homeostasis or adaptive signaling that can actually limit injury. This regulatory role for oxidants is achieved in part by them inducing oxidative post-translational modifications of proteins which may alter their function or interactions. Such mechanisms allow changes in cell oxidant levels to be coupled to regulated alterations in enzymatic function (i.e., signal transduction), which enables “redox signaling.” In this review we focus on the role of cGMP-dependent protein kinase (PKG) Ia disulfide dimerisation, an oxidative modification that is induced by oxidants that directly activates the enzyme, discussing how this impacts on the cardiovascular system. Additionally, how this oxidative activation of PKG may coordinate with or differ from classical activation of this kinase by cGMP is also considered. PMID:26236235

Western Blotting of the Endocannabinoid System.

PubMed

Wager-Miller, Jim; Mackie, Ken

2016-01-01

Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

Supramolecular PEGylation of biopharmaceuticals

PubMed Central

Webber, Matthew J.; Vinciguerra, Brittany; Cortinas, Abel B.; Thapa, Lavanya S.; Jhunjhunwala, Siddharth; Isaacs, Lyle; Langer, Robert; Anderson, Daniel G.

2016-01-01

The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host–guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit[7]uril (CB[7])–PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB[7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy. PMID:27911829

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

PubMed

Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu

2017-11-14

Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.

RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

PubMed

Gasiunas, Giedrius; Siksnys, Virginijus

2013-11-01

Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects. Copyright © 2013 Elsevier Ltd. All rights reserved.

SwissPalm: Protein Palmitoylation database.

PubMed

Blanc, Mathieu; David, Fabrice; Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

SwissPalm: Protein Palmitoylation database

PubMed Central

Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation. PMID:26339475

The Presence of ADP-Ribosylated Fe Protein of Nitrogenase in Rhodobacter capsulatus Is Correlated with Cellular Nitrogen Status

PubMed Central

Yakunin, Alexander F.; Laurinavichene, Tatyana V.; Tsygankov, Anatoly A.; Hallenbeck, Patrick C.

1999-01-01

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions. PMID:10094674

Introduction to the thematic minireview series on redox-active protein modifications and signaling.

PubMed

Banerjee, Ruma

2013-09-13

The dynamics of redox metabolism necessitate cellular strategies for sensing redox changes and for responding to them. A common mechanism for receiving and transmitting redox changes is via reversible modifications of protein cysteine residues. A plethora of cysteine modifications have been described, including sulfenylation, glutathionylation, and disulfide formation. These post-translational modifications have the potential to alter protein structure and/or function and to modulate cellular processes ranging from division to death and from circadian rhythms to secretion. The focus of this thematic minireview series is cysteine modifications in response to reactive oxygen and nitrogen species.

TRIM Family Proteins: Roles in Autophagy, Immunity, and Carcinogenesis.

PubMed

Hatakeyama, Shigetsugu

2017-04-01

Tripartite motif (TRIM) family proteins, most of which have E3 ubiquitin ligase activities, have various functions in cellular processes including intracellular signaling, development, apoptosis, protein quality control, innate immunity, autophagy, and carcinogenesis. The ubiquitin system is one of the systems for post-translational modifications, which play crucial roles not only as markers for degradation of target proteins by the proteasome but also as regulators of protein-protein interactions and of the activation of enzymes. Accumulating evidence has shown that TRIM family proteins have unique, important roles and that their dysregulation causes several diseases classified as cancer, immunological disease, or developmental disorders. In this review we focus on recent emerging topics on TRIM proteins in the regulation of autophagy, innate immunity, and carcinogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recent Achievements in Characterizing the Histone Code and Approaches to Integrating Epigenomics and Systems Biology.

PubMed

Janssen, K A; Sidoli, S; Garcia, B A

2017-01-01

Functional epigenetic regulation occurs by dynamic modification of chromatin, including genetic material (i.e., DNA methylation), histone proteins, and other nuclear proteins. Due to the highly complex nature of the histone code, mass spectrometry (MS) has become the leading technique in identification of single and combinatorial histone modifications. MS has now overcome antibody-based strategies due to its automation, high resolution, and accurate quantitation. Moreover, multiple approaches to analysis have been developed for global quantitation of posttranslational modifications (PTMs), including large-scale characterization of modification coexistence (middle-down and top-down proteomics), which is not currently possible with any other biochemical strategy. Recently, our group and others have simplified and increased the effectiveness of analyzing histone PTMs by improving multiple MS methods and data analysis tools. This review provides an overview of the major achievements in the analysis of histone PTMs using MS with a focus on the most recent improvements. We speculate that the workflow for histone analysis at its state of the art is highly reliable in terms of identification and quantitation accuracy, and it has the potential to become a routine method for systems biology thanks to the possibility of integrating histone MS results with genomics and proteomics datasets. © 2017 Elsevier Inc. All rights reserved.

Protein S-sulfhydration by hydrogen sulfide in cardiovascular system.

PubMed

Meng, Guoliang; Zhao, Shuang; Xie, Liping; Han, Yi; Ji, Yong

2018-04-01

Hydrogen sulfide (H 2 S), independently of any specific transporters, has a number of biological effects on the cardiovascular system. However, until now, the detailed mechanism of H 2 S was not clear. Recently, a novel post-translational modification induced by H 2 S, named S-sulfhydration, has been proposed. S-sulfhydration is the chemical modification of specific cysteine residues of target proteins by H 2 S. There are several methods for detecting S-sulfhydration, such as the modified biotin switch assay, maleimide assay with fluorescent thiol modifying regents, tag-switch method and mass spectrometry. H 2 S induces S-sulfhydration on enzymes or receptors (such as p66Shc, phospholamban, protein tyrosine phosphatase 1B, mitogen-activated extracellular signal-regulated kinase 1 and ATP synthase subunit α), transcription factors (such as specific protein-1, kelch-like ECH-associating protein 1, NF-κB and interferon regulatory factor-1), and ion channels (such as voltage-activated Ca 2+ channels, transient receptor potential channels and ATP-sensitive K + channels) in the cardiovascular system. Although significant progress has been achieved in delineating the role of protein S-sulfhydration by H 2 S in the cardiovascular system, more proteins with detailed cysteine sites of S-sulfhydration as well as physiological function need to be investigated in further studies. This review mainly summarizes the role and possible mechanism of S-sulfhydration in the cardiovascular system. The S-sulfhydrated proteins may be potential novel targets for therapeutic intervention and drug design in the cardiovascular system, which may accelerate the development and application of H 2 S-related drugs in the future. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc. © 2017 The British Pharmacological Society.

Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

PubMed Central

Mendoza, Vanessa Leah; Vachet, Richard W.

2009-01-01

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

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

PubMed

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

2013-07-25

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

Advanced Maillard reaction end products are associated with Alzheimer disease pathology.

PubMed Central

Smith, M A; Taneda, S; Richey, P L; Miyata, S; Yan, S D; Stern, D; Sayre, L M; Monnier, V M; Perry, G

1994-01-01

During aging long-lived proteins accumulate specific post-translational modifications. One family of modifications, termed Maillard reaction products, are initiated by the condensation between amino groups of proteins and reducing sugars. Protein modification by the Maillard reaction is associated with crosslink formation, decreased protein solubility, and increased protease resistance. Here, we present evidence that the characteristic pathological structures associated with Alzheimer disease contain modifications typical of advanced Maillard reaction end products. Specifically, antibodies against two Maillard end products, pyrraline and pentosidine, immunocytochemically label neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease. In contrast, little or no staining is observed in apparently healthy neurons of the same brain. The Maillard-reaction-related modifications described herein could account for the biochemical and insolubility properties of the lesions of Alzheimer disease through the formation of protein crosslinks. Images PMID:8202552

Recording information on protein complexes in an information management system.

PubMed

Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

2011-08-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. Copyright © 2011 Elsevier Inc. All rights reserved.

Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature-triggered hierarchical self-assembly

NASA Astrophysics Data System (ADS)

Mozhdehi, Davoud; Luginbuhl, Kelli M.; Simon, Joseph R.; Dzuricky, Michael; Berger, Rüdiger; Varol, H. Samet; Huang, Fred C.; Buehne, Kristen L.; Mayne, Nicholas R.; Weitzhandler, Isaac; Bonn, Mischa; Parekh, Sapun H.; Chilkoti, Ashutosh

2018-05-01

Post-translational modification of proteins is a strategy widely used in biological systems. It expands the diversity of the proteome and allows for tailoring of both the function and localization of proteins within cells as well as the material properties of structural proteins and matrices. Despite their ubiquity in biology, with a few exceptions, the potential of post-translational modifications in biomaterials synthesis has remained largely untapped. As a proof of concept to demonstrate the feasibility of creating a genetically encoded biohybrid material through post-translational modification, we report here the generation of a family of three stimulus-responsive hybrid materials—fatty-acid-modified elastin-like polypeptides—using a one-pot recombinant expression and post-translational lipidation methodology. These hybrid biomaterials contain an amphiphilic domain, composed of a β-sheet-forming peptide that is post-translationally functionalized with a C14 alkyl chain, fused to a thermally responsive elastin-like polypeptide. They exhibit temperature-triggered hierarchical self-assembly across multiple length scales with varied structure and material properties that can be controlled at the sequence level.

Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chhabra, S.R.; Butland, G.; Elias, D.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications inmore » a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.« less

The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

PubMed Central

Finley, Daniel; Ulrich, Helle D.; Sommer, Thomas; Kaiser, Peter

2012-01-01

Protein modifications provide cells with exquisite temporal and spatial control of protein function. Ubiquitin is among the most important modifiers, serving both to target hundreds of proteins for rapid degradation by the proteasome, and as a dynamic signaling agent that regulates the function of covalently bound proteins. The diverse effects of ubiquitylation reflect the assembly of structurally distinct ubiquitin chains on target proteins. The resulting ubiquitin code is interpreted by an extensive family of ubiquitin receptors. Here we review the components of this regulatory network and its effects throughout the cell. PMID:23028185

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

PubMed

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Tools for phospho- and glycoproteomics of plasma membranes.

PubMed

Wiśniewski, Jacek R

2011-07-01

Analysis of plasma membrane proteins and their posttranslational modifications is considered as important for identification of disease markers and targets for drug treatment. Due to their insolubility in water, studying of plasma membrane proteins using mass spectrometry has been difficult for a long time. Recent technological developments in sample preparation together with important improvements in mass spectrometric analysis have facilitated analysis of these proteins and their posttranslational modifications. Now, large scale proteomic analyses allow identification of thousands of membrane proteins from minute amounts of sample. Optimized protocols for affinity enrichment of phosphorylated and glycosylated peptides have set new dimensions in the depth of characterization of these posttranslational modifications of plasma membrane proteins. Here, I summarize recent advances in proteomic technology for the characterization of the cell surface proteins and their modifications. In the focus are approaches allowing large scale mapping rather than analytical methods suitable for studying individual proteins or non-complex mixtures.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins.

PubMed

Gonsalvez, Graydon B; Tian, Liping; Ospina, Jason K; Boisvert, François-Michel; Lamond, Angus I; Matera, A Gregory

2007-08-27

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells.

Two distinct arginine methyltransferases are required for biogenesis of Sm-class ribonucleoproteins

PubMed Central

Gonsalvez, Graydon B.; Tian, Liping; Ospina, Jason K.; Boisvert, François-Michel; Lamond, Angus I.; Matera, A. Gregory

2007-01-01

Small nuclear ribonucleoproteins (snRNPs) are core components of the spliceosome. The U1, U2, U4, and U5 snRNPs each contain a common set of seven Sm proteins. Three of these Sm proteins are posttranslationally modified to contain symmetric dimethylarginine (sDMA) residues within their C-terminal tails. However, the precise function of this modification in the snRNP biogenesis pathway is unclear. Several lines of evidence suggest that the methyltransferase protein arginine methyltransferase 5 (PRMT5) is responsible for sDMA modification of Sm proteins. We found that in human cells, PRMT5 and a newly discovered type II methyltransferase, PRMT7, are each required for Sm protein sDMA modification. Furthermore, we show that the two enzymes function nonredundantly in Sm protein methylation. Lastly, we provide in vivo evidence demonstrating that Sm protein sDMA modification is required for snRNP biogenesis in human cells. PMID:17709427

Dietary protein restriction causes modification in aluminum-induced alteration in glutamate and GABA system of rat brain

PubMed Central

Nayak, Prasunpriya; Chatterjee, Ajay K

2003-01-01

Background Alteration of glutamate and γ-aminobutyrate system have been reported to be associated with neurodegenerative disorders and have been postulated to be involved in aluminum-induced neurotoxicity as well. Aluminum, an well known and commonly exposed neurotoxin, was found to alter glutamate and γ-aminobutyrate levels as well as activities of associated enzymes with regional specificity. Protein malnutrition also reported to alter glutamate level and some of its metabolic enzymes. Thus the region-wise study of levels of brain glutamate and γ-aminobutyrate system in protein adequacy and inadequacy may be worthwhile to understand the mechanism of aluminum-induced neurotoxicity. Results Protein restriction does not have any significant impact on regional aluminum and γ-aminobutyrate contents of rat brain. Significant interaction of dietary protein restriction and aluminum intoxication to alter regional brain glutamate level was observed in the tested brain regions except cerebellum. Alteration in glutamate α-decarboxylase and γ-aminobutyrate transaminase activities were found to be significantly influenced by interaction of aluminum intoxication and dietary protein restriction in all the tested brain regions. In case of regional brain succinic semialdehyde content, this interaction was significant only in cerebrum and thalamic area. Conclusion The alterations of regional brain glutamate and γ-aminobutyrate levels by aluminum are region specific as well as dependent on dietary protein intake. The impact of aluminum exposure on the metabolism of these amino acid neurotransmitters are also influenced by dietary protein level. Thus, modification of dietary protein level or manipulation of the brain amino acid homeostasis by any other means may be an useful tool to find out a path to restrict amino acid neurotransmitter alterations in aluminum-associated neurodisorders. PMID:12657166

A light-powered bio-capacitor with nanochannel modulation.

PubMed

Rao, Siyuan; Lu, Shanfu; Guo, Zhibin; Li, Yuan; Chen, Deliang; Xiang, Yan

2014-09-03

An artificial bio-capacitor system is established, consisting of the proton-pump protein proteorhodopsin and a modified alumina nanochannel, inspired by the capacitor-like behavior of plasma membranes realized through the cooperation of ion-pump and ion-channel proteins. Capacitor-like features of this simplified system are realized and identified, and the photocurrent duration time can be modulated by nanochannel modification to obtain favorable square-wave currents. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ansong, Charles; Tolic, Nikola; Purvine, Samuel O.

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 codingmore » 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.« less

Modification of structure and pattern of lipid monolayer on water and solid surfaces in presence of globular protein

NASA Astrophysics Data System (ADS)

Sah, Bijay Kumar; Kundu, Sarathi

2017-05-01

Langmuir monolayers of phospholipids at the air-water interface are well-established model systems for mimicking biological membranes and hence are useful for studying lipid-protein interactions. In the present work, phases and phase transformations occurring in the lipid (DMPA) monolayer in the presence of globular protein (BSA) at neutral subphase pH (≈7.0) are highlighted and the corresponding in-plane pattern and morphology are explored from the surface pressure (π) - specific molecular area (A) isotherm, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) both at air-water and air-solid interfaces. Films of pure lipid and lipid-protein complexes are deposited on solid surfaces by Langmuir-Blodgett method. Due to the presence of BSA molecules, phases and domain pattern changes in comparison with that of the pure DMPA. Moreover, accumulations of globular proteins in between lipid domains are also visible through BAM. AFM shows that the mixed film has relatively bigger globular-like morphology in comparison with that of pure DMPA domains. Combination of electrostatic and hydrophobic interactions between protein and lipid are responsible for such modifications.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2010-09-07

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2012-07-10

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation.

PubMed

Eisenhardt, Nathalie; Chaugule, Viduth K; Pichler, Andrea

2016-01-01

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays. These assays provide a fast readout for in vitro SUMO chain formation activity of E3 ligases in a paralog-specific manner. Finally, we critically analyze the application of fluorescent SUMO proteins to study substrate modification in vitro revealing also the drawbacks of the system.

Curcumin/turmeric solubilized in sodium hydroxide inhibits HNE protein modification--an in vitro study.

PubMed

Kurien, Biji T; Scofield, R Hal

2007-03-21

Free radical mediated lipid peroxidation has been implicated in multiple diseases. A major oxidation by-product of this deleterious process is 4-hydroxy-2-nonenal (HNE). HNE is cytotoxic, mutagenic and genotoxic and is involved in disease pathogenesis. Curcumin, a non-steroidal anti-inflammatory agent (occurring as the yellow pigment found in the rhizomes of the perennial herb Curcuma longa known as turmeric), has emerged as the newest "nutraceutical" agent that has been shown to be efficacious against colon cancer and other disorders, including correcting cystic fibrosis defects. Since curcumin has been reported to have anti-oxidant properties we hypothesized that it will inhibit HNE-modification of a protein substrate. Using an ELISA that employed HNE-modification of solid phase antigen following immobilization, we found that the curcumin solubilized in dilute alkali (5mM sodium hydroxide, pH 11) inhibited HNE-protein modification by 65%. Turmeric also inhibited HNE-protein modification similarly (65%) but at a much lower alkali level (130muM sodium hydroxide, pH 7.6). Alkali by itself (5mM sodium hydroxide, pH 11) was found to enhance HNE modification by as much as 267%. Curcumin/turmeric has to inhibit this alkali enhanced HNE-modification prior to inhibiting the normal HNE protein modification induced by HNE. Thus, inhibition of HNE-modification could be a mechanism by which curcumin exerts its antioxidant effects. The pH at which the inhibition of HNE modification of substrate was observed was close to the physiological pH, making this formulation of curcumin potentially useful practically.

Correlation of nucleotides and carbohydrates metabolism with pro-oxidant and antioxidant systems of erythrocytes depending on age in patients with colorectal cancer.

PubMed

Zuikov, S A; Borzenko, B G; Shatova, O P; Bakurova, E M; Polunin, G E

2014-06-01

To examine the relationship between metabolic features of purine nucleotides and antioxidant system depending on the age of patients with colorectal cancer. The activity of adenosine deaminase, xanthine oxidase, glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase, the NOx concentration and the oxidative modification of proteins were determined spectrophotometricaly in 50 apparently healthy people and 26 patients with colorectal cancer stage -III---IV, aged 40 to 79 years. Increase of pro-oxidant system of erythrocytes with the age against decrease in level of antioxidant protection in both healthy individuals and colorectal cancer patients was determined. A significant increase of pro-ducts of oxidative proteins modification in erythrocytes with ageing was shown. Statistically significant correlation between enzymatic and non enzymatic markers pro-oxidant system and the activity of antioxidant defense enzymes in erythrocytes of patient with colorectal cancer was determined. Obtained results have demonstrated the imbalance in the antioxidant system of erythrocytes in colorectal cancer patients that improve the survival of cancer cells that is more distinctly manifested in ageing.

Structure and Modification of Electrode Materials for Protein Electrochemistry.

PubMed

Jeuken, Lars J C

The interactions between proteins and electrode surfaces are of fundamental importance in bioelectrochemistry, including photobioelectrochemistry. In order to optimise the interaction between electrode and redox protein, either the electrode or the protein can be engineered, with the former being the most adopted approach. This tutorial review provides a basic description of the most commonly used electrode materials in bioelectrochemistry and discusses approaches to modify these surfaces. Carbon, gold and transparent electrodes (e.g. indium tin oxide) are covered, while approaches to form meso- and macroporous structured electrodes are also described. Electrode modifications include the chemical modification with (self-assembled) monolayers and the use of conducting polymers in which the protein is imbedded. The proteins themselves can either be in solution, electrostatically adsorbed on the surface or covalently bound to the electrode. Drawbacks and benefits of each material and its modifications are discussed. Where examples exist of applications in photobioelectrochemistry, these are highlighted.

Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

PubMed Central

Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

2016-01-01

Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization.

PubMed

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process. Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications. Copyright © 2011 Elsevier Inc. All rights reserved.

Covalent modification of soy protein isolate by (-)-epigallocatechin-3-gallate: effects on structural and emulsifying properties.

PubMed

Tao, Fei; Jiang, He; Chen, Wenwei; Zhang, Yongyong; Pan, Jiarong; Jiang, Jiaxin; Jia, Zhenbao

2018-05-07

Soy protein isolate (SPI) has promising applications in various food products because of its excellent functional properties and nutritional quality. The structural and emulsifying properties of covalently modified SPI by (-)-epigallocatechin-3-gallate (EGCG) were investigated. SPI was covalently modified by EGCG under alkaline conditions. SDS-PAGE analysis revealed that EGCG modification caused cross-linking of SPI proteins. Circular dichroism spectra demonstrated that the secondary structure of SPI proteins was changed by EGCG modification. In addition, the modifications resulted in the perturbation of the tertiary structure of SPI as evidenced by intrinsic fluorescence spectra and surface hydrophobicity measurements. Oil-in-water emulsions of modified SPI had smaller droplet sizes and better creaming stability compared to those from unmodified SPI. The covalent modification by EGCG improved the emulsifying property of SPI. This study provided an innovative approach for improving the emulsifying properties of proteins. This article is protected by copyright. All rights reserved.

A New Pathway for Protein Haptenation by β-Lactams.

PubMed

Pérez-Ruíz, Raúl; Lence, Emilio; Andreu, Inmaculada; Limones-Herrero, Daniel; González-Bello, Concepción; Miranda, Miguel A; Jiménez, M Consuelo

2017-10-09

The covalent binding of β-lactams to proteins upon photochemical activation has been demonstrated by using an integrated approach that combines photochemical, proteomic and computational studies, selecting human serum albumin (HSA) as a target protein and ezetimibe (1) as a probe. The results have revealed a novel protein haptenation pathway for this family of drugs that is an alternative to the known nucleophilic ring opening of β-lactams by the free amino group of lysine residues. Thus, photochemical ring splitting of the β-lactam ring, following a formal retro-Staudinger reaction, gives a highly reactive ketene intermediate that is trapped by the neighbouring lysine residues, leading to an amide adduct. For the investigated 1/HSA system, covalent modification of residues Lys414 and Lys525, which are located in sub-domains IIIA and IIIB, respectively, occurs. The observed photobinding may constitute the key step in the sequence of events leading to photoallergy. Docking and molecular dynamics simulation studies provide an insight into the molecular basis of the selectivity of 1 for these HSA sub-domains and the covalent modification mechanism. Computational studies also reveal positive cooperative binding of sub-domain IIIB that explains the experimentally observed modification of Lys414, which is located in a barely accessible pocket (sub-domain IIIA). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recording information on protein complexes in an information management system

PubMed Central

Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

2011-01-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

Impaired proteostasis: role in the pathogenesis of diabetes mellitus.

PubMed

Jaisson, Stéphane; Gillery, Philippe

2014-08-01

In living organisms, proteins are regularly exposed to 'molecular ageing', which corresponds to a set of non-enzymatic modifications that progressively cause irreversible damage to proteins. This phenomenon is greatly amplified under pathological conditions, such as diabetes mellitus. For their survival and optimal functioning, cells have to maintain protein homeostasis, also called 'proteostasis'. This process acts to maintain a high proportion of functional and undamaged proteins. Different mechanisms are involved in proteostasis, among them degradation systems (the main intracellular proteolytic systems being proteasome and lysosomes), folding systems (including molecular chaperones), and enzymatic mechanisms of protein repair. There is growing evidence that the disruption of proteostasis may constitute a determining event in pathophysiology. The aim of this review is to demonstrate how such a dysregulation may be involved in the pathogenesis of diabetes mellitus and in the onset of its long-term complications.

Epigenetic Effects of Ethanol on the Liver and Gastrointestinal System

PubMed Central

Shukla, Shivendra D.; Lim, Robert W.

2013-01-01

The widening web of epigenetic regulatory mechanisms also encompasses ethanol-induced changes in the gastrointestinal (GI)–hepatic system. In the past few years, increasing evidence has firmly established that alcohol modifies several epigenetic parameters in the GI tract and liver. The major pathways affected include DNA methylation, different site-specific modifications in histone proteins, and microRNAs. Ethanol metabolism, cell-signaling cascades, and oxidative stress have been implicated in these responses. Furthermore, ethanol-induced fatty liver (i.e., steatohepatitis) and progression of liver cancer (i.e., hepatic carcinoma) may be consequences of the altered epigenetics. Modification of gene and/or protein expression via epigenetic changes also may contribute to the cross-talk among the GI tract and the liver as well as to systemic changes involving other organs. Thus, epigenetic effects of ethanol may have a central role in the various pathophysiological responses induced by ethanol in multiple organs and mediated via the liver–GI axis. PMID:24313164

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections.

PubMed

Adzemovic, Milena Z; Zeitelhofer, Manuel; Leisser, Marianne; Köck, Ulricke; Kury, Angela; Olsson, Tomas

2016-11-14

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS). Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

Chemical methods for encoding and decoding of posttranslational modifications

PubMed Central

Chuh, Kelly N.; Batt, Anna R.; Pratt, Matthew R.

2016-01-01

A large array of posttranslational modifications can dramatically change the properties of proteins and influence different aspects of their biological function such as enzymatic activity, binding interactions, and proteostasis. Despite the significant knowledge that has been gained about the function of posttranslational modifications using traditional biological techniques, the analysis of the site-specific effects of a particular modification, the identification of the full compliment of modified proteins in the proteome, and the detection of new types of modifications remains challenging. Over the years, chemical methods have contributed significantly in both of these areas of research. This review highlights several posttranslational modifications where chemistry-based approaches have made significant contributions to our ability to both prepare homogeneously modified proteins and identify and characterize particular modifications in complex biological settings. As the number and chemical diversity of documented posttranslational modifications continues to rise, we believe that chemical strategies will be essential to advance the field in years to come. PMID:26933738

Mechanism of covalent modification of glyceraldehyde-3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related nitrosating agents.

PubMed

Mohr, S; Stamler, J S; Brüne, B

1994-07-18

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins.

PubMed

Brooks, Susan A

2006-06-01

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

O-GlcNAc profiling: from proteins to proteomes

PubMed Central

2014-01-01

O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics.

PubMed

Chen, Jin-Qiu; Wakefield, Lalage M; Goldstein, David J

2015-06-06

There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

Regulation of cell function by methionine oxidation and reduction

PubMed Central

Hoshi, Toshinori; Heinemann, Stefan H

2001-01-01

Reactive oxygen species (ROS) are generated during normal cellular activity and may exist in excess in some pathophysiological conditions, such as inflammation or reperfusion injury. These molecules oxidize a variety of cellular constituents, but sulfur-containing amino acid residues are especially susceptible. While reversible cysteine oxidation and reduction is part of well-established signalling systems, the oxidation and the enzymatically catalysed reduction of methionine is just emerging as a novel molecular mechanism for cellular regulation. Here we discuss how the oxidation of methionine to methionine sulfoxide in signalling proteins such as ion channels affects the function of these target proteins. Methionine sulfoxide reductase, which reduces methionine sulfoxide to methionine in a thioredoxin-dependent manner, is therefore not only an enzyme important for the repair of age- or degenerative disease-related protein modifications. It is also a potential missing link in the post-translational modification cycle involved in the specific oxidation and reduction of methionine residues in cellular signalling proteins, which may give rise to activity-dependent plastic changes in cellular excitability. PMID:11179387

Ion mobility mass spectrometry of proteins in a modified commercial mass spectrometer

NASA Astrophysics Data System (ADS)

Thalassinos, K.; Slade, S. E.; Jennings, K. R.; Scrivens, J. H.; Giles, K.; Wildgoose, J.; Hoyes, J.; Bateman, R. H.; Bowers, M. T.

2004-08-01

Ion mobility has emerged as an important technique for determining biopolymer conformations in solvent free environments. These experiments have been nearly exclusively performed on home built systems. In this paper we describe modifications to a commercial high performance mass spectrometer, the Waters UK "Ultima" Q-Tof, that allows high sensitivity measurement of peptide and protein cross sections. Arrival time distributions are obtained for a series of peptides (bradykinin, LHRH, substance P, bombesin) and proteins (bovine and equine cytochrome c, myoglobin, [alpha]-lactalbumin) with good agreement found with literature cross sections where available. In complex ATD's, mass spectra can be obtained for each feature confirming assignments. The increased sensitivity of the commercial instrument is retained along with the convenience of the data system, crucial features for analysis of protein misfolding systems.

Didehydrophenylalanine, an abundant modification in the beta subunit of plant polygalacturonases.

PubMed

Sergeant, Kjell; Printz, Bruno; Gutsch, Annelie; Behr, Marc; Renaut, Jenny; Hausman, Jean-Francois

2017-01-01

The structure and the activity of proteins are often regulated by transient or stable post- translational modifications (PTM). Different from well-known, abundant modifications such as phosphorylation and glycosylation some modifications are limited to one or a few proteins across a broad range of related species. Although few examples of the latter type are known, the evolutionary conservation of these modifications and the enzymes responsible for their synthesis suggest an important physiological role. Here, the first observation of a new, fold-directing PTM is described. During the analysis of alfalfa cell wall proteins a -2Da mass shift was observed on phenylalanine residues in the repeated tetrapeptide FxxY of the beta-subunit of polygalacturonase. This modular protein is known to be involved in developmental and stress-responsive processes. The presence of this modification was confirmed using in-house and external datasets acquired by different commonly used techniques in proteome studies. Based on these analyses it was found that all identified phenylalanine residues in the sequence FxxY of this protein were modified to α,β-didehydro-Phe (ΔPhe). Besides showing the reproducible identification of ΔPhe in different species arguments that substantiate the fold-determining role of ΔPhe are given.

Sequence tagging reveals unexpected modifications in toxicoproteomics

PubMed Central

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

2010-01-01

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

Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Qian, Weijun

2013-02-01

Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

Molecular Genetic and Gene Therapy Studies of the Musculoskeletal System

DTIC Science & Technology

2004-10-01

H3023F07 Cct5 GO:0006457 protein folding H3023D07 D630041 K24Rik GO:0006464 protein modification H3066E06 C430014H23Rik GO:0006468 protein amino acid ...phosphorylation H3057F01 Csnkle GO:0006468 protein amino acid phosphorylation H3063A08 Lgmn GO:0006508 proteolysis and peptidolysis H3048C09 Fbxl12 GO...H3001E07 0.45 UNKNOWN H3057F01 Csnkle 0.44 Mus musculus casein circadian kinase 1, epsilon rhythm(GO:0007623);protein (Csnkle), mRNA amino acid

Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

DOEpatents

Wang, Jiangyun; Schultz, Peter G.

2013-03-12

The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

Biochemical quantitation of the eIF5A hypusination in Arabidopsis thaliana uncovers ABA-dependent regulation

PubMed Central

Belda-Palazón, Borja; Nohales, María A.; Rambla, José L.; Aceña, José L.; Delgado, Oscar; Fustero, Santos; Martínez, M. Carmen; Granell, Antonio; Carbonell, Juan; Ferrando, Alejandro

2014-01-01

The eukaryotic translation elongation factor eIF5A is the only protein known to contain the unusual amino acid hypusine which is essential for its biological activity. This post-translational modification is achieved by the sequential action of the enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The crucial molecular function of eIF5A during translation has been recently elucidated in yeast and it is expected to be fully conserved in every eukaryotic cell, however the functional description of this pathway in plants is still sparse. The genetic approaches with transgenic plants for either eIF5A overexpression or antisense have revealed some activities related to the control of cell death processes but the molecular details remain to be characterized. One important aspect of fully understanding this pathway is the biochemical description of the hypusine modification system. Here we have used recombinant eIF5A proteins either modified by hypusination or non-modified to establish a bi-dimensional electrophoresis (2D-E) profile for the three eIF5A protein isoforms and their hypusinated or unmodified proteoforms present in Arabidopsis thaliana. The combined use of the recombinant 2D-E profile together with 2D-E/western blot analysis from whole plant extracts has provided a quantitative approach to measure the hypusination status of eIF5A. We have used this information to demonstrate that treatment with the hormone abscisic acid produces an alteration of the hypusine modification system in Arabidopsis thaliana. Overall this study presents the first biochemical description of the post-translational modification of eIF5A by hypusination which will be functionally relevant for future studies related to the characterization of this pathway in Arabidopsis thaliana. PMID:24904603

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output

PubMed Central

Gresham, Kenneth S.; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin

2017-01-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress. PMID:27909224

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

PubMed

Gresham, Kenneth S; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin; Stelzer, Julian E

2017-03-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/d t max and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca 2+ activation compared with SAL-treated mice but unaltered myofilament Ca 2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress. Copyright © 2017 the American Physiological Society.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins

PubMed Central

Yao, Chunxiang; Behring, Jessica B.; Shao, Di; Sverdlov, Aaron L.; Whelan, Stephen A.; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A.; Seta, Francesca; Costello, Catherine E.; Cohen, Richard A.; Matsui, Reiko; Colucci, Wilson S.; McComb, Mark E.; Bachschmid, Markus M.

2015-01-01

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a ‘Tandem Mass Tag’ (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation. PMID:26642319

Chemical Posttranslational Modification with Designed Rhodium(II) Catalysts.

PubMed

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

2016-01-01

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

Re-analysis of protein data reveals the germination pathway and up accumulation mechanism of cell wall hydrolases during the radicle protrusion step of seed germination in Podophyllum hexandrum- a high altitude plant

PubMed Central

Dogra, Vivek; Bagler, Ganesh; Sreenivasulu, Yelam

2015-01-01

Podophyllum hexandrum Royle is an important high-altitude plant of Himalayas with immense medicinal value. Earlier, it was reported that the cell wall hydrolases were up accumulated during radicle protrusion step of Podophyllum seed germination. In the present study, Podophyllum seed Germination protein interaction Network (PGN) was constructed by using the differentially accumulated protein (DAP) data set of Podophyllum during the radicle protrusion step of seed germination, with reference to Arabidopsis protein–protein interaction network (AtPIN). The developed PGN is comprised of a giant cluster with 1028 proteins having 10,519 interactions and a few small clusters with relevant gene ontological signatures. In this analysis, a germination pathway related cluster which is also central to the topology and information dynamics of PGN was obtained with a set of 60 key proteins. Among these, eight proteins which are known to be involved in signaling, metabolism, protein modification, cell wall modification, and cell cycle regulation processes were found commonly highlighted in both the proteomic and interactome analysis. The systems-level analysis of PGN identified the key proteins involved in radicle protrusion step of seed germination in Podophyllum. PMID:26579141

Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

PubMed

Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

2017-04-01

Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated. Copyright © 2017 Elsevier B.V. All rights reserved.

The rational parameterization theorem for multisite post-translational modification systems.

PubMed

Thomson, Matthew; Gunawardena, Jeremy

2009-12-21

Post-translational modification of proteins plays a central role in cellular regulation but its study has been hampered by the exponential increase in substrate modification forms ("modforms") with increasing numbers of sites. We consider here biochemical networks arising from post-translational modification under mass-action kinetics, allowing for multiple substrates, having different types of modification (phosphorylation, methylation, acetylation, etc.) on multiple sites, acted upon by multiple forward and reverse enzymes (in total number L), using general enzymatic mechanisms. These assumptions are substantially more general than in previous studies. We show that the steady-state modform concentrations constitute an algebraic variety that can be parameterized by rational functions of the L free enzyme concentrations, with coefficients which are rational functions of the rate constants. The parameterization allows steady states to be calculated by solving L algebraic equations, a dramatic reduction compared to simulating an exponentially large number of differential equations. This complexity collapse enables analysis in contexts that were previously intractable and leads to biological predictions that we review. Our results lay a foundation for the systems biology of post-translational modification and suggest deeper connections between biochemical networks and algebraic geometry.

Methylated nucleosides in tRNA and tRNA methyltransferases

PubMed Central

Hori, Hiroyuki

2014-01-01

To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

Identification of Sumoylated Proteins in the Silkworm Bombyx mori

PubMed Central

Tang, Xudong; Fu, Xuliang; Hao, Bifang; Zhu, Feng; Xiao, Shengyan; Xu, Li; Shen, Zhongyuan

2014-01-01

Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori. PMID:25470021

Nanoparticle-macrophage interactions: A balance between clearance and cell-specific targeting

PubMed Central

Rattan, Rahul; Bhattacharjee, Somnath; Zong, Hong; Swain, Corban; Siddiqui, Muneeb A.; Visovatti, Scott H.; Kanthi, Yogendra; Desai, Sajani; Pinsky, David J.; Goonewardena, Sascha N.

2017-01-01

The surface properties of nanoparticles (NPs) are a major factor that influences how these nanomaterials interact with biological systems. Interactions between NPs and macrophages of the reticuloendothelial system (RES) can reduce the efficacy of NP diagnostics and therapeutics. Traditionally, to limit NP clearance by the RES system, the NP surface is neutralized with molecules like poly(ethylene glycol) (PEG) which are known to resist protein adsorption and RES clearance. Unfortunately, PEG modification is not without drawbacks including difficulties with the synthesis and associations with immune reactions. To overcome some of these obstacles, we neutralized the NP surface by acetylation and compared this modification to PEGylation for RES clearance and tumor-specific targeting. We found that acetylation was comparable to PEGylation in reducing RES clearance. Additionally, we found that dendrimer acetylation did not impact folic acid (FA)-mediated targeting of tumor cells whereas PEG surface modification reduced the targeting ability of the NP. These results clarify the impact of different NP surface modifications on RES clearance and cell-specific targeting and provide insights into the design of more effective NPs. PMID:28705434

A Pictet-Spengler ligation for protein chemical modification

PubMed Central

Agarwal, Paresh; van der Weijden, Joep; Sletten, Ellen M.; Rabuka, David; Bertozzi, Carolyn R.

2013-01-01

Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C–C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications. PMID:23237853

Novel, fluorescent, SSB protein chimeras with broad utility

PubMed Central

Liu, Juan; Choi, Meerim; Stanenas, Adam G; Byrd, Alicia K; Raney, Kevin D; Cohan, Christopher; Bianco, Piero R

2011-01-01

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use. PMID:21462278

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity

PubMed Central

Basila, Megan; Kelley, Melissa L.

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing. PMID:29176845

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

PubMed

Basila, Megan; Kelley, Melissa L; Smith, Anja van Brabant

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

DELIVERY OF THERAPEUTIC PROTEINS

PubMed Central

Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

2009-01-01

The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

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

PubMed Central

Wells, Lance

2016-01-01

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

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

PubMed Central

Mahajan, Rohit; Gerace, Larry; Melchior, Frauke

1998-01-01

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role. PMID:9442102

Gold nanoparticle-DNA aptamer composites as a universal carrier for in vivo delivery of biologically functional proteins.

PubMed

Ryou, Sang-Mi; Yeom, Ji-Hyun; Kang, Hyo Jung; Won, Miae; Kim, Jin-Sik; Lee, Boeun; Seong, Maeng-Je; Ha, Nam-Chul; Bae, Jeehyeon; Lee, Kangseok

2014-12-28

Although the delivery of biologically functional protein(s) into mammalian cells could be of tremendous value to biomedical research, the development of such technology has been hindered by the lack of a safe and effective delivery method. Here, we present a simple, efficient, and versatile gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system, with nanoblock-like properties, that allows any recombinant protein to be loaded without additional modifications and delivered into mammalian living systems. AuNP-Apt-based protein delivery system was able to deliver various proteins into variety of cell types in vitro without showing cytotoxicity. This AuNP-Apt system was also effective for the local and systemic targeted delivery of proteins in vivo. A local injection of the AuNP-Apt loaded with the apoptosis-inducing BIM protein efficiently inhibited the growth of xenograft tumors in mice. Furthermore, an intravenous injection of AuNP-Apt loaded with both epidermal growth factor (EGF) and BIM resulted in the targeted delivery of BIM into a xenograft tumor derived from EGF receptor-overexpressing cancer cells with no detectable systemic toxicity. Our findings show that this system can serve as an innovative platform for the development of protein-based biomedical applications. Copyright © 2014 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells.more » As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.« less

Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

PubMed

Leurs, Melanie; Tiller, Joerg C

2017-01-01

The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chhabra, Swapnil; Butland, Gareth; Elias, Dwayne A

The ability to conduct advanced functional genomic studies of the thousands of 38 sequenced bacteria has been hampered by the lack of available tools for making high39 throughput chromosomal manipulations in a systematic manner that can be applied across 40 diverse species. In this work, we highlight the use of synthetic biological tools to 41 assemble custom suicide vectors with reusable and interchangeable DNA parts to 42 facilitate chromosomal modification at designated loci. These constructs enable an array 43 of downstream applications including gene replacement and creation of gene fusions with 44 affinity purification or localization tags. We employed thismore » approach to engineer 45 chromosomal modifications in a bacterium that has previously proven difficult to 46 manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of 47 662 strains. Furthermore, we demonstrate how these modifications can be used for 48 examining metabolic pathways, protein-protein interactions, and protein localization. The 49 ubiquity of suicide constructs in gene replacement throughout biology suggests that this 50 approach can be applied to engineer a broad range of species for a diverse array of 51 systems biological applications and is amenable to high-throughput implementation.« less

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis

PubMed Central

Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang

2017-01-01

Summary The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population. PMID:26360626

Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis.

PubMed

Xu, Shou-Ling; Chalkley, Robert J; Maynard, Jason C; Wang, Wenfei; Ni, Weimin; Jiang, Xiaoyue; Shin, Kihye; Cheng, Ling; Savage, Dasha; Hühmer, Andreas F R; Burlingame, Alma L; Wang, Zhi-Yong

2017-02-21

Genetic studies have shown essential functions of O-linked N -acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

PubMed

Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

2015-09-01

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Comparative examination of adsorption of serum proteins on HSA- and PLGA-based nanoparticles using SDS-PAGE and LC-MS.

PubMed

Gossmann, R; Fahrländer, E; Hummel, M; Mulac, D; Brockmeyer, J; Langer, K

2015-06-01

The behavior of nanosized drug carrier systems under cell culture conditions and therefore also the destiny in the body are highly influenced by the protein corona, which is formed upon entering a biological environment. Some of the adsorbed proteins, named opsonins, lead to a shortened plasma circulation half-life of the nanoparticles. Others are attributed to promote the transport of nanoparticles into other compartments of the body, just to mention two examples. Hence, detailed knowledge concerning the composition of the protein corona is of great importance. The aim of this work was to investigate the influence of the nanoparticle starting material and the surface modification on the composition of the adsorbed serum proteins in a cell culture environment. Therefore, positively charged nanoparticles based on the biodegradable polymer poly(dl-lactide-co-glycolide) (PLGA) stabilized with didodecyldimethylammonium bromide (DMAB) and negatively charged nanoparticles based on human serum albumin (HSA) were prepared and modified with hydrophilic polymers. By incubating the nanoparticles with fetal bovine serum (FBS) the adsorption of serum proteins on the colloidal system was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a semi-quantitative analysis of the protein corona was performed and after enzymatic in-solution-digestion the adsorbed proteins were identified using high resolution LC-MS. Our study accentuates the influence of the core material, surface charge, and surface modification on the amount and nature of the adsorbed proteins. The combination of SDS-PAGE and LC-MS turns out to be a simple and reliable method to investigate the protein corona of nanoparticles. Copyright © 2015 Elsevier B.V. All rights reserved.

Nitric Oxide-Dependent Posttranslational Modification in Plants: An Update

PubMed Central

Astier, Jeremy; Lindermayr, Christian

2012-01-01

Nitric oxide (NO) has been demonstrated as an essential regulator of several physiological processes in plants. The understanding of the molecular mechanism underlying its critical role constitutes a major field of research. NO can exert its biological function through different ways, such as the modulation of gene expression, the mobilization of second messengers, or interplays with protein kinases. Besides this signaling events, NO can be responsible of the posttranslational modifications (PTM) of target proteins. Several modifications have been identified so far, whereas metal nitrosylation, the tyrosine nitration and the S-nitrosylation can be considered as the main ones. Recent data demonstrate that these PTM are involved in the control of a wide range of physiological processes in plants, such as the plant immune system. However, a great deal of effort is still necessary to pinpoint the role of each PTM in plant physiology. Taken together, these new advances in proteomic research provide a better comprehension of the role of NO in plant signaling. PMID:23203119

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability

PubMed Central

Khan, Niamat; Lenz, Christof; Binder, Lutz; Pantakani, Dasaradha Venkata Krishna; Asif, Abdul R.

2016-01-01

Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea. Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability. Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability. Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients. PMID:27104530

A view inside the nature of protein crystals

NASA Astrophysics Data System (ADS)

Oswald, R.; Pietzsch, M.; Ulrich, J.

2017-07-01

In this work a fundamental analysis of protein crystal modifications was presented to compare and confirm the components of protein crystal modifications. The result is that a protein crystal contains besides the protein, the precipitant and water. A mass spectrometer coupled to a thermogravimetry device was used to confirm the different waters (free water -the chosen buffer- and bound water) inside the crystals. Here the biggest amount of water is the free water (the buffer) with an amount of approximately 35%. The bound water (in the sense of a hydrate) has only an amount of about 1-1.5%. Furthermore, an x-ray analysis to confirm the influence range of pH value on the stability of one crystal modification for the understanding of effects on dissolution mechanism of protein crystals was investigated. The crystals of the tetragonal modification crystallized at pH 4.7, 4.85, 5.0, 5.15 and 5.3 maintain according to the x-ray measurements the same lattice parameters. The measured data are discussed.

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

PubMed

Liang, Lunxi; Wang, Huanbin; Shi, Hubing; Li, Zhaoli; Yao, Han; Bu, Zhigao; Song, Ningning; Li, Chushu; Xiang, Dabin; Zhang, Yao; Wang, Jilin; Hu, Ye; Xu, Qi; Ma, Yanlei; Cheng, Zhongyi; Wang, Yingchao; Zhao, Shuliang; Qian, Jin; Chen, Yingxuan; Fang, Jing-Yuan; Xu, Jie

2018-06-21

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule. Copyright © 2018 Elsevier Ltd. All rights reserved.

Permuting the PGF Signature Motif Blocks both Archaeosortase-Dependent C-Terminal Cleavage and Prenyl Lipid Attachment for the Haloferax volcanii S-Layer Glycoprotein.

PubMed

Abdul Halim, Mohd Farid; Karch, Kelly R; Zhou, Yitian; Haft, Daniel H; Garcia, Benjamin A; Pohlschroder, Mechthild

2015-12-28

For years, the S-layer glycoprotein (SLG), the sole component of many archaeal cell walls, was thought to be anchored to the cell surface by a C-terminal transmembrane segment. Recently, however, we demonstrated that the Haloferax volcanii SLG C terminus is removed by an archaeosortase (ArtA), a novel peptidase. SLG, which was previously shown to be lipid modified, contains a C-terminal tripartite structure, including a highly conserved proline-glycine-phenylalanine (PGF) motif. Here, we demonstrate that ArtA does not process an SLG variant where the PGF motif is replaced with a PFG motif (slg(G796F,F797G)). Furthermore, using radiolabeling, we show that SLG lipid modification requires the PGF motif and is ArtA dependent, lending confirmation to the use of a novel C-terminal lipid-mediated protein-anchoring mechanism by prokaryotes. Similar to the case for the ΔartA strain, the growth, cellular morphology, and cell wall of the slg(G796F,F797G) strain, in which modifications of additional H. volcanii ArtA substrates should not be altered, are adversely affected, demonstrating the importance of these posttranslational SLG modifications. Our data suggest that ArtA is either directly or indirectly involved in a novel proteolysis-coupled, covalent lipid-mediated anchoring mechanism. Given that archaeosortase homologs are encoded by a broad range of prokaryotes, it is likely that this anchoring mechanism is widely conserved. Prokaryotic proteins bound to cell surfaces through intercalation, covalent attachment, or protein-protein interactions play critical roles in essential cellular processes. Unfortunately, the molecular mechanisms that anchor proteins to archaeal cell surfaces remain poorly characterized. Here, using the archaeon H. volcanii as a model system, we report the first in vivo studies of a novel protein-anchoring pathway involving lipid modification of a peptidase-processed C terminus. Our findings not only yield important insights into poorly understood aspects of archaeal biology but also have important implications for key bacterial species, including those of the human microbiome. Additionally, insights may facilitate industrial applications, given that photosynthetic cyanobacteria encode uncharacterized homologs of this evolutionarily conserved enzyme, or may spur development of unique drug delivery systems. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

PubMed

Deming, Timothy J

2017-03-15

Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

Biological Chemistry and Functionality of Protein Sulfenic Acids and Related Thiol Modifications

PubMed Central

Devarie-Baez, Nelmi O.; Silva Lopez, Elsa I.; Furdui, Cristina M.

2016-01-01

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease. PMID:26340608

Thioredoxin 1-Mediated Post-Translational Modifications: Reduction, Transnitrosylation, Denitrosylation, and Related Proteomics Methodologies

PubMed Central

Wu, Changgong; Parrott, Andrew M.; Fu, Cexiong; Liu, Tong; Marino, Stefano M.; Gladyshev, Vadim N.; Jain, Mohit R.; Baykal, Ahmet T.; Li, Qing; Oka, Shinichi; Sadoshima, Junichi; Beuve, Annie; Simmons, William J.

2011-01-01

Abstract Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction. Antioxid. Redox Signal. 15, 2565–2604. PMID:21453190

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system.

PubMed

Kajino, T; Saito, Y; Asami, O; Yamada, Y; Hirai, M; Udata, S

1997-10-01

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L-1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

Synapse formation and plasticity: recent insights from the perspective of the ubiquitin proteasome system.

PubMed

Patrick, Gentry N

2006-02-01

The formation of synaptic connections during the development of the nervous system requires the precise targeting of presynaptic and postsynaptic compartments. Furthermore, synapses are continually modified in the brain by experience. Recently, the ubiquitin proteasome system has emerged as a key regulator of synaptic development and function. The modification of proteins by ubiquitin, and in many cases their subsequent proteasomal degradation, has proven to be an important mechanism to control protein stability, activity and localization at synapses. Recent work has highlighted key questions of the UPS during the development and remodeling of synaptic connections in the nervous system.

On the biomarkers and mechanisms of konzo, a distinct upper motor neuron disease associated with food (cassava) cyanogenic exposure.

PubMed

Kassa, Roman M; Kasensa, Nyamabo L; Monterroso, Victor H; Kayton, Robert J; Klimek, John E; David, Larry L; Lunganza, Kalala R; Kayembe, Kazadi T; Bentivoglio, Marina; Juliano, Sharon L; Tshala-Katumbay, Desire D

2011-03-01

Konzo is a self-limiting central motor-system disease associated with food dependency on cassava and low dietary intake of sulfur amino acids (SAA). Under conditions of SAA-deficiency, ingested cassava cyanogens yield metabolites that include thiocyanate and cyanate, a protein-carbamoylating agent. We studied the physical and biochemical modifications of rat serum and spinal cord proteins arising from intoxication of young adult rats with 50-200mg/kg linamarin, or 200mg/kg sodium cyanate (NaOCN), or vehicle (saline) and fed either a normal amino acid- or SAA-deficient diet for up to 2 weeks. Animals under SAA-deficient diet and treatment with linamarin or NaOCN developed hind limb tremors or motor weakness, respectively. LC/MS-MS analysis revealed differential albumin carbamoylation in animals treated with NaOCN, vs. linamarin/SAA-deficient diet, or vehicle. 2D-DIGE and MALDI-TOF/MS-MS analysis of the spinal cord proteome showed differential expression of proteins involved in oxidative mechanisms (e.g. peroxiredoxin 6), endocytic vesicular trafficking (e.g. dynamin 1), protein folding (e.g. protein disulfide isomerase), and maintenance of the cytoskeleton integrity (e.g. α-spectrin). Studies are needed to elucidate the role of the aformentioned modifications in the pathogenesis of cassava-associated motor-system disease. Copyright © 2010 Elsevier Ltd. All rights reserved.

Functional analysis of proteins and protein species using shotgun proteomics and linear mathematics.

PubMed

Hoehenwarter, Wolfgang; Chen, Yanmei; Recuenco-Munoz, Luis; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Covalent post-translational modification of proteins is the primary modulator of protein function in the cell. It greatly expands the functional potential of the proteome compared to the genome. In the past few years shotgun proteomics-based research, where the proteome is digested into peptides prior to mass spectrometric analysis has been prolific in this area. It has determined the kinetics of tens of thousands of sites of covalent modification on an equally large number of proteins under various biological conditions and uncovered a transiently active regulatory network that extends into diverse branches of cellular physiology. In this review, we discuss this work in light of the concept of protein speciation, which emphasizes the entire post-translationally modified molecule and its interactions and not just the modification site as the functional entity. Sometimes, particularly when considering complex multisite modification, all of the modified molecular species involved in the investigated condition, the protein species must be completely resolved for full understanding. We present a mathematical technique that delivers a good approximation for shotgun proteomics data.

Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2016-09-01

Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein breakdown and release of β-casomorphins during in vitro gastro-intestinal digestion of sterilised model systems of liquid infant formula.

PubMed

Cattaneo, Stefano; Stuknytė, Milda; Masotti, Fabio; De Noni, Ivano

2017-02-15

Protein modifications occurring during sterilisation of infant formulas can affect protein digestibility and release of bioactive peptides. The effect of glycation and cross-linking on protein breakdown and release of β-casomorphins was evaluated during in vitro gastro-intestinal digestion (GID) of six sterilised model systems of infant formula. Protein degradation during in vitro GID was evaluated by SDS-PAGE and by measuring the nitrogen content of ultrafiltration (3kDa) permeates before and after in vitro GID of model IFs. Glycation strongly hindered protein breakdown, whereas cross-linking resulting from β-elimination reactions had a negligible effect. Only β-casomorphin 7 (β-CM7) was detected (0.187-0.858mgL(-1)) at the end of the intestinal digestion in all untreated IF model systems. The level of β-CM7 in the sterilised model systems prepared without addition of sugars ranged from 0.256 to 0.655mgL(-1). The release of this peptide during GID was hindered by protein glycation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

PubMed

Jantsch, Michael F; Quattrone, Alessandro; O'Connell, Mary; Helm, Mark; Frye, Michaela; Macias-Gonzales, Manuel; Ohman, Marie; Ameres, Stefan; Willems, Luc; Fuks, Francois; Oulas, Anastasis; Vanacova, Stepanka; Nielsen, Henrik; Bousquet-Antonelli, Cecile; Motorin, Yuri; Roignant, Jean-Yves; Balatsos, Nikolaos; Dinnyes, Andras; Baranov, Pavel; Kelly, Vincent; Lamm, Ayelet; Rechavi, Gideon; Pelizzola, Mattia; Liepins, Janis; Holodnuka Kholodnyuk, Irina; Zammit, Vanessa; Ayers, Duncan; Drablos, Finn; Dahl, John Arne; Bujnicki, Janusz; Jeronimo, Carmen; Almeida, Raquel; Neagu, Monica; Costache, Marieta; Bankovic, Jasna; Banovic, Bojana; Kyselovic, Jan; Valor, Luis Miguel; Selbert, Stefan; Pir, Pinar; Demircan, Turan; Cowling, Victoria; Schäfer, Matthias; Rossmanith, Walter; Lafontaine, Denis; David, Alexandre; Carre, Clement; Lyko, Frank; Schaffrath, Raffael; Schwartz, Schraga; Verdel, Andre; Klungland, Arne; Purta, Elzbieta; Timotijevic, Gordana; Cardona, Fernando; Davalos, Alberto; Ballana, Ester; O Carroll, Donal; Ule, Jernej; Fray, Rupert

2018-05-09

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

Modifications of Glycans: Biological Significance and Therapeutic Opportunities

PubMed Central

Muthana, Saddam M.; Campbell, Christopher; Gildersleeve, Jeffrey C.

2012-01-01

Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate’s overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as postglycosylational modifications. PMID:22195988

Arraying proteins by cell-free synthesis.

PubMed

He, Mingyue; Wang, Ming-Wei

2007-10-01

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

[Prokaryotic expression systems].

PubMed

Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

2013-03-01

For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

Molecular piracy: manipulation of the ubiquitin system by Kaposi's sarcoma-associated herpesvirus.

PubMed

Fujimuro, Masahiro; Hayward, S Diane; Yokosawa, Hideyoshi

2007-01-01

Ubiquitination, one of several post-translational protein modifications, plays a key role in the regulation of cellular events, including protein degradation, signal transduction, endocytosis, protein trafficking, apoptosis and immune responses. Ubiquitin attachment at the lysine residue of cellular factors acts as a signal for endocytosis and rapid degradation by the 26S proteasome. It has recently been observed that viruses, especially oncogenic herpesviruses, utilise molecular piracy by encoding their own proteins to interfere with regulation of cell signalling. Kaposi's sarcoma- associated herpesvirus (KSHV) manipulates the ubiquitin system to facilitate cell proliferation, anti-apoptosis and evasion from immunity. In this review, we will describe the strategies used by KSHV at distinct stages of the viral life-cycle to control the ubiquitin system and promote oncogenesis and viral persistence. (c) 2007 John Wiley & Sons, Ltd.

Examining protein-lipid interactions in model systems with a new squarylium fluorescent dye.

PubMed

Ioffe, Valeriya M; Gorbenko, Galyna P; Tatarets, Anatoliy L; Patsenker, Leonid D; Terpechnig, Ewald A

2006-07-01

The applicability of newly synthesized squarylium dye Sq to probing the changes in physical characteristics of lipid bilayer on the formation of protein-lipid complexes has been evaluated. Lipid vesicles composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and cholesterol (Chol) were employed as lipid component of model membrane systems while protein constituent was represented by lysozyme (Lz). Fluorescence intensity of Sq was found to decrease on Lz association with lipid bilayer. This effect was observed in all kinds of model systems suggesting that Sq is sensitive to modification of lipid bilayer physical properties on hydrophobic protein-lipid interactions. It was found that Sq spectral response to variations in Chol content depends on relative contributions of electrostatic and hydrophobic components of Lz-membrane binding.

Chemical Methods for the Direct Detection and Labeling of S-Nitrosothiols

PubMed Central

Bechtold, Erika

2012-01-01

Abstract Significance: Posttranslational modification of proteins through phosphorylation, glycosylation, and oxidation adds complexity to the proteome by reversibly altering the structure and function of target proteins in a highly controlled fashion. Recent Advances: The study of reversible cysteine oxidation highlights a role for this oxidative modification in complex signal transduction pathways. Nitric oxide (NO), and its respective metabolites (including reactive nitrogen species), participates in a variety of these cellular redox processes, including the reversible oxidation of cysteine to S-nitrosothiols (RSNOs). RSNOs act as endogenous transporters of NO, but also possess beneficial effects independent of NO-related signaling, which suggests a complex and versatile biological role. In this review, we highlight the importance of RSNOs as a required posttranslational modification and summarize the current methods available for detecting S-nitrosation. Critical Issues: Given the limitations of these indirect detection methods, the review covers recent developments toward the direct detection of RSNOs by phosphine-based chemical probes. The intrinsic properties that dictate this phosphine/RSNO reactivity are summarized. In general, RSNOs (both small molecule and protein) react with phosphines to yield reactive S-substituted aza-ylides that undergo further reactions leading to stable RSNO-based adducts. Future Directions: This newly explored chemical reactivity forms the basis of a number of exciting potential chemical methods for protein RSNO detection in biological systems. Antioxid. Redox Signal. 17, 981–991. PMID:22356122

The structural damages of lens crystallins induced by peroxynitrite and methylglyoxal, two causative players in diabetic complications and preventive role of lens antioxidant components.

PubMed

Moghadam, Sogand Sasan; Oryan, Ahmad; Kurganov, Boris I; Tamaddon, Ali-Mohammad; Alavianehr, Mohammad Mehdi; Moosavi-Movahedi, Ali Akbar; Yousefi, Reza

2017-10-01

Peroxynitrite (PON) and methylglyoxal (MGO), two diabetes-associated compounds, are believed to be important causative players in development of diabetic cataracts. In the current study, different spectroscopic methods, gel electrophoresis, lens culture and microscopic assessments were applied to examine the impact of individual, subsequent or simultaneous modification of lens crystallins with MGO and PON on their structure, oligomerization and aggregation. The protein modifications were confirmed with detection of the significantly increased quantity of carbonyl groups and decreased levels of sulfhydryl, tyrosine and tryptophan. Also, lens proteins modification with these chemical agents was accompanied with important structural alteration, oligomerization, disulfide/chromophore mediated protein crosslinking and important proteolytic instability. All these structural damages were more pronounced when the lens proteins were modified in the presence of both mentioned chemical agents, either in sequential or simultaneous manner. Ascorbic acid and glutathione, as the main components of lens antioxidant defense mechanism, were also capable to markedly prevent the damaging effects of PON and MGO on lens crystallins, as indicated by gel electrophoresis. The results of this study may highlight the importance of lens antioxidant defense system in protection of crystallins against the structural insults induced by PON and MGO during chronic hyperglycemia in the diabetic patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamic regulation of mitochondrial fission through modification of the dynamin-related protein Drp1

PubMed Central

Chang, Chuang-Rung; Blackstone, Craig

2017-01-01

Mitochondria in cells comprise a tubulovesicular network shaped continuously by complementary fission and fusion events. The mammalian Drp1 protein plays a key role in fission, while Mfn1, Mfn2, and OPA1 are required for fusion. Shifts in the balance between these opposing processes can occur rapidly, indicating that modifications to these proteins may regulate mitochondrial membrane dynamics. We highlight posttranslational modifications of the mitochondrial fission protein Drp1, for which these regulatory mechanisms are best characterized. This dynamin-related GTPase undergoes a number of steps to mediate mitochondrial fission, including translocation from cytoplasm to the mitochondrial outer membrane, higher-order assembly into spirals, GTP hydrolysis associated with a conformational change and membrane deformation, and ultimately disassembly. Many of these steps may be influenced by covalent modification of Drp1. We discuss the dynamic nature of Drp1 modifications and how they contribute not only to the normal regulation of mitochondrial division, but also to neuropathologic processes. PMID:20649536

Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins.

PubMed

Vásquez-Garzón, Verónica R; Rouimi, Patrick; Jouanin, Isabelle; Waeg, Georg; Zarkovic, Neven; Villa-Treviño, Saul; Guéraud, Françoise

2012-05-01

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.

Regulation of Protein Degradation by O-GlcNAcylation: Crosstalk with Ubiquitination*

PubMed Central

Ruan, Hai-Bin; Nie, Yongzhan; Yang, Xiaoyong

2013-01-01

The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Misfolded, damaged, and unwanted proteins are tagged with a chain of ubiquitin moieties for degradation by the proteasome, which is critical for cellular homeostasis. In this review, we summarize the current knowledge of the interplay between O-GlcNAcylation and ubiquitination in the control of protein degradation. Understanding the mechanisms of action of O-GlcNAcylation in the ubiquitin-proteosome system shall facilitate the development of therapeutics for human diseases such as cancer, metabolic syndrome, and neurodegenerative diseases. PMID:23824911

Protein O-GlcNAcylation: a new signaling paradigm for the cardiovascular system

PubMed Central

Laczy, Boglarka; Hill, Bradford G.; Wang, Kai; Paterson, Andrew J.; White, C. Roger; Xing, Dongqi; Chen, Yiu-Fai; Darley-Usmar, Victor; Oparil, Suzanne; Chatham, John C.

2009-01-01

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demonstrating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GlcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function. PMID:19028792

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

PubMed

Drake, Walter R; Hou, Ching-Wen; Zachara, Natasha E; Grimes, Catherine Leimkuhler

2018-06-01

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

Prediction of Protein Modification Sites of Pyrrolidone Carboxylic Acid Using mRMR Feature Selection and Analysis

PubMed Central

Zheng, Lu-Lu; Niu, Shen; Hao, Pei; Feng, KaiYan; Cai, Yu-Dong; Li, Yixue

2011-01-01

Pyrrolidone carboxylic acid (PCA) is formed during a common post-translational modification (PTM) of extracellular and multi-pass membrane proteins. In this study, we developed a new predictor to predict the modification sites of PCA based on maximum relevance minimum redundancy (mRMR) and incremental feature selection (IFS). We incorporated 727 features that belonged to 7 kinds of protein properties to predict the modification sites, including sequence conservation, residual disorder, amino acid factor, secondary structure and solvent accessibility, gain/loss of amino acid during evolution, propensity of amino acid to be conserved at protein-protein interface and protein surface, and deviation of side chain carbon atom number. Among these 727 features, 244 features were selected by mRMR and IFS as the optimized features for the prediction, with which the prediction model achieved a maximum of MCC of 0.7812. Feature analysis showed that all feature types contributed to the modification process. Further site-specific feature analysis showed that the features derived from PCA's surrounding sites contributed more to the determination of PCA sites than other sites. The detailed feature analysis in this paper might provide important clues for understanding the mechanism of the PCA formation and guide relevant experimental validations. PMID:22174779

Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

PubMed Central

Konrad, Zvia; Eichler, Jerry

2002-01-01

Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

Analysis of Cysteine Redox Post-Translational Modifications in Cell Biology and Drug Pharmacology.

PubMed

Wani, Revati; Murray, Brion W

2017-01-01

Reversible cysteine oxidation is an emerging class of protein post-translational modification (PTM) that regulates catalytic activity, modulates conformation, impacts protein-protein interactions, and affects subcellular trafficking of numerous proteins. Redox PTMs encompass a broad array of cysteine oxidation reactions with different half-lives, topographies, and reactivities such as S-glutathionylation and sulfoxidation. Recent studies from our group underscore the lesser known effect of redox protein modifications on drug binding. To date, biological studies to understand mechanistic and functional aspects of redox regulation are technically challenging. A prominent issue is the lack of tools for labeling proteins oxidized to select chemotype/oxidant species in cells. Predictive computational tools and curated databases of oxidized proteins are facilitating structural and functional insights into regulation of the network of oxidized proteins or redox proteome. In this chapter, we discuss analytical platforms for studying protein oxidation, suggest computational tools currently available in the field to determine redox sensitive proteins, and begin to illuminate roles of cysteine redox PTMs in drug pharmacology.

Biophysical analysis of the effect of chemical modification by 4-oxononenal on the structure, stability, and function of binding immunoglobulin protein (BiP)

PubMed Central

Shah, Dinen D.; Singh, Surinder M.; Dzieciatkowska, Monika

2017-01-01

Binding immunoglobulin protein (BiP) is a molecular chaperone important for the folding of numerous proteins, which include millions of immunoglobulins in human body. It also plays a key role in the unfolded protein response (UPR) in the endoplasmic reticulum. Free radical generation is a common phenomenon that occurs in cells under healthy as well as under stress conditions such as ageing, inflammation, alcohol consumption, and smoking. These free radicals attack the cell membranes and generate highly reactive lipid peroxidation products such as 4-oxononenal (4-ONE). BiP is a key protein that is modified by 4-ONE. In this study, we probed how such chemical modification affects the biophysical properties of BiP. Upon modification, BiP shows significant tertiary structural changes with no changes in its secondary structure. The protein loses its thermodynamic stability, particularly, that of the nucleotide binding domain (NBD) where ATP binds. In terms of function, the modified BiP completely loses its ATPase activity with decreased ATP binding affinity. However, modified BiP retains its immunoglobulin binding function and its chaperone activity of suppressing non-specific protein aggregation. These results indicate that 4-ONE modification can significantly affect the structure-function of key proteins such as BiP involved in cellular pathways, and provide a molecular basis for how chemical modifications can result in the failure of quality control mechanisms inside the cell. PMID:28886061

Tailored HIV-1 vectors for genetic modification of primary human dendritic cells and monocytes.

PubMed

Durand, Stéphanie; Nguyen, Xuan-Nhi; Turpin, Jocelyn; Cordeil, Stephanie; Nazaret, Nicolas; Croze, Séverine; Mahieux, Renaud; Lachuer, Joël; Legras-Lachuer, Catherine; Cimarelli, Andrea

2013-01-01

Monocyte-derived dendritic cells (MDDCs) play a key role in the regulation of the immune system and are the target of numerous gene therapy applications. The genetic modification of MDDCs is possible with human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LVs) but requires high viral doses to bypass their natural resistance to viral infection, and this in turn affects their physiological properties. To date, a single viral protein is able to counter this restrictive phenotype, Vpx, a protein derived from members of the HIV-2/simian immunodeficiency virus SM lineage that counters at least two restriction factors present in myeloid cells. By tagging Vpx with a short heterologous membrane-targeting domain, we have obtained HIV-1 LVs incorporating high levels of this protein (HIV-1-Src-Vpx). These vectors efficiently transduce differentiated MDDCs and monocytes either as previously purified populations or as populations within unsorted peripheral blood mononuclear cells (PBMCs). In addition, these vectors can be efficiently pseudotyped with receptor-specific envelopes, further restricting their cellular tropism almost uniquely to MDDCs. Compared to conventional HIV-1 LVs, these novel vectors allow for an efficient genetic modification of MDDCs and, more importantly, do not cause their maturation or affect their survival, which are unwanted side effects of the transduction process. This study describes HIV-1-Src-Vpx LVs as a novel potent tool for the genetic modification of differentiated MDDCs and of circulating monocyte precursors with strong potential for a wide range of gene therapy applications.

PROTEOMIC IDENTIFICATION OF CARBONYLATED PROTEINS AND THEIR OXIDATION SITES

PubMed Central

Madian, Ashraf G.; Regnier, Fred E.

2011-01-01

Excessive oxidative stress leaves a protein carbonylation fingerprint in biological systems. Carbonylation is an irreversible post translational modification (PTM) that often leads to the loss of protein function and can be a component of multiple diseases. Protein carbonyl groups can be generated directly (by amino acids oxidation and the a-amidation pathway) or indirectly by forming adducts with lipid peroxidation products or glycation and advanced glycation end-products. Studies of oxidative stress are complicated by the low concentration of oxidation products and wide array of routes by which proteins are carbonylated. The development of new selection and enrichment techniques coupled with advances in mass spectrometry are allowing identification of hundreds of new carbonylated protein products from a broad range of proteins located at many sites in biological systems. The focus of this review is on the use of proteomics tools and methods to identify oxidized proteins along with specific sites of oxidative damage and the consequences of protein oxidation. PMID:20521848

Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

PubMed

Lee, Chia-Fang; Paull, Tanya T; Person, Maria D

2013-10-04

Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

RNA-modifying proteins as anticancer drug targets.

PubMed

Boriack-Sjodin, P Ann; Ribich, Scott; Copeland, Robert A

2018-06-01

All major biological macromolecules (DNA, RNA, proteins and lipids) undergo enzyme-catalysed covalent modifications that impact their structure, function and stability. A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA stability and translation to proteins; these mechanisms of translational control have been termed epitranscriptomics. Emerging data suggest that some epitranscriptomic mechanisms are altered in human cancers as well as other human diseases. In this Review, we examine the current understanding of RNA modifications with a focus on mRNA methylation, highlight their possible roles in specific cancer indications and discuss the emerging potential of RNA-modifying proteins as therapeutic targets.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

Beyond histones - the expanding roles of protein lysine methylation.

PubMed

Wu, Zhouran; Connolly, Justin; Biggar, Kyle K

2017-09-01

A robust signaling network is essential for cell survival. At the molecular level, this is often mediated by as many as 200 different types of post-translational modifications (PTMs) that are made to proteins. These include well-documented examples such as phosphorylation, ubiquitination, acetylation and methylation. Of these modifications, non-histone protein lysine methylation has only recently emerged as a prevalent modification occurring on numerous proteins, thus extending its role well beyond the histone code. To date, this modification has been found to regulate protein activity, protein-protein interactions and interplay with other PTMs. As a result, lysine methylation is now known to be a coordinator of protein function and is a key driver in several cellular signaling events. Recent advances in mass spectrometry have also allowed the characterization of a growing number of lysine methylation events on an increasing number of proteins. As a result, we are now beginning to recognize lysine methylation as a dynamic event that is involved in a number of biological processes, including DNA damage repair, cell growth, metabolism and signal transduction among others. In light of current research advances, the stage is now set to study the extent of lysine methylation that exists within the entire proteome, its dynamics, and its association with physiological and pathological processes. © 2017 Federation of European Biochemical Societies.

Cysteine Oxidative Post-translational Modifications: Emerging Regulation in the Cardiovascular System

PubMed Central

Chung, Heaseung S.; Wang, Sheng-Bing; Venkatraman, Vidya; Murray, Christopher I.; Van Eyk, Jennifer E.

2014-01-01

In the cardiovascular system, changes in the oxidative balance can affect many aspects of cellular physiology through redox-signaling. Depending on the magnitude, fluctuations in the cell's production of reactive oxygen and nitrogen species can regulate normal metabolic processes, activate protective mechanisms, or be cytotoxic. Reactive oxygen and nitrogen species can have many effects including the post-translational modification of proteins at critical cysteine (Cys) thiols. A subset can act as redox-switches, which elicit functional effects in response to changes in oxidative state. While the general concepts of redox-signaling have been established, the identity and function of many regulatory switches remains unclear. Characterizing the effects of individual modifications is the key to understanding how the cell interprets oxidative signals under physiological and pathological conditions. Here, we review the various Cys oxidative post-translational modifications (Ox-PTMs) and their ability to function as redox-switches that regulate the cell's response to oxidative stimuli. In addition, we discuss how these modifications have the potential to influence other post-translational modifications' signaling pathways though cross-talk. Finally, we review the growing number of tools being developed to identify and quantify the various Cys Ox-PTMs and how this will advance our understanding of redox-regulation. PMID:23329793

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

PubMed

Merabet, Samir; Litim-Mecheri, Isma; Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-10-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

PubMed Central

Karlsson, Daniel; Dixit, Richa; Saadaoui, Mehdi; Monier, Bruno; Brun, Christine; Thor, Stefan; Vijayraghavan, K.; Perrin, Laurent; Pradel, Jacques; Graba, Yacine

2011-01-01

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences. PMID:22046139

Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

PubMed

Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

2015-01-01

Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

Proteomic analyses of the environmental toxicity of carcinogenic chemicals

EPA Science Inventory

Protein expression and posttranslational modifications consistently change in response to the exposure to environmental chemicals. Recent technological advances in proteomics provide new tools for more efficient characterization of protein expression and posttranslational modific...

Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

PubMed Central

Liu, Pei; Zhang, Huoming; Yu, Boying; Xiong, Liming; Xia, Yiji

2015-01-01

Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response. PMID:25720653

Further Characterization of the Target of a Potential Aptamer Biomarker for Pancreatic Cancer: Cyclophilin B and Its Posttranslational Modifications

PubMed Central

Sullenger, Bruce A.

2013-01-01

Posttranslational modifications on proteins can serve as useful biomarkers for disease. However, their discovery and detection in biological fluids is challenging. Aptamers are oligonucleotide ligands that demonstrate high affinity toward their target proteins and can discriminate closely related proteins with superb specificity. Previously, we generated a cyclophilin B aptamer (M9-5) that could discriminate sera from pancreatic cancer patients and healthy volunteers with high specificity and sensitivity. In our present work we further characterize the aptamer and the target protein, cyclophilin B, and demonstrate that the aptamer could discriminate between cyclophilin B expressed in human cells versus bacteria. Using mass-spectrometric analysis, we discovered post-translational modifications on cyclophilin B that might be responsible for the M9-5 selectivity. The ability to distinguish between forms of the same protein with differing post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers. PMID:24152208

N(epsilon)-carboxymethyllysine-modified proteins are unable to bind to RAGE and activate an inflammatory response.

PubMed

Buetler, Timo M; Leclerc, Estelle; Baumeyer, Alexandra; Latado, Helia; Newell, John; Adolfsson, Oskar; Parisod, Véronique; Richoz, Janique; Maurer, Sarah; Foata, Francis; Piguet, Dominique; Junod, Sylviane; Heizmann, Claus W; Delatour, Thierry

2008-03-01

Advanced glycation endproducts (AGEs) containing carboxymethyllysine (CML) modifications are generally thought to be ligands of the receptor for AGEs, RAGEs. It has been argued that this results in the activation of pro-inflammatory pathways and diseases. However, it has not been shown conclusively that a CML-modified protein can interact directly with RAGE. Here, we have analyzed whether beta-lactoglobulin (bLG) or human serum albumin (HSA) modified chemically to contain only CML (10-40% lysine modification) can (i) interact with RAGE in vitro and (ii) interact with and activate RAGE in lung epithelial cells. Our results show that CML-modified bLG or HSA are unable to bind to RAGE in a cell-free assay system (Biacore). Furthermore, they are unable to activate pro-inflammatory signaling in the cellular system. Thus, CML probably does not form the necessary structure(s) to interact with RAGE and activate an inflammatory signaling cascade in RAGE-expressing cells.

System-wide identification of wild-type SUMO-2 conjugation sites

PubMed Central

Hendriks, Ivo A.; D'Souza, Rochelle C.; Chang, Jer-Gung; Mann, Matthias; Vertegaal, Alfred C. O.

2015-01-01

SUMOylation is a reversible post-translational modification (PTM) regulating all nuclear processes. Identification of SUMOylation sites by mass spectrometry (MS) has been hampered by bulky tryptic fragments, which thus far necessitated the use of mutated SUMO. Here we present a SUMO-specific protease-based methodology which circumvents this problem, dubbed Protease-Reliant Identification of SUMO Modification (PRISM). PRISM allows for detection of SUMOylated proteins as well as identification of specific sites of SUMOylation while using wild-type SUMO. The method is generic and could be widely applied to study lysine PTMs. We employ PRISM in combination with high-resolution MS to identify SUMOylation sites from HeLa cells under standard growth conditions and in response to heat shock. We identified 751 wild-type SUMOylation sites on endogenous proteins, including 200 dynamic SUMO sites in response to heat shock. Thus, we have developed a method capable of quantitatively studying wild-type mammalian SUMO at the site-specific and system-wide level. PMID:26073453

Hsp70 Forms Antiparallel Dimers Stabilized by Post-translational Modifications to Position Clients for Transfer to Hsp90

PubMed Central

Morgner, Nina; Schmidt, Carla; Beilsten-Edmands, Victoria; Ebong, Ima-obong; Patel, Nisha A.; Clerico, Eugenia M.; Kirschke, Elaine; Daturpalli, Soumya; Jackson, Sophie E.; Agard, David; Robinson, Carol V.

2015-01-01

Summary Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90. PMID:25921532

Posttranscriptional RNA Modifications: playing metabolic games in a cell's chemical Legoland.

PubMed

Helm, Mark; Alfonzo, Juan D

2014-02-20

Nature combines existing biochemical building blocks, at times with subtlety of purpose. RNA modifications are a prime example of this, where standard RNA nucleosides are decorated with chemical groups and building blocks that we recall from our basic biochemistry lectures. The result: a wealth of chemical diversity whose full biological relevance has remained elusive despite being public knowledge for some time. Here, we highlight several modifications that, because of their chemical intricacy, rely on seemingly unrelated pathways to provide cofactors for their synthesis. Besides their immediate role in affecting RNA function, modifications may act as sensors and transducers of information that connect a cell's metabolic state to its translational output, carefully orchestrating a delicate balance between metabolic rate and protein synthesis at a system's level. Copyright © 2014 Elsevier Ltd. All rights reserved.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons

PubMed Central

Noh, Kyung-Min; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W.; Shen, Li; Li, Haitao; Allis, C. David

2015-01-01

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. PMID:25538301

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons.

PubMed

Noh, Kyung-Min; Maze, Ian; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W; Shen, Li; Li, Haitao; Allis, C David

2015-06-02

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Effects of oxidative modification on thermal aggregation and gel properties of soy protein by malondialdehyde.

PubMed

Wu, Wei; Hua, Yufei; Lin, Qinlu

2014-03-01

Malondialdehyde (MDA) was selected as a representative of lipid peroxidation products to investigate the effects of oxidative modification on thermal aggregation and gel properties of soy protein by lipid peroxidation products. Incubation of soy protein with increasing concentration of MDA resulted in gradual decrease of particle size and content of thermal aggregates during heat denaturation. Oxidative modification by MDA resulted in a decrease in water holding capacity, gel hardness, and gel strength of soy protein gel. An increase in coarseness and interstice of MDA modified protein gel network was accompanied by uneven distribution of interstice as MDA concentration increased. The results showed that degree of thermal aggregation of MDA-modified soy protein gradually decreased as MDA concentration increased, which contributed to a decrease in water holding capacity, gel hardness, and gel strength of MDA-modified soy protein gel.

Physical, Chemical and Biochemical Modifications of Protein-Based Films and Coatings: An Extensive Review

PubMed Central

Zink, Joël; Wyrobnik, Tom; Prinz, Tobias; Schmid, Markus

2016-01-01

Protein-based films and coatings are an interesting alternative to traditional petroleum-based materials. However, their mechanical and barrier properties need to be enhanced in order to match those of the latter. Physical, chemical, and biochemical methods can be used for this purpose. The aim of this article is to provide an overview of the effects of various treatments on whey, soy, and wheat gluten protein-based films and coatings. These three protein sources have been chosen since they are among the most abundantly used and are well described in the literature. Similar behavior might be expected for other protein sources. Most of the modifications are still not fully understood at a fundamental level, but all the methods discussed change the properties of the proteins and resulting products. Mastering these modifications is an important step towards the industrial implementation of protein-based films. PMID:27563881

Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

PubMed Central

Tanase, Maya; Urbanska, Aleksandra M.; Zolla, Valerio; Clement, Cristina C.; Huang, Liling; Morozova, Kateryna; Follo, Carlo; Goldberg, Michael; Roda, Barbara; Reschiglian, Pierluigi; Santambrogio, Laura

2016-01-01

Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. PMID:26776680

Non-specific protein modifications by a phytochemical induce heat shock response for self-defense.

PubMed

Ohnishi, Kohta; Ohkura, Shinya; Nakahata, Erina; Ishisaka, Akari; Kawai, Yoshichika; Terao, Junji; Mori, Taiki; Ishii, Takeshi; Nakayama, Tsutomu; Kioka, Noriyuki; Matsumoto, Shinya; Ikeda, Yasutaka; Akiyama, Minoru; Irie, Kazuhiro; Murakami, Akira

2013-01-01

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities.

Non-Specific Protein Modifications by a Phytochemical Induce Heat Shock Response for Self-Defense

PubMed Central

Ohnishi, Kohta; Ohkura, Shinya; Nakahata, Erina; Ishisaka, Akari; Kawai, Yoshichika; Terao, Junji; Mori, Taiki; Ishii, Takeshi; Nakayama, Tsutomu; Kioka, Noriyuki; Matsumoto, Shinya; Ikeda, Yasutaka; Akiyama, Minoru; Irie, Kazuhiro; Murakami, Akira

2013-01-01

Accumulated evidence shows that some phytochemicals provide beneficial effects for human health. Recently, a number of mechanistic studies have revealed that direct interactions between phytochemicals and functional proteins play significant roles in exhibiting their bioactivities. However, their binding selectivities to biological molecules are considered to be lower due to their small and simple structures. In this study, we found that zerumbone, a bioactive sesquiterpene, binds to numerous proteins with little selectivity. Similar to heat-denatured proteins, zerumbone-modified proteins were recognized by heat shock protein 90, a constitutive molecular chaperone, leading to heat shock factor 1-dependent heat shock protein induction in hepa1c1c7 mouse hepatoma cells. Furthermore, oral administration of this phytochemical up-regulated heat shock protein expressions in the livers of Sprague-Dawley rats. Interestingly, pretreatment with zerumbone conferred a thermoresistant phenotype to hepa1c1c7 cells as well as to the nematode Caenorhabditis elegans. It is also important to note that several phytochemicals with higher hydrophobicity or electrophilicity, including phenethyl isothiocyanate and curcumin, markedly induced heat shock proteins, whereas most of the tested nutrients did not. These results suggest that non-specific protein modifications by xenobiotic phytochemicals cause mild proteostress, thereby inducing heat shock response and leading to potentiation of protein quality control systems. We considered these bioactivities to be xenohormesis, an adaptation mechanism against xenobiotic chemical stresses. Heat shock response by phytochemicals may be a fundamental mechanism underlying their various bioactivities. PMID:23536805

In-situ Click Reaction Coupled with Quantitative Proteomics for Identifying Protein Targets of Catechol Estrogens.

PubMed

Liang, Huei-Chen; Liu, Yi-Chen; Chen, Hsin; Ku, Ming Chun; Do, Quynh-Trang; Wang, Chih-Yen; Tzeng, Shun-Fen; Chen, Shu-Hui

2018-06-13

Catechol estrogens (CEs) are metabolic electrophiles that actively undergo covalent interaction with cellular proteins, influencing molecular function. There is no feasible method to identify their binders in a living system. Herein, we developed a click chemistry-based approach using ethinylestradiol (EE2) as the precursor probe coupled with quantitative proteomics to identify protein targets of CEs and classify their binding strengths. Using in-situ metabolic conversion and click reaction in liver microsomes, CEs-protein complex was captured by the probe, digested by trypsin, stable isotope labeled via reductive amination, and analyzed by liquid chromatography-mass spectrometry (LC-MS). A total of 334 liver proteins were repeatedly identified (n  2); 274 identified proteins were classified as strong binders based on precursor mass mapping. The binding strength was further scaled by D/H ratio (activity probe/solvent): 259 strong binders had D/H > 5.25; 46 weak binders had 5.25 > D/H > 1; 5 non-specific binders (keratins) had D/H < 1. These results were confirmed using spiked covalent control (strong binder) and noncovalent control (weak binder), as well as in vitro testing of cytochrome c (D/H = 5.9) which showed covalent conjugation with CEs. Many identified strong binders, such as glutathione transferase, catechol-O-methyl transferase, superoxide dismutase, catalase, glutathione peroxidase, and cytochrome c, are involved in cellular redox processes or detoxification activities. CE conjugation was shown to suppress the superoxide oxidase activity of cytochrome c, suggesting that CEs modification may alter the redox action of cellular proteins. Due to structural similarity and inert alkyne group, EE2 probe is very likely to capture protein targets of CEs in general. Thus, this strategy can be adopted to explore the biological impact of CEs modification in living systems.

Nanochemistry of Protein-Based Delivery Agents

PubMed Central

Rajendran, Subin R. C. K.; Udenigwe, Chibuike C.; Yada, Rickey Y.

2016-01-01

The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior. PMID:27489854

Nanochemistry of protein-based delivery agents

NASA Astrophysics Data System (ADS)

Rajendran, Subin; Udenigwe, Chibuike; Yada, Rickey

2016-07-01

The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

Proteomic technology for biomarker profiling in cancer: an update*

PubMed Central

Alaoui-Jamali, Moulay A.; Xu, Ying-jie

2006-01-01

The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers. PMID:16625706

Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.

PubMed

Hodgson, Derek J; Aubin, Yves

2017-05-10

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

PubMed

Iyer, Lakshminarayan M; Zhang, Dapeng; Burroughs, A Maxwell; Aravind, L

2013-09-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

PubMed Central

Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

2013-01-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology. PMID:23814188

Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions.

PubMed

Zehender, Hartmut; Mayr, Lorenz M

2007-10-01

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.

Sulfenic acid chemistry, detection and cellular lifetime☆

PubMed Central

Gupta, Vinayak; Carroll, Kate S.

2014-01-01

Background Reactive oxygen species-mediated cysteine sulfenic acid modification has emerged as an important regulatory mechanism in cell signaling. The stability of sulfenic acid in proteins is dictated by the local microenvironment and ability of antioxidants to reduce this modification. Several techniques for detecting this cysteine modification have been developed, including direct and in situ methods. Scope of review This review presents a historical discussion of sulfenic acid chemistry and highlights key examples of this modification in proteins. A comprehensive survey of available detection techniques with advantages and limitations is discussed. Finally, issues pertaining to rates of sulfenic acid formation, reduction, and chemical trapping methods are also covered. Major conclusions Early chemical models of sulfenic acid yielded important insights into the unique reactivity of this species. Subsequent pioneering studies led to the characterization of sulfenic acid formation in proteins. In parallel, the discovery of oxidant-mediated cell signaling pathways and pathological oxidative stress has led to significant interest in methods to detect these modifications. Advanced methods allow for direct chemical trapping of protein sulfenic acids directly in cells and tissues. At the same time, many sulfenic acids are short-lived and the reactivity of current probes must be improved to sample these species, while at the same time, preserving their chemical selectivity. Inhibitors with binding scaffolds can be rationally designed to target sulfenic acid modifications in specific proteins. General significance Ever increasing roles for protein sulfenic acids have been uncovered in physiology and pathology. A more complete understanding of sulfenic acid-mediated regulatory mechanisms will continue to require rigorous and new chemical insights. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. PMID:23748139

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

PubMed

Portolano, Nicola; Watson, Peter J; Fairall, Louise; Millard, Christopher J; Milano, Charles P; Song, Yun; Cowley, Shaun M; Schwabe, John W R

2014-10-16

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

PubMed

Gizak, Agnieszka; Rakus, Dariusz

2016-01-11

Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

Redox proteomic evaluation of oxidative modification and recovery in a 3D reconstituted human skin tissue model exposed to UVB.

PubMed

Dyer, J M; Haines, S R; Thomas, A; Wang, W; Walls, R J; Clerens, S; Harland, D P

2017-04-01

Exposure to UV in humans resulting in sunburn triggers a complex series of events that are a mix of immediate and delayed damage mediation and healing. While studies on the effects of UV exposure on DNA damage and repair have been reported, changes in the oxidative modification of skin proteins are poorly understood at the molecular level, despite the important role played by structural proteins in skin tissue, and the effect of the integrity of these proteins on skin appearance and health. Proteomic molecular mapping of oxidation was here applied to try to enhance understanding of skin damage and recovery from oxidative damage and UVB exposure. A redox proteomic-based approach was applied to evaluating skin protein modification when exposed to varying doses of UVB after initial oxidative stress, via tracking changes in protein oxidation during the healing process in vitro using a full-thickness reconstituted human skin tissue model. Bioassays and structural evaluation confirmed that our cultured skin tissues underwent a normal physiological response to UVB exposure. A set of potential skin marker peptides was generated, for use in tracking skin protein oxidative modification. Exposure to UVB after thermal oxidative stress was found to result in higher levels of skin protein oxidation than a non-irradiated control for up to seven days after exposure. Recovery of the skin proteins from oxidative stress, as assessed by the overall protein oxidation levels, was found to be impaired by UVB exposure. Oxidative modification was largely observed in skin structural proteins. Exposure of skin proteins to UVB exacerbates oxidative damage to structural skin proteins, with higher exposure levels leading to increasingly impaired recovery from this damage. This has potential implications for the functional performance of the proteins and inter-related skin health and cosmetic appearance. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

PubMed Central

Xu, Tao; Johnson, Cole A; Gestwicki, Jason E; Kumar, Anuj

2016-01-01

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. one chimera consists of a FK506-binding protein (FKBp12) fused to a cellular ‘address’ (nuclear localization signal or nuclear export sequence). the second chimera consists of a target protein fused to a fluorescent protein and the FKBp12-rapamycin-binding (FrB) domain from FKBp-12-rapamycin associated protein 1 (Frap1, also known as mtor). rapamycin induces dimerization of the FKBp12- and FrB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment. PMID:21030958

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization.

PubMed

Xu, Tao; Johnson, Cole A; Gestwicki, Jason E; Kumar, Anuj

2010-11-01

We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.

Epigenetic Mechanisms in Developmental Alcohol-Induced Neurobehavioral Deficits

PubMed Central

Basavarajappa, Balapal S.; Subbanna, Shivakumar

2016-01-01

Alcohol consumption during pregnancy and its damaging consequences on the developing infant brain are significant public health, social, and economic issues. The major distinctive features of prenatal alcohol exposure in humans are cognitive and behavioral dysfunction due to damage to the central nervous system (CNS), which results in a continuum of disarray that is collectively called fetal alcohol spectrum disorder (FASD). Many rodent models have been developed to understand the mechanisms of and to reproduce the human FASD phenotypes. These animal FASD studies have provided several molecular pathways that are likely responsible for the neurobehavioral abnormalities that are associated with prenatal alcohol exposure of the developing CNS. Recently, many laboratories have identified several immediate, as well as long-lasting, epigenetic modifications of DNA methylation, DNA-associated histone proteins and microRNA (miRNA) biogenesis by using a variety of epigenetic approaches in rodent FASD models. Because DNA methylation patterns, DNA-associated histone protein modifications and miRNA-regulated gene expression are crucial for synaptic plasticity and learning and memory, they can therefore offer an answer to many of the neurobehavioral abnormalities that are found in FASD. In this review, we briefly discuss the current literature of DNA methylation, DNA-associated histone proteins modification and miRNA and review recent developments concerning epigenetic changes in FASD. PMID:27070644

Analysis of sDMA modifications of PIWI proteins

PubMed Central

Honda, Shozo; Kirino, Yoriko; Kirino, Yohei

2015-01-01

Summary Arginine methylation is an important post-translational protein modification that modulates protein function for a wide range of biological processes. PIWI proteins, a subclade of the Argonaute family proteins, contain evolutionarily conserved symmetrical dimethylarginines (sDMAs). It has become increasingly apparent that the sDMAs of PIWI proteins serve as binding elements for TUDOR-domain containing proteins and that sDMA-dependent protein interactions play crucial roles in the biogenesis and function of PIWI-interacting RNAs (piRNAs). We describe a method for detecting PIWI sDMAs and purifying PIWI/piRNA complexes using anti-sDMA antibodies. PMID:24178562

In Cell Footprinting Coupled with Mass Spectrometry for the Structural Analysis of Proteins in Live Cells.

PubMed

Espino, Jessica A; Mali, Vishaal S; Jones, Lisa M

2015-08-04

Protein footprinting coupled with mass spectrometry has become a widely used tool for the study of protein-protein and protein-ligand interactions and protein conformational change. These methods provide residue-level analysis on protein interaction sites and have been successful in studying proteins in vitro. The extension of these methods for in cell footprinting would open an avenue to study proteins that are not amenable for in vitro studies and would probe proteins in their native environment. Here we describe the application of an oxidative-based footprinting approach inside cells in which hydroxyl radicals are used to oxidatively modify proteins. Mass spectrometry is used to detect modification sites and to calculate modification levels. The method is probing biologically relevant proteins in live cells, and proteins in various cellular compartments can be oxdiatively modified. Several different amino acid residues are modified making the method a general labeling strategy for the study of a variety of proteins. Further, comparison of the extent of oxidative modification with solvent accessible surface area reveals the method successfully probes solvent accessibility. This marks the first time protein footprinting has been performed in live cells.

Understanding the Role of O-GlcNAc Modifications in Plant Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olszewski, Neil, E.

2011-06-16

This project has contributed towards understanding the role of O-GlcNAc (O-linked N-acetylglucosamine) transferases (OGTs) in plants. Through analyses of single and double mutants, we have investigated the unique and overlapping functions of SECRET AGENT (SEC) and SPINDLY (SPY), the arabidopsis OGTs. This work showed that SEC functions as negative regulators of the long-day flowering pathway. SEC also has a positive role in regulation of rosette. An E. coli co-expression system that allows potential substrates to be co-expressed with and O-GlcNAc modified by SEC was developed. We showed that SEC is a bona fide OGT that modifies itself with single O-linkedmore » GlcNAc(s). Using this system, we tested a number of proteins that were hypothesized to be substrates of SEC and identified a number of substrates include GIGANTEA (GI), a component of the long day flowering pathway. The hypothesis that O-GlcNAc modification controls GI activity was tested by first mapping where E. coli-expressed SEC modifies GI and then assessing the activity of a non-modifiable mutant form of GI. The activity of the mutant form of GI was indistinguishable from that of wild type suggesting that either O-GlcNAc does not regulate GI activity or that additional modification sites exist on GI. In collaboration with Dr. Juan Antonio Garcia at Universidad Autónoma de Madrid the role of O-GlcNAc modification of the plum pox virus coat protein (PPV-CP) was investigated. SEC was shown to O-GlcNAc modify PPV-CP and the modification was shown to facilitate the infection process. E. coli-expressed SEC was shown to modify the same PPV-CP sites that are modified in plants. SEC has a large protein interaction domain called the TPR domain that has been hypothesized to have a role in determining the substrate specificity of the enzyme and/or to regulate its activity. A mutational analysis of the TPR domain did not find evidence for a role in substrate specificity but did obtain evidence that the domain regulates enzyme activity.« less

Identifying DNA methylation in a nanochannel

NASA Astrophysics Data System (ADS)

Sun, Xiaoyin; Yasui, Takao; Yanagida, Takeshi; Kaji, Noritada; Rahong, Sakon; Kanai, Masaki; Nagashima, Kazuki; Kawai, Tomoji; Baba, Yoshinobu

2016-01-01

DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24-96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h.

Biological particle analysis by mass spectrometry

NASA Technical Reports Server (NTRS)

Vilker, V. L.; Platz, R. M.

1983-01-01

An instrument that analyzes the chemical composition of biological particles in aerosol or hydrosol form was developed. Efforts were directed toward the acquisition of mass spectra from aerosols of biomolecules and bacteria. The filament ion source was installed on the particle analysis by mass spectrometry system. Modifications of the vacuum system improved the sensitivity of the mass spectrometer. After the modifications were incorporated, detailed mass spectra of simple compounds from the three major classes of biomolecules, proteins, nucleic acids, and carbohydrates were obtained. A method of generating bacterial aerosols was developed. The aerosols generated were collected and examined in the scanning electron microscope to insure that the bacteria delivered to the mass spectrometer were intact and free from debris.

Development of Scaffolds for Light Harvesting and Photocatalysis from the Coat Protein of Tobacco Mosaic Virus

NASA Astrophysics Data System (ADS)

Dedeo, Michel Toussaint

The utility of a previously developed TMV-based light harvesting system has been dramatically expanded through the introduction of reactive handles for the site-specific modification of the interior and exterior surfaces. Further experiments to reengineer the coat protein have produced structures with unique, unexpected, and useful assembly properties that complement the newly available surface modifications. Energy transfer from chromophores in the RNA channel of self-assembled TMV structures to the exterior was made possible by conjugation of acceptor dyes and porphyrins to the N-terminus. By repositioning the N-terminus to the pore through circular permutation, this process was repeated to create structures that mimic the light harvesting 1 complex of photosynthetic bacteria. To study and improve upon natural photosynthesis, closely packed chromophore arrays and gold nanoparticles were tethered to the pore of stabilized TMV disks through introduction of a uniquely reactive lysine. Finally, a dimeric TMV coat protein was produced to control the distribution and arrangement of synthetic groups with synergistic activity.

Receptor Tyrosine Kinase Signaling – A Proteomic Perspective

PubMed Central

Biarc, Jordane; Chalkley, Robert J.; Burlingame, A. L.; Bradshaw, Ralph A.

2011-01-01

The stimulation of various cellular processes through extracellular signals is of paramount importance in biological systems and is a central focus in the diagnosis, treatment and prevention of disease. The information transfer is accomplished in a variety of ways by the interaction of soluble, matrix-associated and cell bound ligands that either bind specifically to plasma membrane-associated proteins that act as receptors, or penetrate to the cytoplasmic/nuclear compartments to bind and activate receptors located there. The former class of entities generates intracellular signals that are transmitted and amplified by chemical modifications that are manifested as protein post-translational modifications (PTMs). These are both reversible and irreversible and range from phosphorylation of tyrosine, threonine and serine residues to endoproteolytic cleavages. Although the PTMs alter the activity and functions of many of the proteins in these cascades, the major outcomes of most of the signaling pathways are the activation/deactivation of transcriptional regulators with the concomitant changes in gene expression that generally underlie biological responses. PMID:21056590

Mucin-Type O-Glycosylation in Invertebrates.

PubMed

Staudacher, Erika

2015-06-09

O-Glycosylation is one of the most important posttranslational modifications of proteins. It takes part in protein conformation, protein sorting, developmental processes and the modulation of enzymatic activities. In vertebrates, the basics of the biosynthetic pathway of O-glycans are already well understood. However, the regulation of the processes and the molecular aspects of defects, especially in correlation with cancer or developmental abnormalities, are still under investigation. The knowledge of the correlating invertebrate systems and evolutionary aspects of these highly conserved biosynthetic events may help improve the understanding of the regulatory factors of this pathway. Invertebrates display a broad spectrum of glycosylation varieties, providing an enormous potential for glycan modifications which may be used for the design of new pharmaceutically active substances. Here, overviews of the present knowledge of invertebrate mucin-type O-glycan structures and the currently identified enzymes responsible for the biosynthesis of these oligosaccharides are presented, and the few data dealing with functional aspects of O-glycans are summarised.

Monotopic modifications derived from in vitro glycation of albumin with ribose.

PubMed

Pataridis, Statis; Stastná, Zdeňka; Sedláková, Pavla; Mikšík, Ivan

2013-06-01

Post-translational modifications are significant reactions that occur to proteins. One of these modifications is a non-enzymatic reaction between the oxo-group(s) of sugars and amino-group(s) of protein - glycation. This reaction plays an important role in the chronic complications of diabetes mellitus, or in the aging process of organisms, that is, it has an important role in the pathophysiology and "normal" physiology of animals. In the work presented here, we studied the glycation of albumins (HSA and BSA). Methodologically, we used nano-LC coupled to a QTOF mass spectrometer. In vitro-modified proteins were cleaved by trypsin and the arising peptides were separated on a C(18) nano column with a trap-column. Peptides and their modifications were analysed with a high-resolution QTOF mass spectrometer with a mass determination precision of better than 5 ppm. Non-enzymatic in vitro reaction products between albumin and ribose were identified. Besides well-known carboxymethyl lysine, new modifications were determined - creating mass shifts of 78 and 218. The origin of the first modification is discussed and its possible structure is presented. In addition, a mass shift of 132 belonging to a Schiff base was also identified. The location of all the modifications within the structure of the proteins was determined and their reactivity to various oxo-compounds was also examined. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Ubiquitination as an efficient molecular strategy employed in salmonella infection

USDA-ARS?s Scientific Manuscript database

The ubiquitin modification has various functions in the host innate immune system in response to the bacterial infection. To counteract the host immunity, Salmonella can specifically target ubiquitin pathways by its effector proteins. In this review, we describe the multiple facets of ubiquitin func...

Baking a mass-spectrometry data PIE with McMC and simulated annealing: predicting protein post-translational modifications from integrated top-down and bottom-up data.

PubMed

Jefferys, Stuart R; Giddings, Morgan C

2011-03-15

Post-translational modifications are vital to the function of proteins, but are hard to study, especially since several modified isoforms of a protein may be present simultaneously. Mass spectrometers are a great tool for investigating modified proteins, but the data they provide is often incomplete, ambiguous and difficult to interpret. Combining data from multiple experimental techniques-especially bottom-up and top-down mass spectrometry-provides complementary information. When integrated with background knowledge this allows a human expert to interpret what modifications are present and where on a protein they are located. However, the process is arduous and for high-throughput applications needs to be automated. This article explores a data integration methodology based on Markov chain Monte Carlo and simulated annealing. Our software, the Protein Inference Engine (the PIE) applies these algorithms using a modular approach, allowing multiple types of data to be considered simultaneously and for new data types to be added as needed. Even for complicated data representing multiple modifications and several isoforms, the PIE generates accurate modification predictions, including location. When applied to experimental data collected on the L7/L12 ribosomal protein the PIE was able to make predictions consistent with manual interpretation for several different L7/L12 isoforms using a combination of bottom-up data with experimentally identified intact masses. Software, demo projects and source can be downloaded from http://pie.giddingslab.org/

Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis.

PubMed

Aroca, Angeles; Benito, Juan M; Gotor, Cecilia; Romero, Luis C

2017-10-13

Hydrogen sulfide-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, a process called protein persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves using the tag-switch method. The 2015 identified persulfidated proteins were isolated from plants grown under controlled conditions, and therefore, at least 5% of the entire Arabidopsis proteome may undergo persulfidation under baseline conditions. Bioinformatic analysis revealed that persulfidated cysteines participate in a wide range of biological functions, regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein persulfidation is mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Modify the Histone to Win the Battle: Chromatin Dynamics in Plant–Pathogen Interactions

PubMed Central

Ramirez-Prado, Juan S.; Piquerez, Sophie J. M.; Bendahmane, Abdelhafid; Hirt, Heribert; Raynaud, Cécile; Benhamed, Moussa

2018-01-01

Relying on an immune system comes with a high energetic cost for plants. Defense responses in these organisms are therefore highly regulated and fine-tuned, permitting them to respond pertinently to the attack of a microbial pathogen. In recent years, the importance of the physical modification of chromatin, a highly organized structure composed of genomic DNA and its interacting proteins, has become evident in the research field of plant–pathogen interactions. Several processes, including DNA methylation, changes in histone density and variants, and various histone modifications, have been described as regulators of various developmental and defense responses. Herein, we review the state of the art in the epigenomic aspects of plant immunity, focusing on chromatin modifications, chromatin modifiers, and their physiological consequences. In addition, we explore the exciting field of understanding how plant pathogens have adapted to manipulate the plant epigenomic regulation in order to weaken their immune system and thrive in their host, as well as how histone modifications in eukaryotic pathogens are involved in the regulation of their virulence. PMID:29616066

Peptide and protein delivery using new drug delivery systems.

PubMed

Jain, Ashish; Jain, Aviral; Gulbake, Arvind; Shilpi, Satish; Hurkat, Pooja; Jain, Sanjay K

2013-01-01

Pharmaceutical and biotechnological research sorts protein drug delivery systems by importance based on their various therapeutic applications. The effective and potent action of the proteins/peptides makes them the drugs of choice for the treatment of numerous diseases. Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate target-specific protein carriers. Many efforts have been made for effective delivery of proteins/peptidal drugs through various routes of administrations for successful therapeutic effects. Nanoparticles made of biodegradable polymers such as poly lactic acid, polycaprolactone, poly(lactic-co-glycolic acid), the poly(fumaric-co-sebacic) anhydride chitosan, and modified chitosan, as well as solid lipids, have shown great potential in the delivery of proteins/peptidal drugs. Moreover, scientists also have used liposomes, PEGylated liposomes, niosomes, and aquasomes, among others, for peptidal drug delivery. They also have developed hydrogels and transdermal drug delivery systems for peptidal drug delivery. A receptor-mediated delivery system is another attractive strategy to overcome the limitation in drug absorption that enables the transcytosis of the protein across the epithelial barrier. Modification such as PEGnology is applied to various proteins and peptides of the desired protein and peptides also increases the circulating life, solubility and stability, pharmacokinetic properties, and antigenicity of protein. This review focuses on various approaches for effective protein/peptidal drug delivery, with special emphasis on insulin delivery.

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Modifications in structure and interaction of nanoparticle-protein-surfactant complexes in electrolyte solution

NASA Astrophysics Data System (ADS)

Mehan, Sumit; Kumar, S.; Aswal, V. K.; Schweins, R.

2016-05-01

SANS experiments of three-component system of anionic silica nanoparticles, anionic BSA protein and anionic SDS surfactants have been carried out without and with electrolyte in aqueous solution. In both the cases, the interaction of surfactant with protein results in formation of bead-necklace structure of protein-surfactant complexes in solution. These protein-surfactant complexes interact very differently with nanoparticles in absence and presence of electrolyte. In absence of electrolyte, nanoparticles remain in dispersed phase in solution, whereas with the addition of electrolyte the nanoparticles fractal aggregates are formed. SANS describes the phase behavior to be governed by competition of electrostatic and depletion interactions among the components solution.

Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy

PubMed Central

Sieg, Kelsey M.; Shallow, Keegan D.; Sorenson, Christine M.; Sheibani, Nader

2013-01-01

Purpose Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation. Methods The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2Akita/+ mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays. Results We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2Akita/+ mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of retinal pericytes, but not retinal endothelial cells, was attenuated by increased O-GlcNAc modification. Conclusions The O-GlcNAc modification pattern changes during postnatal retinal vascular development and neovascularization, and its dysregulation under hyperglycemia and/or ischemia may contribute to the pathogenesis of the diabetic retinopathy and retinal neovascularization. PMID:23734074

Proteome complexity and the forces that drive proteome imbalance.

PubMed

Harper, J Wade; Bennett, Eric J

2016-09-15

The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer.

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links.

PubMed

Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

2008-07-01

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.

Interpretive Reporting of Protein Electrophoresis Data by Microcomputer

PubMed Central

Talamo, Thomas S.; Losos, Frank J.; Kessler, G. Frederick

1982-01-01

A microcomputer based system for interpretive reporting of protein electrophoretic data has been developed. Data for serum, urine and cerebrospinal fluid protein electrophoreses as well as immunoelectrophoresis can be entered. Patient demographic information is entered through the keyboard followed by manual entry of total and fractionated protein levels obtained after densitometer scanning of the electrophoretic strip. The patterns are then coded, interpreted, and final reports generated. In most cases interpretation time is less than one second. Misinterpretation by computer is uncommon and can be corrected by edit functions within the system. These discrepancies between computer and pathologist interpretation are automatically stored in a data file for later review and possible program modification. Any or all previous tests on a patient may be reviewed with graphic display of the electrophoretic pattern. The system has been in use for several months and is presently well accepted by both laboratory and clinical staff. It also allows rapid storage, retrieval and analysis of protein electrophoretic datab.

Functionalization of protein-based nanocages for drug delivery applications.

PubMed

Schoonen, Lise; van Hest, Jan C M

2014-07-07

Traditional drug delivery strategies involve drugs which are not targeted towards the desired tissue. This can lead to undesired side effects, as normal cells are affected by the drugs as well. Therefore, new systems are now being developed which combine targeting functionalities with encapsulation of drug cargo. Protein nanocages are highly promising drug delivery platforms due to their perfectly defined structures, biocompatibility, biodegradability and low toxicity. A variety of protein nanocages have been modified and functionalized for these types of applications. In this review, we aim to give an overview of different types of modifications of protein-based nanocontainers for drug delivery applications.

Retrovirus-based vectors for transient and permanent cell modification.

PubMed

Schott, Juliane W; Hoffmann, Dirk; Schambach, Axel

2015-10-01

Retroviral vectors are commonly employed for long-term transgene expression via integrating vector technology. However, three alternative retrovirus-based platforms are currently available that allow transient cell modification. Gene expression can be mediated from either episomal DNA or RNA templates, or selected proteins can be directly transferred through retroviral nanoparticles. The different technologies are functionally graded with respect to safety, expression magnitude and expression duration. Improvement of the initial technologies, including modification of vector designs, targeted increase in expression strength and duration as well as improved safety characteristics, has allowed maturation of retroviral systems into efficient and promising tools that meet the technological demands of a wide variety of potential application areas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.

PubMed

Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang

2016-02-01

The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Alkylation sensitivity screens reveal a conserved cross-species functionome

PubMed Central

Svilar, David; Dyavaiah, Madhu; Brown, Ashley R.; Tang, Jiang-bo; Li, Jianfeng; McDonald, Peter R.; Shun, Tong Ying; Braganza, Andrea; Wang, Xiao-hong; Maniar, Salony; St Croix, Claudette M.; Lazo, John S.; Pollack, Ian F.; Begley, Thomas J.; Sobol, Robert W.

2013-01-01

To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases UNG and MYH as well as MPG to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in E. coli and S. cerevisiae. The conserved biological processes across all three species composes an Alkylation Functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance. PMID:23038810

Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins.

PubMed

Chatgilialoglu, Chryssostomos; Ferreri, Carla; Torreggiani, Armida; Salzano, Anna Maria; Renzone, Giovanni; Scaloni, Andrea

2011-10-19

The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field. Copyright © 2011 Elsevier B.V. All rights reserved.

HIstome--a relational knowledgebase of human histone proteins and histone modifying enzymes.

PubMed

Khare, Satyajeet P; Habib, Farhat; Sharma, Rahul; Gadewal, Nikhil; Gupta, Sanjay; Galande, Sanjeev

2012-01-01

Histones are abundant nuclear proteins that are essential for the packaging of eukaryotic DNA into chromosomes. Different histone variants, in combination with their modification 'code', control regulation of gene expression in diverse cellular processes. Several enzymes that catalyze the addition and removal of multiple histone modifications have been discovered in the past decade, enabling investigations of their role(s) in normal cellular processes and diverse pathological conditions. This sudden influx of data, however, has resulted in need of an updated knowledgebase that compiles, organizes and presents curated scientific information to the user in an easily accessible format. Here, we present HIstome, a browsable, manually curated, relational database that provides information about human histone proteins, their sites of modifications, variants and modifying enzymes. HIstome is a knowledgebase of 55 human histone proteins, 106 distinct sites of their post-translational modifications (PTMs) and 152 histone-modifying enzymes. Entries have been grouped into 5 types of histones, 8 types of post-translational modifications and 14 types of enzymes that catalyze addition and removal of these modifications. The resource will be useful for epigeneticists, pharmacologists and clinicians. HIstome: The Histone Infobase is available online at http://www.iiserpune.ac.in/∼coee/histome/ and http://www.actrec.gov.in/histome/.

Proteomic methods for analysis of S-nitrosation⋄

PubMed Central

Kettenhofen, Nicholas; Broniowska, Katarzyna; Keszler, Agnes; Zhang, Yanhong; Hogg, Neil

2007-01-01

This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology. PMID:17360249

Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

PubMed

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-03-06

Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

Modification of the Creator recombination system for proteomics applications – improved expression by addition of splice sites

PubMed Central

Colwill, Karen; Wells, Clark D; Elder, Kelly; Goudreault, Marilyn; Hersi, Kadija; Kulkarni, Sarang; Hardy, W Rod; Pawson, Tony; Morin, Gregg B

2006-01-01

Background Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. Results To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. Conclusion The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics. PMID:16519801

Action of microbial transglutaminase (MTGase) in the modification of food proteins: a review.

PubMed

Gaspar, Ana Luisa Camolezi; de Góes-Favoni, Silvana Pedroso

2015-03-15

Microbial transglutaminase (MTGase) is an enzyme of the class of transferases widely known to modify protein functional properties in food systems. The main mechanisms of action involved are polymerisations, which result in changes in the molecule's hydrophobicity. Among the functional properties, MTGase affects solubility and hence gelation, emulsification, foaming, viscosity and water-holding capacity, which all depend on protein solubility. Although the enzyme's benefits for protein functionality in a variety of food systems are widely reported in the literature, few studies have focussed on its mechanisms. The purpose of this review is therefore to investigate the mechanisms of action of MTGase and consider its influence on the functional properties with different protein substrates. Understanding these mechanisms is the first step in the development and application, in food production, of new and better functional properties with improved and/or innovative characteristics that can satisfy consumer expectations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Discovery of a novel protein modification: alpha-glycerophosphate is a substituent of meningococcal pilin.

PubMed Central

Stimson, E; Virji, M; Barker, S; Panico, M; Blench, I; Saunders, J; Payne, G; Moxon, E R; Dell, A; Morris, H R

1996-01-01

Pili, which are filamentous protein structures on the surface of the meningitis-causing organism Neisseria meningitidis, are known to be post-translationally modified with substituents that affect their mobility in SDS/PAGE and which might play a crucial role in adherence and bloodstream invasion. Tryptic digests of pili were analysed by fast atom bombardment and electrospray MS to identify putative modifications. Serine-93 was found to carry a novel modification of alpha-glycerophosphate. This is the first time that alpha-glycerophosphate has been observed as a substituent of a prokaryotic or eukaryotic protein. PMID:8645220

Transgenic bovine as bioreactors: Challenges and perspectives

PubMed Central

Monzani, Paulo S.; Adona, Paulo R.; Ohashi, Otávio M.; Meirelles, Flávio V.; Wheeler, Matthew B.

2016-01-01

ABSTRACT The use of recombinant proteins has increased in diverse commercial sectors. Various systems for protein production have been used for the optimization of production and functional protein expression. The mammary gland is considered to be a very interesting system for the production of recombinant proteins due to its high level of expression and its ability to perform post-translational modifications. Cows produce large quantities of milk over a long period of lactation, and therefore this species is an important candidate for recombinant protein expression in milk. However, transgenic cows are more difficult to generate due to the inefficiency of transgenic methodologies, the long periods for transgene detection, recombinant protein expression and the fact that only a single calf is obtained at the end of each pregnancy. An increase in efficiency for transgenic methodologies for cattle is a big challenge to overcome. Promising methodologies have been proposed that can help to overcome this obstacle, enabling the use of transgenic cattle as bioreactors for protein production in milk for industry. PMID:27166649

Synthesis of chlorophyll-amino acid conjugates as models for modification of proteins with chromo/fluorophores.

PubMed

Tamiaki, Hitoshi; Isoda, Yasuaki; Tanaka, Takuya; Machida, Shinnosuke

2014-02-15

A chlorophyll-a derivative bonded directly with epoxide at the peripheral position of the chlorin π-system was reacted with N-urethane and C-ester protected amino acids bearing an alcoholic or phenolic hydroxy group as well as a carboxy group at the residue to give chlorophyll-amino acid conjugates. The carboxy residues of N,C-protected aspartic and glutamic acids were esterified with the epoxide in high yields. The synthetic conjugates in dichloromethane had absorption bands throughout the visible region including intense red-side Qy and blue-side Soret bands. By their excitation at the visible bands, strong and efficient fluorescence emission was observed up to the near-infrared region. The chromo/fluorophores are promising for preparation of functional peptides and modification of proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Epigenetic modifications unlock the milk protein gene loci during mouse mammary gland development and differentiation

USDA-ARS?s Scientific Manuscript database

Unlike with other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organiz...

Identification and characterization of a novel ISG15-ubiquitin mixed chain and its role in regulating protein homeostasis

PubMed Central

Fan, Jun-Bao; Arimoto, Kei-lchiro; Motamedchaboki, Khatereh; Yan, Ming; Wolf, Dieter A.; Zhang, Dong-Er

2015-01-01

As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site. Using a model substrate, we demonstrate that ISG15 can modify ubiquitin, which is immobilized on its substrate, to form ISG15-ubiquitin mixed chains. Furthermore, our results indicate that ISG15-ubiquitin mixed chains do not serve as degradation signals for a ubiquitin fusion degradation substrate. Accordingly, an ISG15-ubiquitin fusion protein, which mimics an ISG15-ubiquitin mixed chain, negatively regulates cellular turnover of ubiquitylated proteins. In addition, ISG15-ubiquitin mixed chains, which are detectable on endogenously ubiquitylated proteins, dampen cellular turnover of these proteins. Thus, our studies unveil an unanticipated interplay between two protein modification systems and highlight its role in coordinating protein homeostasis. PMID:26226047

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

PubMed Central

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

2012-01-01

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

Restriction-Modification systems interplay causes avoidance of GATC site in prokaryotic genomes.

PubMed

Ershova, Anna; Rusinov, Ivan; Vasiliev, Mikhail; Spirin, Sergey; Karyagina, Anna

2016-04-01

Palindromes are frequently underrepresented in prokaryotic genomes. Palindromic 5[Formula: see text]-GATC-3[Formula: see text] site is a recognition site of different Restriction-Modification (R-M) systems, as well as solitary methyltransferase Dam. Classical GATC-specific R-M systems methylate GATC and cleave unmethylated GATC. On the contrary, methyl-directed Type II restriction endonucleases cleave methylated GATC. Methylation of GATC by Dam methyltransferase is involved in the regulation of different cellular processes. The diversity of functions of GATC-recognizing proteins makes GATC sequence a good model for studying the reasons of palindrome avoidance in prokaryotic genomes. In this work, the influence of R-M systems and solitary proteins on the GATC site avoidance is described by a mathematical model. GATC avoidance is strongly associated with the presence of alternate (methyl-directed or classical Type II R-M system) genes in different strains of the same species, as we have shown for Streptococcus pneumoniae, Neisseria meningitidis, Eubacterium rectale, and Moraxella catarrhalis. We hypothesize that GATC avoidance can result from a DNA exchange between strains with different methylation status of GATC site within the process of natural transformation. If this hypothesis is correct, the GATC avoidance is a sign of a DNA exchange between bacteria with different methylation status in a mixed population.

Protein tyrosine nitration in plants: Present knowledge, computational prediction and future perspectives.

PubMed

Kolbert, Zsuzsanna; Feigl, Gábor; Bordé, Ádám; Molnár, Árpád; Erdei, László

2017-04-01

Nitric oxide (NO) and related molecules (reactive nitrogen species) regulate diverse physiological processes mainly through posttranslational modifications such as protein tyrosine nitration (PTN). PTN is a covalent and specific modification of tyrosine (Tyr) residues resulting in altered protein structure and function. In the last decade, great efforts have been made to reveal candidate proteins, target Tyr residues and functional consequences of nitration in plants. This review intends to evaluate the accumulated knowledge about the biochemical mechanism, the structural and functional consequences and the selectivity of plants' protein nitration and also about the decomposition or conversion of nitrated proteins. At the same time, this review emphasizes yet unanswered or uncertain questions such as the reversibility/irreversibility of tyrosine nitration, the involvement of proteasomes in the removal of nitrated proteins or the effect of nitration on Tyr phosphorylation. The different NO producing systems of algae and higher plants raise the possibility of diversely regulated protein nitration. Therefore studying PTN from an evolutionary point of view would enrich our present understanding with novel aspects. Plant proteomic research can be promoted by the application of computational prediction tools such as GPS-YNO 2 and iNitro-Tyr software. Using the reference Arabidopsis proteome, Authors performed in silico analysis of tyrosine nitration in order to characterize plant tyrosine nitroproteome. Nevertheless, based on the common results of the present prediction and previous experiments the most likely nitrated proteins were selected thus recommending candidates for detailed future research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Reciprocal phosphorylation and glycosylation recognition motifs control NCAPP1 interaction with pumpkin phloem proteins and their cell-to-cell movement.

PubMed

Taoka, Ken-Ichiro; Ham, Byung-Kook; Xoconostle-Cázares, Beatriz; Rojas, Maria R; Lucas, William J

2007-06-01

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein-protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)-Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36-amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

PubMed Central

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J.

2016-01-01

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. PMID:26771603

Diversity and functions of protein glycosylation in insects.

PubMed

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

PubMed

Pitzschke, Andrea; Xue, Hui; Persak, Helene; Datta, Sneha; Seifert, Georg J

2016-01-12

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

PubMed

Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

2015-12-07

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

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

PubMed Central

Arif, Rabia; Akram, Faiza; Jamil, Tazeen; Lee, Siu Fai

2017-01-01

Posttranslational modifications (PTMs) occur in all essential proteins taking command of their functions. There are many domains inside proteins where modifications take place on side-chains of amino acids through various enzymes to generate different species of proteins. In this manuscript we have, for the first time, predicted posttranslational modifications of frequency clock and mating type a-1 proteins in Sordaria fimicola collected from different sites to see the effect of environment on proteins or various amino acids pickings and their ultimate impact on consensus sequences present in mating type proteins using bioinformatics tools. Furthermore, we have also measured and walked through genomic DNA of various Sordaria strains to determine genetic diversity by genotyping the short sequence repeats (SSRs) of wild strains of S. fimicola collected from contrasting environments of two opposing slopes (harsh and xeric south facing slope and mild north facing slope) of Evolution Canyon (EC), Israel. Based on the whole genome sequence of S. macrospora, we targeted 20 genomic regions in S. fimicola which contain short sequence repeats (SSRs). Our data revealed genetic variations in strains from south facing slope and these findings assist in the hypothesis that genetic variations caused by stressful environments lead to evolution. PMID:28717646

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

PubMed

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

2017-01-01

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

Nanodiscs in Membrane Biochemistry and Biophysics.

PubMed

Denisov, Ilia G; Sligar, Stephen G

2017-03-22

Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodiscs for structural and mechanistic studies of membrane proteins.

Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production.

PubMed

Roointan, Amir; Morowvat, Mohammad Hossein

The rising potential for CRISPR-Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR-Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR-Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.

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

PubMed Central

Kontaxi, Christiana; Piccardo, Pedro; Gill, Andrew C.

2017-01-01

Tau is a microtubule-associated protein responsible mainly for stabilizing the neuronal microtubule network in the brain. Under normal conditions, tau is highly soluble and adopts an “unfolded” conformation. However, it undergoes conformational changes resulting in a less soluble form with weakened microtubule stabilizing properties. Altered tau forms characteristic pathogenic inclusions in Alzheimer's disease and related tauopathies. Although, tau hyperphosphorylation is widely considered to be the major trigger of tau malfunction, tau undergoes several post-translational modifications at lysine residues including acetylation, methylation, ubiquitylation, SUMOylation, and glycation. We are only beginning to define the site-specific impact of each type of lysine modification on tau biology as well as the possible interplay between them, but, like phosphorylation, these modifications are likely to play critical roles in tau's normal and pathobiology. This review summarizes the latest findings focusing on lysine post-translational modifications that occur at both endogenous tau protein and pathological tau forms in AD and other tauopathies. In addition, it highlights the significance of a site-dependent approach of studying tau post-translational modifications under normal and pathological conditions. PMID:28848737

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

PubMed

Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

2016-01-01

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

Partners in crime: The role of tandem modules in gene transcription.

PubMed

Sharma, Rajal; Zhou, Ming-Ming

2015-09-01

Histones and their modifications play an important role in the regulation of gene transcription. Numerous modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and SUMOylation, have been described. These modifications almost always co-occur and thereby increase the combinatorial complexity of post-translational modification detection. The domains that recognize these histone modifications often occur in tandem in the context of larger proteins and complexes. The presence of multiple modifications can positively or negatively regulate the binding of these tandem domains, influencing downstream cellular function. Alternatively, these tandem domains can have novel functions from their independent parts. Here we summarize structural and functional information known about major tandem domains and their histone binding properties. An understanding of these interactions is key for the development of epigenetic therapy. © 2015 The Protein Society.

Diagonal chromatography to study plant protein modifications.

PubMed

Walton, Alan; Tsiatsiani, Liana; Jacques, Silke; Stes, Elisabeth; Messens, Joris; Van Breusegem, Frank; Goormachtig, Sofie; Gevaert, Kris

2016-08-01

An interesting asset of diagonal chromatography, which we have introduced for contemporary proteome research, is its high versatility concerning proteomic applications. Indeed, the peptide modification or sorting step that is required between consecutive peptide separations can easily be altered and thereby allows for the enrichment of specific, though different types of peptides. Here, we focus on the application of diagonal chromatography for the study of modifications of plant proteins. In particular, we show how diagonal chromatography allows for studying proteins processed by proteases, protein ubiquitination, and the oxidation of protein-bound methionines. We discuss the actual sorting steps needed for each of these applications and the obtained results. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families*

PubMed Central

Dewhurst, Henry M.; Choudhury, Shilpa; Torres, Matthew P.

2015-01-01

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. PMID:26070665

The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

PubMed

Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

2010-04-01

We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

PubMed

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO 2 affects plant growth and productivity. CO 2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO 2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO 2 ) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO 2 responses.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

PubMed Central

Yin, Zepeng; Balmant, Kelly; Geng, Sisi; Zhu, Ning; Zhang, Tong; Dufresne, Craig; Dai, Shaojun; Chen, Sixue

2017-01-01

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses. PMID:28184230

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

PubMed

Feltes, Bruno César; Bonatto, Diego

2015-01-01

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

Modification and Functional Inhibition of Regulator of G-Protein Signaling 4 (RGS4) by 4-Hydroxy-2-nonenal

PubMed Central

Monroy, C. Aaron; Doorn, Jonathan A.; Roman, David L.

2015-01-01

Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of Gα subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of Δ51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4. PMID:24229325

Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chavez, Juan; Chung, Woon-Gye; Miranda, Cristobal L.

2010-01-18

The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction,more » and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. Finally, this study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in d-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.« less

Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

PubMed

Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

2015-10-21

Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

Biosynthesis of the Urease Metallocenter*

PubMed Central

Farrugia, Mark A.; Macomber, Lee; Hausinger, Robert P.

2013-01-01

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview. PMID:23539618

Mapping the O-Mannose Glycoproteome in Saccharomyces cerevisiae *

PubMed Central

Neubert, Patrick; Halim, Adnan; Zauser, Martin; Essig, Andreas; Joshi, Hiren J.; Zatorska, Ewa; Larsen, Ida Signe Bohse; Loibl, Martin; Castells-Ballester, Joan; Aebi, Markus; Clausen, Henrik; Strahl, Sabine

2016-01-01

O-Mannosylation is a vital protein modification conserved from fungi to humans. Yeast is a perfect model to study this post-translational modification, because in contrast to mammals O-mannosylation is the only type of O-glycosylation. In an essential step toward the full understanding of protein O-mannosylation we mapped the O-mannose glycoproteome in baker's yeast. Taking advantage of an O-glycan elongation deficient yeast strain to simplify sample complexity, we identified over 500 O-glycoproteins from all subcellular compartments for which over 2300 O-mannosylation sites were mapped by electron-transfer dissociation (ETD)-based MS/MS. In this study, we focus on the 293 O-glycoproteins (over 1900 glycosylation sites identified by ETD-MS/MS) that enter the secretory pathway and are targets of ER-localized protein O-mannosyltransferases. We find that O-mannosylation is not only a prominent modification of cell wall and plasma membrane proteins, but also of a large number of proteins from the secretory pathway with crucial functions in protein glycosylation, folding, quality control, and trafficking. The analysis of glycosylation sites revealed that O-mannosylation is favored in unstructured regions and β-strands. Furthermore, O-mannosylation is impeded in the proximity of N-glycosylation sites suggesting the interplay of these types of post-translational modifications. The detailed knowledge of the target proteins and their O-mannosylation sites opens for discovery of new roles of this essential modification in eukaryotes, and for a first glance on the evolution of different types of O-glycosylation from yeast to mammals. PMID:26764011

Data-Driven Approach To Determine Popular Proteins for Targeted Proteomics Translation of Six Organ Systems.

PubMed

Lam, Maggie P Y; Venkatraman, Vidya; Xing, Yi; Lau, Edward; Cao, Quan; Ng, Dominic C M; Su, Andrew I; Ge, Junbo; Van Eyk, Jennifer E; Ping, Peipei

2016-11-04

Amidst the proteomes of human tissues lie subsets of proteins that are closely involved in conserved pathophysiological processes. Much of biomedical research concerns interrogating disease signature proteins and defining their roles in disease mechanisms. With advances in proteomics technologies, it is now feasible to develop targeted proteomics assays that can accurately quantify protein abundance as well as their post-translational modifications; however, with rapidly accumulating number of studies implicating proteins in diseases, current resources are insufficient to target every protein without judiciously prioritizing the proteins with high significance and impact for assay development. We describe here a data science method to prioritize and expedite assay development on high-impact proteins across research fields by leveraging the biomedical literature record to rank and normalize proteins that are popularly and preferentially published by biomedical researchers. We demonstrate this method by finding priority proteins across six major physiological systems (cardiovascular, cerebral, hepatic, renal, pulmonary, and intestinal). The described method is data-driven and builds upon the collective knowledge of previous publications referenced on PubMed to lend objectivity to target selection. The method and resulting popular protein lists may also be useful for exploring biological processes associated with various physiological systems and research topics, in addition to benefiting ongoing efforts to facilitate the broad translation of proteomics technologies.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells.

PubMed

Malina, Halina Z

2011-01-19

The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases.

System in biology leading to cell pathology: stable protein-protein interactions after covalent modifications by small molecules or in transgenic cells

PubMed Central

2011-01-01

Background The physiological processes in the cell are regulated by reversible, electrostatic protein-protein interactions. Apoptosis is such a regulated process, which is critically important in tissue homeostasis and development and leads to complete disintegration of the cell. Pathological apoptosis, a process similar to apoptosis, is associated with aging and infection. The current study shows that pathological apoptosis is a process caused by the covalent interactions between the signaling proteins, and a characteristic of this pathological network is the covalent binding of calmodulin to regulatory sequences. Results Small molecules able to bind covalently to the amino group of lysine, histidine, arginine, or glutamine modify the regulatory sequences of the proteins. The present study analyzed the interaction of calmodulin with the BH3 sequence of Bax, and the calmodulin-binding sequence of myristoylated alanine-rich C-kinase substrate in the presence of xanthurenic acid in primary retinal epithelium cell cultures and murine epithelial fibroblast cell lines transformed with SV40 (wild type [WT], Bid knockout [Bid-/-], and Bax-/-/Bak-/- double knockout [DKO]). Cell death was observed to be associated with the covalent binding of calmodulin, in parallel, to the regulatory sequences of proteins. Xanthurenic acid is known to activate caspase-3 in primary cell cultures, and the results showed that this activation is also observed in WT and Bid-/- cells, but not in DKO cells. However, DKO cells were not protected against death, but high rates of cell death occurred by detachment. Conclusions The results showed that small molecules modify the basic amino acids in the regulatory sequences of proteins leading to covalent interactions between the modified sequences (e.g., calmodulin to calmodulin-binding sites). The formation of these polymers (aggregates) leads to an unregulated and, consequently, pathological protein network. The results suggest a mechanism for the involvement of small molecules in disease development. In the knockout cells, incorrect interactions between proteins were observed without the protein modification by small molecules, indicating the abnormality of the protein network in the transgenic system. The irreversible protein-protein interactions lead to protein aggregation and cell degeneration, which are observed in all aging-associated diseases. PMID:21247434

Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system.

PubMed

Babiker, E E; Azakami, H; Ogawa, T; Kato, A

2000-02-01

To elucidate the molecular mechanism of the allergenicity of soybean P34 protein recognized as the most allergenic protein in soybean, the protein was expressed in Escherichia coli transformed with a plasmid carrying P34 cDNA. SDS-PAGE pattern showed that the molecular weight of the recombinant P34 was approximately 2 kDa less than that of the native soybean P34. The difference in the molecular mass between these two proteins could be due to the native P34 in soybean being glycosylated at position Asn(170), whereas the recombinant protein generated in E. coli lacks this post-translational modification. Immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with patients' sera. The results suggest that the recombinant P34 is immunologically reactive, indicating that both proteins have similar epitope structures. Thus, the recombinant P34 produced by the E. coli expression system can be used as a standard allergen for molecular design to reduce the allergenic structure.

Circles within circles: crosstalk between protein Ser/Thr/Tyr-phosphorylation and Met oxidation

USDA-ARS?s Scientific Manuscript database

Background: Reversible posttranslational protein modifications such as phosphorylation of Ser/Thr/Tyr and Met oxidation are critical for both metabolic regulation and cellular signalling. Although these modifications are typically studied individually, herein we describe the potential for cross-talk...

Plasma Proteins Modified by Advanced Glycation End Products (AGEs) Reveal Site-specific Susceptibilities to Glycemic Control in Patients with Type 2 Diabetes.

PubMed

Greifenhagen, Uta; Frolov, Andrej; Blüher, Matthias; Hoffmann, Ralf

2016-04-29

Protein glycation refers to the reversible reaction between aldoses (or ketoses) and amino groups yielding relatively stable Amadori (or Heyns) products. Consecutive oxidative cleavage reactions of these products or the reaction of amino groups with other reactive substances (e.g. α-dicarbonyls) yield advanced glycation end products (AGEs) that can alter the structures and functions of proteins. AGEs have been identified in all organisms, and their contents appear to rise with some diseases, such as diabetes and obesity. Here, we report a pilot study using highly sensitive and specific proteomics approach to identify and quantify AGE modification sites in plasma proteins by reversed phase HPLC mass spectrometry in tryptic plasma digests. In total, 19 AGE modification sites corresponding to 11 proteins were identified in patients with type 2 diabetes mellitus under poor glycemic control. The modification degrees of 15 modification sites did not differ among cohorts of normoglycemic lean or obese and type 2 diabetes mellitus patients under good and poor glycemic control. The contents of two amide-AGEs in human serum albumin and apolipoprotein A-II were significantly higher in patients with poor glycemic control, although the plasma levels of both proteins were similar among all plasma samples. These two modification sites might be useful to predict long term, AGE-related complications in diabetic patients, such as impaired vision, increased arterial stiffness, or decreased kidney function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack?

PubMed

Boys, Brian L; Kuprowski, Mark C; Noël, James J; Konermann, Lars

2009-05-15

The exposure of solution-phase proteins to reactive oxygen species (ROS) causes oxidative modifications, giving rise to the formation of covalent +16 Da adducts. Electrospray ionization (ESI) mass spectrometry (MS) is the most widely used method for monitoring the extent of these modifications. Unfortunately, protein oxidation can also take place as an experimental artifact during ESI, such that it may be difficult to assess the actual level of oxidation in bulk solution. Previous work has demonstrated that ESI-induced oxidation is highly prevalent when operating at strongly elevated capillary voltage V(0) (e.g., +8 kV) and with oxygen nebulizer gas in the presence of a clearly visible corona discharge. Protein oxidation under these conditions is commonly attributed to OH radicals generated in the plasma of the discharge. On the other hand, charge balancing oxidation reactions are known to take place at the metal/liquid interface of the emitter. Previous studies have not systematically explored whether such electrochemical processes could be responsible for the formation of oxidative +16 Da adducts instead of (or in combination with) plasma-generated ROS. Using hemoglobin as a model system, this work illustrates the occurrence of extensive protein oxidation even under typical operating conditions (e.g., V(0) = 3.5 kV, N(2) nebulizer gas). Surprisingly, measurements of the current flowing in the ESI circuit demonstrate that a weak corona discharge persists for these relatively gentle settings. On the basis of comparative experiments with nebulizer gases of different dielectric strength, it is concluded that ROS generated under discharge conditions are solely responsible for ESI-induced protein oxidation. This result is corroborated through off-line electrolysis experiments designed to mimic the electrochemical processes taking place during ESI. Our findings highlight the necessity of using easily oxidizable internal standards in biophysical or biomedical ESI-MS studies where knowledge of protein oxidation in bulk solution is desired. Strategies for eliminating ESI-induced oxidation artifacts are discussed.

Albumin modification and fragmentation in renal disease.

PubMed

Donadio, Carlo; Tognotti, Danika; Donadio, Elena

2012-02-18

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

Tumor Targeting and Drug Delivery by Anthrax Toxin.

PubMed

Bachran, Christopher; Leppla, Stephen H

2016-07-01

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery.

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

PubMed Central

Proietto, Marco; Bianchi, Michele Maria; Ballario, Paola; Brenna, Andrea

2015-01-01

Blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. One of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. Circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. Neurospora crassa provides an excellent model for studying the molecular mechanisms involved in these phenomena. The White Collar Complex (WCC), a blue-light receptor and transcription factor of the circadian oscillator, and Frequency (FRQ), the circadian clock pacemaker, are at the core of the Neurospora circadian system. The eukaryotic circadian clock relies on transcriptional/translational feedback loops: some proteins rhythmically repress their own synthesis by inhibiting the activity of their transcriptional factors, generating self-sustained oscillations over a period of about 24 h. One of the basic mechanisms that perpetuate self-sustained oscillations is post translation modification (PTM). The acronym PTM generically indicates the addition of acetyl, methyl, sumoyl, or phosphoric groups to various types of proteins. The protein can be regulatory or enzymatic or a component of the chromatin. PTMs influence protein stability, interaction, localization, activity, and chromatin packaging. Chromatin modification and PTMs have been implicated in regulating circadian clock function in Neurospora. Research into the epigenetic control of transcription factors such as WCC has yielded new insights into the temporal modulation of light-dependent gene transcription. Here we report on epigenetic and protein PTMs in the regulation of the Neurospora crassa circadian clock. We also present a model that illustrates the molecular mechanisms at the basis of the blue light control of the circadian clock. PMID:26198228

Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

PubMed Central

Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

2015-01-01

Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Modifications in nanoparticle-protein interactions by varying the protein conformation

NASA Astrophysics Data System (ADS)

Kumar, Sugam; Yadav, I.; Aswal, V. K.; Kohlbrecher, J.

2017-05-01

Small-angle neutron scattering has been used to study the interaction of silica nanoparticle with Bovine Serum Albumin (BSA) protein without and with a protein denaturing agent urea. The measurements have been carried out at pH 7 where both the components (nanoparticle and protein) are similarly charged. We show that the interactions in nanoparticle-protein system can be modified by changing the conformation of protein through the presence of urea. In the absence of urea, the strong electrostatic repulsion between the nanoparticle and protein prevents protein adsorption on nanoparticle surface. This non-adsorption, in turn gives rise to depletion attraction between nanoparticles. However, with addition of urea the depletion attraction is completely suppressed. Urea driven denaturation of protein is utilized to expose the positively charged patched of the BSA molecules which eventually leads to adsorption of BSA on nanoparticles eliminating the depletion interaction.

Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography

PubMed Central

Ito, Yoichiro; Clary, Robert

2016-01-01

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1–2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate. PMID:27790621

Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography.

PubMed

Ito, Yoichiro; Clary, Robert

2016-12-01

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1-2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate.

Ionic liquid-regenerated macroporous cellulose monolith: Fabrication, characterization and its protein chromatography.

PubMed

Du, Kaifeng

2017-04-21

Macroporous cellulose monolith as chromatographic support was successfully fabricated from an ionic liquid dissolved cellulose solution by an emulsification method and followed by the cross-linking reaction and DEAE modification. With the physical characterization, the cellulose monolith featured by both the interconnected macropores in range of 0.5-2.5μm and the diffusion pores centered at about 10nm. Given the bimodal pore system, the monolith possessed the specific surface area of 36.4m 2 g -1 and the column permeability of about 7.45×10 -14 m 2 . After the DEAE modification, the anion cellulose monolith was evaluated for its chromatography performances. It demonstrated that the static and dynamic adsorption capacity of BSA reached about 66.7mgmL -1 and 43.9mgmL -1 at 10% breakthrough point, respectively. The results were comparable to other chromatographic adsorbent. In addition, the proteins mixture with different pI was well separated at high flow velocity (611.0cmh -1 ) and high protein recovery (over 97%), proving the macroporous cellulose monolith had excellent separation performance. In this way, the prepared cellulose monolith with bimodal pores system is expected for the potential application in high-speed chromatography. Copyright © 2017 Elsevier B.V. All rights reserved.

Human T-lymphotropic virus proteins and post-translational modification pathways

PubMed Central

Bidoia, Carlo

2012-01-01

Cell life from the cell cycle to the signaling transduction and response to stimuli is finely tuned by protein post-translational modifications (PTMs). PTMs alter the conformation, the stability, the localization, and hence the pattern of interactions of the targeted protein. Cell pathways involve the activation of enzymes, like kinases, ligases and transferases, that, once activated, act on many proteins simultaneously, altering the state of the cell and triggering the processes they are involved in. Viruses enter a balanced system and hijack the cell, exploiting the potential of PTMs either to activate viral encoded proteins or to alter cellular pathways, with the ultimate consequence to perpetuate through their replication. Human T-lymphotropic virus type 1 (HTLV-1) is known to be highly oncogenic and associates with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis and other inflammatory pathological conditions. HTLV-1 protein activity is controlled by PTMs and, in turn, viral activity is associated with the modulation of cellular pathways based on PTMs. More knowledge is acquired about the PTMs involved in the activation of its proteins, like Tax, Rex, p12, p13, p30, HTLV-I basic leucine zipper factor and Gag. However, more has to be understood at the biochemical level in order to counteract the associated fatal outcomes. This review will focus on known PTMs that directly modify HTLV-1 components and on enzymes whose activity is modulated by viral proteins. PMID:24175216

Reciprocal Phosphorylation and Glycosylation Recognition Motifs Control NCAPP1 Interaction with Pumpkin Phloem Proteins and Their Cell-to-Cell Movement[W

PubMed Central

Taoka, Ken-ichiro; Ham, Byung-Kook; Xoconostle-Cázares, Beatriz; Rojas, Maria R.; Lucas, William J.

2007-01-01

In plants, cell-to-cell trafficking of non-cell-autonomous proteins (NCAPs) involves protein–protein interactions, and a role for posttranslational modification has been implicated. In this study, proteins contained in pumpkin (Cucurbita maxima cv Big Max) phloem sap were used as a source of NCAPs to further explore the molecular basis for selective NCAP trafficking. Protein overlay assays and coimmunoprecipitation experiments established that phosphorylation and glycosylation, on both Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (Nt-NCAPP1) and the phloem NCAPs, are essential for their interaction. Detailed molecular analysis of a representative phloem NCAP, Cm-PP16-1, identified the specific residues on which glycosylation and phosphorylation must occur for effective binding to NCAPP1. Microinjection studies confirmed that posttranslational modification on these residues is essential for cell-to-cell movement of Cm-PP16-1. Lastly, a glutathione S-transferase (GST)–Cm-PP16-1 fusion protein system was employed to test whether the peptide region spanning these residues was required for cell-to-cell movement. These studies established that a 36–amino acid peptide was sufficient to impart cell-to-cell movement capacity to GST, a normally cell-autonomous protein. These findings are consistent with the hypothesis that a phosphorylation-glycosylation recognition motif functions to control the binding of a specific subset of phloem NCAPs to NCAPP1 and their subsequent transport through plasmodesmata. PMID:17601822

Tyrosine sulfation in a Gram-negative bacterium

PubMed Central

Han, Sang-Wook; Lee, Sang-Won; Bahar, Ofir; Schwessinger, Benjamin; Robinson, Michelle R.; Shaw, Jared B.; Madsen, James A.; Brodbelt, Jennifer S.; Ronald, Pamela C.

2015-01-01

Tyrosine sulfation, a well-characterized post-translation modification in eukaryotes, has not previously been reported in prokaryotes. Here we demonstrate that the RaxST protein from the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae, is a tyrosine sulfotransferase. We used a newly developed sulfotransferase assay and ultraviolet photodissociation mass spectrometry (UVPD) to demonstrate that RaxST catalyzes sulfation of tyrosine 22 of the Xoo Ax21 (activator of XA21-mediated immunity). These results demonstrate a previously undescribed post-translational modification in a prokaryotic species with implications extending to host immune response and bacterial cell-cell communication system. PMID:23093190

Lentiviral gene transduction of mouse and human hematopoietic stem cells.

PubMed

van Til, Niek P; Wagemaker, Gerard

2014-01-01

Lentiviral vectors can be used to genetically modify a broad range of cells. Hematopoietic stem cells (HSCs) are particularly suitable for lentiviral gene augmentation, because these cells can be enriched with relative ease from mouse bone marrow and human hematopoietic sources, and in principle require relatively limited cell numbers to completely reconstitute the hematopoietic system in vivo. Furthermore, lentiviral vectors are very efficient if pseudotyped with broad tropism envelope proteins. This chapter focuses on gene modification by the use of self-inactivating third-generation human immunodeficiency virus-derived lentiviral vectors for ex vivo HSC modification for both mouse and human application.

Comparison of in vitro systems of protein digestion using either mammal or fish proteolytic enzymes.

PubMed

Moyano, F J; Savoie, L

2001-02-01

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.

A Class of Reactive Acyl-CoA Species Reveals the Non-Enzymatic Origins of Protein Acylation

PubMed Central

Wagner, Gregory R.; Bhatt, Dhaval P.; O’Connell, Thomas M.; Thompson, J. Will; Dubois, Laura G.; Backos, Donald S.; Yang, Hao; Mitchell, Grant A.; Ilkayeva, Olga R.; Stevens, Robert D.; Grimsrud, Paul A.; Hirschey, Matthew D.

2017-01-01

SUMMARY The mechanisms underlying the formation of acyl protein modifications remain poorly understood. By investigating the reactivity of endogenous acyl-CoA metabolites, we found a class of acyl-CoAs that undergoes intramolecular catalysis to form reactive intermediates which non-enzymatically modify proteins. Based on this mechanism, we predicted, validated, and characterized a protein modification: 3-hydroxy-3-methylglutaryl(HMG)-lysine. In a model of altered HMG-CoA metabolism, we found evidence of two additional protein modifications: 3-methylglutaconyl(MGc)-lysine and 3-methylglutaryl(MG)-lysine. Using quantitative proteomics, we compared the ‘acylomes’ of two reactive acyl-CoA species, namely HMG-CoA and glutaryl-CoA, which are generated in different pathways. We found proteins that are uniquely modified by each reactive metabolite, as well as common proteins and pathways. We identified the tricarboxylic acid cycle as a pathway commonly regulated by acylation, and validated malate dehydrogenase as a key target. These data uncover a fundamental relationship between reactive acyl-CoA species and proteins, and define a new regulatory paradigm in metabolism. PMID:28380375

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

PubMed Central

Williams, Kerstin J.; Bennett, Mark H.; Barton, Geraint R.; Jenkins, Victoria A.; Robertson, Brian D.

2013-01-01

Summary PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation. PMID:23352854

Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition*

PubMed Central

Matafora, Vittoria; D'Amato, Alfonsina; Mori, Silvia; Blasi, Francesco; Bachi, Angela

2009-01-01

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. PMID:19596686

The PA influenza virus polymerase subunit is a phosphorylated protein.

PubMed

Sanz-Ezquerro, J J; Fernández Santarén, J; Sierra, T; Aragón, T; Ortega, J; Ortín, J; Smith, G L; Nieto, A

1998-03-01

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins.

PubMed

Hess, Gaelen T; Cragnolini, Juan J; Popp, Maximilian W; Allen, Mark A; Dougan, Stephanie K; Spooner, Eric; Ploegh, Hidde L; Belcher, Angela M; Guimaraes, Carla P

2012-07-18

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.

An M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins

PubMed Central

Hess, Gaelen T.; Cragnolini, Juan J.; Popp, Maximilian W.; Allen, Mark A.; Dougan, Stephanie K.; Spooner, Eric; Ploegh, Hidde L.; Belcher, Angela M.; Guimaraes, Carla P.

2013-01-01

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool. PMID:22759232

Year 2 Report: Protein Function Prediction Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, C E

2012-04-27

Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fullymore » automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.« less

Parasites, proteomes and systems: has Descartes' clock run out of time?

PubMed

Wastling, J M; Armstrong, S D; Krishna, R; Xia, D

2012-08-01

Systems biology aims to integrate multiple biological data types such as genomics, transcriptomics and proteomics across different levels of structure and scale; it represents an emerging paradigm in the scientific process which challenges the reductionism that has dominated biomedical research for hundreds of years. Systems biology will nevertheless only be successful if the technologies on which it is based are able to deliver the required type and quality of data. In this review we discuss how well positioned is proteomics to deliver the data necessary to support meaningful systems modelling in parasite biology. We summarise the current state of identification proteomics in parasites, but argue that a new generation of quantitative proteomics data is now needed to underpin effective systems modelling. We discuss the challenges faced to acquire more complete knowledge of protein post-translational modifications, protein turnover and protein-protein interactions in parasites. Finally we highlight the central role of proteome-informatics in ensuring that proteomics data is readily accessible to the user-community and can be translated and integrated with other relevant data types.

Parasites, proteomes and systems: has Descartes’ clock run out of time?

PubMed Central

WASTLING, J. M.; ARMSTRONG, S. D.; KRISHNA, R.; XIA, D.

2012-01-01

SUMMARY Systems biology aims to integrate multiple biological data types such as genomics, transcriptomics and proteomics across different levels of structure and scale; it represents an emerging paradigm in the scientific process which challenges the reductionism that has dominated biomedical research for hundreds of years. Systems biology will nevertheless only be successful if the technologies on which it is based are able to deliver the required type and quality of data. In this review we discuss how well positioned is proteomics to deliver the data necessary to support meaningful systems modelling in parasite biology. We summarise the current state of identification proteomics in parasites, but argue that a new generation of quantitative proteomics data is now needed to underpin effective systems modelling. We discuss the challenges faced to acquire more complete knowledge of protein post-translational modifications, protein turnover and protein-protein interactions in parasites. Finally we highlight the central role of proteome-informatics in ensuring that proteomics data is readily accessible to the user-community and can be translated and integrated with other relevant data types. PMID:22828391

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

PLMD: An updated data resource of protein lysine modifications.

PubMed

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

2017-05-20

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

Role of O-GlcNAcylation in nutritional sensing, insulin resistance and in mediating the benefits of exercise.

PubMed

Myslicki, Jason P; Belke, Darrell D; Shearer, Jane

2014-11-01

The purpose of this review is to highlight the role of O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification in metabolic disease states and to summarize current knowledge of how exercise affects this important post-translational signalling pathway. O-GlcNAc modification is an intracellular tool capable of integrating energy supply with demand. The accumulation of excess energy associated with obesity and insulin resistance is mediated, in part, by the hexosamine biosynthetic pathway (HBP), which results in the O-GlcNAcylation of a myriad of proteins, thereby affecting their respective function, stability, and localization. Insulin resistance is related to the excessive O-GlcNAcylation of key metabolic proteins causing a chronic blunting of insulin signalling pathways and precipitating the accompanying pathologies, such as heart and kidney disease. Lifestyle modifications such as diet and exercise also modify the pathway. Exercise is a front-line and cost-effective therapeutic approach for insulin resistance, and recent work shows that the intervention can alter O-GlcNAc gene expression, signalling, and protein modification. However, there is currently no consensus on the effect of frequency, intensity, type, and duration of exercise on O-GlcNAc modification, the HBP, and its related enzymes. On one end of the spectrum, mild, prolonged swim training reduces O-GlcNAcylation, while on the other end, higher intensity treadmill running increases cardiac protein O-GlcNAc modification. Clearly, a balance between acute and chronic stress of exercise is needed to reap the benefits of the intervention on O-GlcNAc signalling.

Mass spectrometric determination of early and advanced glycation in biology.

PubMed

Rabbani, Naila; Ashour, Amal; Thornalley, Paul J

2016-08-01

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1-5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N(ε)-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them - amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine - particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.

Genetic engineering and chemical conjugation of potato virus X.

PubMed

Lee, Karin L; Uhde-Holzem, Kerstin; Fischer, Rainer; Commandeur, Ulrich; Steinmetz, Nicole F

2014-01-01

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle

DOE PAGES

Kramer, Philip A.; Duan, Jicheng; Gaffrey, Matthew J.; ...

2018-05-23

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 minmore » after the last stimulation and processed for redox proteomics assay of S-glutathionylation.« less

Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kramer, Philip A.; Duan, Jicheng; Gaffrey, Matthew J.

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 minmore » after the last stimulation and processed for redox proteomics assay of S-glutathionylation.« less

Modification of ruminal fermentation and methane production by adding legumes containing condensed tannins to an orchardgrass diet in continuous culture systems

USDA-ARS?s Scientific Manuscript database

Condensed tannins (CT) can alter ruminal fermentation and enteric methane (CH4) production in ruminants; however, research is lacking on how increased CT levels affect nutrient digestibility, volatile fatty acid (VFA) production, bacterial protein synthesis, fatty acid (FA) profiles, protozoal popul...

Proteome complexity and the forces that drive proteome imbalance

PubMed Central

Harper, J. Wade; Bennett, Eric J.

2016-01-01

Summary The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer. PMID:27629639

O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

PubMed Central

Lima, Victor V.; Spitler, Kathryn; Choi, Hyehun; Webb, R. Clinton; Tostes, Rita C.

2012-01-01

O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury. PMID:22757958

Macrophage Migration Inhibitory Factor is subjected to glucose modification and oxidation in Alzheimer’s Disease

PubMed Central

Kassaar, Omar; Pereira Morais, Marta; Xu, Suying; Adam, Emily L.; Chamberlain, Rosemary C.; Jenkins, Bryony; James, Tony; Francis, Paul T.; Ward, Stephen; Williams, Robert J.; van den Elsen, Jean

2017-01-01

Glucose and glucose metabolites are able to adversely modify proteins through a non-enzymatic reaction called glycation, which is associated with the pathology of Alzheimer’s Disease (AD) and is a characteristic of the hyperglycaemia induced by diabetes. However, the precise protein glycation profile that characterises AD is poorly defined and the molecular link between hyperglycaemia and AD is unknown. In this study, we define an early glycation profile of human brain using fluorescent phenylboronate gel electrophoresis and identify early glycation and oxidation of macrophage migration inhibitory factor (MIF) in AD brain. This modification inhibits MIF enzyme activity and ability to stimulate glial cells. MIF is involved in immune response and insulin regulation, hyperglycaemia, oxidative stress and glycation are all implicated in AD. Our study indicates that glucose modified and oxidised MIF could be a molecular link between hyperglycaemia and the dysregulation of the innate immune system in AD. PMID:28230058

Albumin based versatile multifunctional nanocarriers for cancer therapy: Fabrication, surface modification, multimodal therapeutics and imaging approaches.

PubMed

Kudarha, Ritu R; Sawant, Krutika K

2017-12-01

Albumin is a versatile protein used as a carrier system for cancer therapeutics. As a carrier it can provide tumor specificity, reduce drug related toxicity, maintain therapeutic concentration of the active moiety like drug, gene, peptide, protein etc. for long period of time and also reduce drug related toxicities. Apart from cancer therapy, it is also utilized in the imaging and multimodal therapy of cancer. This review highlights the important properties, structure and types of albumin based nanocarriers with regards to their use for cancer targeting. It also provides brief discussion on methods of preparation of these nanocarriers and their surface modification. Applications of albumin nanocarriers for cancer therapy, gene delivery, imaging, phototherapy and multimodal therapy have also been discussed. This review also provides brief discussion about albumin based marketed nano formulations and those under clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Post-translational modification of host proteins in pathogen-triggered defence signalling in plants.

PubMed

Stulemeijer, Iris J E; Joosten, Matthieu H A J

2008-07-01

Microbial plant pathogens impose a continuous threat to global food production. Similar to animals, an innate immune system allows plants to recognize pathogens and swiftly activate defence. To activate a rapid response, receptor-mediated pathogen perception and subsequent downstream signalling depends on post-translational modification (PTM) of components essential for defence signalling. We discuss different types of PTMs that play a role in mounting plant immunity, which include phosphorylation, glycosylation, ubiquitination, sumoylation, nitrosylation, myristoylation, palmitoylation and glycosylphosphatidylinositol (GPI)-anchoring. PTMs are rapid, reversible, controlled and highly specific, and provide a tool to regulate protein stability, activity and localization. Here, we give an overview of PTMs that modify components essential for defence signalling at the site of signal perception, during secondary messenger production and during signalling in the cytoplasm. In addition, we discuss effectors from pathogens that suppress plant defence responses by interfering with host PTMs.

Quantification of Cysteinyl-S-Nitrosylation by Fluorescence in Unbiased Proteomic Studies*

PubMed Central

Wiktorowicz, John E.; Stafford, Susan; Rea, Harriet; Urvil, Petri; Soman, Kizhake; Kurosky, Alexander; Perez-Polo, J. Regino; Savidge, Tor C.

2011-01-01

Cysteinyl-S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins due to physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia reperfusion model, and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique’s power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses. PMID:21615140

Comparative glycoproteomics of stem cells identifies new players in ricin toxicity.

PubMed

Stadlmann, Johannes; Taubenschmid, Jasmin; Wenzel, Daniel; Gattinger, Anna; Dürnberger, Gerhard; Dusberger, Frederico; Elling, Ulrich; Mach, Lukas; Mechtler, Karl; Penninger, Josef M

2017-09-28

Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.

Label-free proteomics assisted by affinity enrichment for elucidating the chemical reactivity of the liver mitochondrial proteome toward adduction by the lipid electrophile 4-hydroxy-2-nonenal (HNE)

NASA Astrophysics Data System (ADS)

Maier, Claudia

2016-03-01

The analysis of oxidative stress-induced post-translational modifications remains challenging due to the chemical diversity of these modifications, the possibility of the presence of positional isomers and the low stoichiometry of the modified proteins present in a cell or tissue proteome. Alcoholic liver disease (ALD) is a multifactorial disease in which mitochondrial dysfunction and oxidative stress have been identified as being critically involved in the progression of the disease from steatosis to cirrhosis. Ethanol metabolism leads to increased levels of reactive oxygen species (ROS), glutathione depletion and lipid peroxidation. Posttranslational modification of proteins by electrophilic products of lipid peroxidation has been associated with governing redox-associated signaling mechanisms, but also as contributing to protein dysfunction leading to organelle and liver injury. In particular the prototypical α,β-unsaturated aldehyde, 4-hydroxy-2-nonenal (HNE), has been extensively studied as marker of increased oxidative stress in hepatocytes. In this study, we combined a LC-MS label-free quantification method and affinity enrichment to assess the dose-dependent insult by HNE on the proteome of rat liver mitochondria. We used a carbonyl-selective probe, the ARP probe, to label HNE-protein adducts and to perform affinity capture at the protein level. Using LC-MS to obtain protein abundance estimates, a list of protein targets was obtained with increasing concentration of HNE used in the exposure studies. In parallel, we performed affinity capture at the peptide level to acquire site-specific information. Examining the concentration-dependence of the protein modifications, we observed distinct reactivity profiles for HNE-protein adduction. Pathway analysis indicated that proteins associated with metabolic processes, including amino acid, fatty acid and glyoxylate and dicarboxylate metabolism, bile acid synthesis and TCA cycle, showed enhanced reactivity to HNE adduction. Whereas, proteins associated with oxidative phosphorylation displayed retardation toward HNE adduction. We provide a list of 31 protein targets with a total of 61 modification sites that may guide future targeted LC-MS assays to monitor disease progression and/or intervention in preclinical models of ALD and possibly other liver diseases with oxidative stress component.

Protein arginine methylation: a prominent modification and its demethylation.

PubMed

Wesche, Juste; Kühn, Sarah; Kessler, Benedikt M; Salton, Maayan; Wolf, Alexander

2017-09-01

Arginine methylation of histones is one mechanism of epigenetic regulation in eukaryotic cells. Methylarginines can also be found in non-histone proteins involved in various different processes in a cell. An enzyme family of nine protein arginine methyltransferases catalyses the addition of methyl groups on arginines of histone and non-histone proteins, resulting in either mono- or dimethylated-arginine residues. The reversibility of histone modifications is an essential feature of epigenetic regulation to respond to changes in environmental factors, signalling events, or metabolic alterations. Prominent histone modifications like lysine acetylation and lysine methylation are reversible. Enzyme family pairs have been identified, with each pair of lysine acetyltransferases/deacetylases and lysine methyltransferases/demethylases operating complementarily to generate or erase lysine modifications. Several analyses also indicate a reversible nature of arginine methylation, but the enzymes facilitating direct removal of methyl moieties from arginine residues in proteins have been discussed controversially. Differing reports have been seen for initially characterized putative candidates, like peptidyl arginine deiminase 4 or Jumonji-domain containing protein 6. Here, we review the most recent cellular, biochemical, and mass spectrometry work on arginine methylation and its reversible nature with a special focus on putative arginine demethylases, including the enzyme superfamily of Fe(II) and 2-oxoglutarate-dependent oxygenases.

The tomato UV-damaged DNA binding protein 1 (DDB1) plays a role in organ size control via an epigenetic manner

USDA-ARS?s Scientific Manuscript database

Epigenetic regulation, including various covalent modifications of histone proteins and methylation of cytosine bases in DNA, participates broadly in many fundamentally physiological and developmental processes. The repressed or active states of transcription resulted from epigenetic modifications a...

Simultaneous quantification of oil and protein in cottonseed by low-field time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cottonseed quality traits is likely to be achieved through a combination of genetic modification, manipulation of nutrient allocation and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relation...

Non-destructive quantification of oil and protein in cottonseed by time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cotton seed quality traits is likely to be achieved through a combination of genetic modification, nutrient allocation, and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relationship between f...

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications.

PubMed

Porter, Joseph J; Mehl, Ryan A

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described.

Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2016-12-01

Prokaryotic expression systems are superior in producing valuable recombinant proteins, enzymes and therapeutic products. Conventional microbial technology is evolving gradually and amalgamated with advanced technologies in order to give rise to improved processes for the production of metabolites, recombinant biopharmaceuticals and industrial enzymes. Recently, several novel approaches have been employed in a bacterial expression platform to improve recombinant protein expression. These approaches involve metabolic engineering, use of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, high-throughput devices for cloning and process screening as well as fermentation technologies. Advancement of the novel technologies in E. coli systems led to the production of "difficult to express" complex products including small peptides, antibody fragments, few proteins and full-length aglycosylated monoclonal antibodies in considerable large quantity. Wacker's secretion technologies, Pfenex system, inducers, cell-free systems, strain engineering for post-translational modification, such as disulfide bridging and bacterial N-glycosylation, are still under evaluation for the production of complex proteins and peptides in E. coli in an efficient manner. This appraisal provides an impression of expression technologies developed in recent times for enhanced production of heterologous proteins in E. coli which are of foremost importance for diverse applications in microbiology and biopharmaceutical production.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pincus, David; Ryan, Christopher J.; Smith, Richard D.

2013-03-12

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

Influence of xanthan gum on the structural characteristics of myofibrillar proteins treated by high pressure.

PubMed

Villamonte, Gina; Jury, Vanessa; Jung, Stéphanie; de Lamballerie, Marie

2015-03-01

The effects of xanthan gum on the structural modifications of myofibrillar proteins (0.3 M NaCl, pH 6) induced by high pressure (200, 400, and 600 MPa, 6 min) were investigated. The changes in the secondary and tertiary structures of myofibrillar proteins were analyzed by circular dichroism. The protein denaturation was also evaluated by differential scanning calorimetry. Likewise, the protein surface hydrophobicity and the solubility of myofibrillar proteins were measured. High pressure (600 MPa) induced the loss of α-helix structures and an increase of β-sheet structures. However, the presence of xanthan gum hindered the former mechanism of protein denaturation by high pressure. In fact, changes in the secondary (600 MPa) and the tertiary structure fingerprint of high-pressure-treated myofibrillar proteins (400 to 600 MPa) were observed in the presence of xanthan gum. These modifications were confirmed by the thermal analysis, the thermal transitions of high-pressure (400 to 600 MPa)-treated myofibrillar proteins were modified in systems containing xanthan gum. As consequence, the high-pressure-treated myofibrillar proteins with xanthan gum showed increased solubility from 400 MPa, in contrast to high-pressure treatment (600 MPa) without xanthan gum. Moreover, the surface hydrophobicity of high-pressure-treated myofibrillar proteins was enhanced in the presence of xanthan gum. These effects could be due to the unfolding of myofibrillar proteins at high-pressure levels, which exposed sites that most likely interacted with the anionic polysaccharide. This study suggests that the role of food additives could be considered for the development of meat products produced by high-pressure processing. © 2015 Institute of Food Technologists®

Using ion exchange chromatography to purify a recombinantly expressed protein.

PubMed

Duong-Ly, Krisna C; Gabelli, Sandra B

2014-01-01

Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. © 2014 Elsevier Inc. All rights reserved.

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

PubMed

Hwang, Hongik; Rhim, Hyewhon

2018-03-01

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

Microbial factories for recombinant pharmaceuticals

PubMed Central

Ferrer-Miralles, Neus; Domingo-Espín, Joan; Corchero, José Luis; Vázquez, Esther; Villaverde, Antonio

2009-01-01

Most of the hosts used to produce the 151 recombinant pharmaceuticals so far approved for human use by the Food and Drug Administration (FDA) and/or by the European Medicines Agency (EMEA) are microbial cells, either bacteria or yeast. This fact indicates that despite the diverse bottlenecks and obstacles that microbial systems pose to the efficient production of functional mammalian proteins, namely lack or unconventional post-translational modifications, proteolytic instability, poor solubility and activation of cell stress responses, among others, they represent convenient and powerful tools for recombinant protein production. The entering into the market of a progressively increasing number of protein drugs produced in non-microbial systems has not impaired the development of products obtained in microbial cells, proving the robustness of the microbial set of cellular systems (so far Escherichia coli and Saccharomyces cerevisae) developed for protein drug production. We summarize here the nature, properties and applications of all those pharmaceuticals and the relevant features of the current and potential producing hosts, in a comparative way. PMID:19317892

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

PubMed

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

PubMed

Güray, Melda Z; Zheng, Shi; Doucette, Alan A

2017-02-03

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions.

PubMed

Suzuki, D; Miyata, T; Saotome, N; Horie, K; Inagi, R; Yasuda, Y; Uchida, K; Izuhara, Y; Yagame, M; Sakai, H; Kurokawa, K

1999-04-01

Advanced glycation end products (AGE) include a variety of protein adducts whose accumulation has been implicated in tissue damage associated with diabetic nephropathy (DN). It was recently demonstrated that among AGE, glycoxidation products, whose formation is closely linked to oxidation, such as carboxymethyllysine (CML) and pentosidine, accumulate in expanded mesangial matrix and nodular lesions in DN, in colocalization with malondialdehyde-lysine (MDA-lysine), a lipoxidation product, whereas pyrraline, another AGE structure whose deposition is rather independent from oxidative stress, was not found within diabetic glomeruli. Because CML, pentosidine, and MDA-lysine are all formed under oxidative stress by carbonyl amine chemistry between protein amino group and carbonyl compounds, their colocalization suggests a local oxidative stress and increased protein carbonyl modification in diabetic glomerular lesions. To address this hypothesis, human renal tissues from patients with DN or IgA nephropathy were examined with specific antibodies to characterize most, if not all, carbonyl modifications of proteins by autoxidation products of carbohydrates, lipids, and amino acids: CML (derived from carbohydrates, lipids, and amino acid), pentosidine (derived from carbohydrates), MDA-lysine (derived from lipids), 4-hydroxynonenal-protein adduct (derived from lipids), and acrolein-protein adduct (derived from lipids and amino acid). All of the protein adducts were identified in expanded mesangial matrix and nodular lesions in DN. In IgA nephropathy, another primary glomerular disease leading to end-stage renal failure, despite positive staining for MDA-lysine and 4-hydroxynonenal-protein adduct in the expanded mesangial area, CML, pentosidine, and acrolein-protein adduct immunoreactivities were only faint in glomeruli. These data suggest a broad derangement in nonenzymatic biochemistry in diabetic glomerular lesions, and implicate an increased local oxidative stress and carbonyl modification of proteins in diabetic glomerular tissue damage ("carbonyl stress").

Genome-based analysis of heme biosynthesis and uptake in prokaryotic systems.

PubMed

Cavallaro, Gabriele; Decaria, Leonardo; Rosato, Antonio

2008-11-01

Heme is the prosthetic group of many proteins that carry out a variety of key biological functions. In addition, for many pathogenic organisms, heme (acquired from the host) may constitute a very important source of iron. Organisms can meet their heme demands by taking it up from external sources, by producing the cofactor through a dedicated biosynthetic pathway, or both. Here we analyzed the distribution of proteins specifically involved in the processes of heme biosynthesis and heme uptake in 474 prokaryotic organisms. These data allowed us to identify which organisms are capable of performing none, one, or both processes, based on the similarity to known systems. Some specific instances where one or more proteins along the pathways had unusual modifications were singled out. For two key protein domains involved in heme uptake, we could build a series of structural models, which suggested possible alternative modes of heme binding. Future directions for experimental work are given.

Covalent modification of proteins by cocaine

NASA Astrophysics Data System (ADS)

Deng, Shi-Xian; Bharat, Narine; Fischman, Marian C.; Landry, Donald W.

2002-03-01

Cocaine covalently modifies proteins through a reaction in which the methyl ester of cocaine acylates the -amino group of lysine residues. This reaction is highly specific in vitro, because no other amino acid reacts with cocaine, and only cocaine's methyl ester reacts with the lysine side chain. Covalently modified proteins were present in the plasma of rats and human subjects chronically exposed to cocaine. Modified endogenous proteins are immunogenic, and specific antibodies were elicited in mouse and detected in the plasma of human subjects. Covalent modification of proteins could explain cocaine's autoimmune effects and provide a new biochemical approach to cocaine's long-term actions.

Heme oxygenase-1 posttranslational modifications in the brain of subjects with Alzheimer disease and mild cognitive impairment.

PubMed

Barone, Eugenio; Di Domenico, Fabio; Sultana, Rukhsana; Coccia, Raffaella; Mancuso, Cesare; Perluigi, Marzia; Butterfield, D Allan

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Oxidative and nitrosative stress plays a principal role in the pathogenesis of AD. The induction of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species. Although initially proposed as a neuroprotective system in AD brain, the HO-1/BVR-A pathophysiological features are under debate. We previously reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative posttranslational modifications in the brain of subjects with AD and those with mild cognitive impairment (MCI). Furthermore, other groups proposed the observed increase in HO-1 in AD brain as a possible neurotoxic mechanism. Here we provide new insights about HO-1 in the brain of subjects with AD and MCI, the latter condition being the transitional phase between normal aging and early AD. HO-1 protein levels were significantly increased in the hippocampus of AD subjects, whereas HO-2 protein levels were significantly decreased in both AD and MCI hippocampi. In addition, significant increases in Ser-residue phosphorylation together with increased oxidative posttranslational modifications were found in the hippocampus of AD subjects. Interestingly, despite the lack of oxidative stress-induced AD neuropathology in cerebellum, HO-1 demonstrated increased Ser-residue phosphorylation and oxidative posttranslational modifications in this brain area, suggesting HO-1 as a target of oxidative damage even in the cerebellum. The significance of these findings is profound and opens new avenues into the comprehension of the role of HO-1 in the pathogenesis of AD. Copyright © 2012 Elsevier Inc. All rights reserved.

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

The movement of proteins across the insect and tick digestive system.

PubMed

Jeffers, Laura A; Michael Roe, R

2008-02-01

The movement of intact proteins across the digestive system was shown in a number of different blood-feeding and non-blood-feeding insects in the orders Blattaria, Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Neuroptera and Siphonaptera, as well as in two tick families Ixodidae and Argasidae. Protein movement was observed for both normal dietary and xenobiotic proteins, which suggest that the mechanism for transfer is not substrate specific. The number of studies on the mechanism of movement is limited. The research so far suggests that movement can occur by either a transcellular or an intercellular pathway in the ventriculus with most of the research describing the former. Transfer is by continuous diffusion with no evidence of pinocytosis or vesicular transport common in mammalian systems. Proteins can move across the digestive system without modification of their primary or multimeric structure and with retention of their functional characteristics. Accumulation in the hemolymph is the result of the protein degradation rate in the gut and hemolymph and transfer rate across the digestive system and can be highly variable depending on species. Research on the development of delivery systems to enhance protein movement across the insect digestive system is in its infancy. The approaches so far considered with some success include the use of lipophilic-polyethylene glycol (PEG) polymers, the development of fusion proteins with lectins, reduced gut protease activity and the development of amphiphilic peptidic analogs. Additional research on understanding the basic mechanisms of protein delivery across the insect digestive system, the importance of structure activity in this transfer and the development of technology to improve movement across the gut could be highly significant to the future of protein and nucleic acid-based insecticide development as well as traditional chemical insecticidal technologies.

Rab protein evolution and the history of the eukaryotic endomembrane system

PubMed Central

Brighouse, Andrew; Dacks, Joel B.

2010-01-01

Spectacular increases in the quantity of sequence data genome have facilitated major advances in eukaryotic comparative genomics. By exploiting homology with classical model organisms, this makes possible predictions of pathways and cellular functions currently impossible to address in intractable organisms. Echoing realization that core metabolic processes were established very early following evolution of life on earth, it is now emerging that many eukaryotic cellular features, including the endomembrane system, are ancient and organized around near-universal principles. Rab proteins are key mediators of vesicle transport and specificity, and via the presence of multiple paralogues, alterations in interaction specificity and modification of pathways, contribute greatly to the evolution of complexity of membrane transport. Understanding system-level contributions of Rab proteins to evolutionary history provides insight into the multiple processes sculpting cellular transport pathways and the exciting challenges that we face in delving further into the origins of membrane trafficking specificity. PMID:20582450

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging*

PubMed Central

Ramallo Guevara, Carina; Philipp, Oliver; Hamann, Andrea; Werner, Alexandra; Osiewacz, Heinz D.; Rexroth, Sascha; Rögner, Matthias; Poetsch, Ansgar

2016-01-01

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. PMID:26884511

Palmitoylation as a Functional Regulator of Neurotransmitter Receptors

PubMed Central

Naumenko, Vladimir S.

2018-01-01

The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation) within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LICs). From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior. PMID:29849559

Protein Tyrosine Nitration: Role in Aging.

PubMed

Chakravarti, Bulbul; Chakravarti, Deb N

2017-01-01

Aging is the inevitable fate of all living organisms, but the molecular basis of physiological aging is poorly understood. Oxidative stress is believed to play a key role in the aging process. In addition to Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS) are generated during aerobic metabolism in living organisms. Although protein damage and functional modification by ROS have been demonstrated in details, fewer studies have been reported on protein damage by RNS and its implication in the aging process. Proteins undergoing tyrosine nitration are associated with pathophysiology of several diseases, as well as physiological aging. The purpose of the current review article is to provide a brief summary of the biochemical mechanisms of tyrosine nitration, methodologies used for the detection of these modified proteins, effect of RNS induced post translational modification on biological functions and the putative role of tyrosine nitrated proteins in the aging process. Published studies on the role of RNS in age related functional alteration of various organs/ tissues were critically reviewed and evaluated. Covalent modification of various proteins by tyrosine nitration is associated with modification of biological functions of various organs/tissues such as skeletal muscle, heart, brain and liver due to aging. This information will be helpful to further investigate the interplay of different biochemical pathways and networks involved in the tyrosine nitration of various proteins due to aging with the ultimate goal to prevent the detrimental effects of RNS on the functional activities of these proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

The Expanding Landscape of the Thiol Redox Proteome*

PubMed Central

Yang, Jing; Carroll, Kate S.; Liebler, Daniel C.

2016-01-01

Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO2H), S-sulfonylation (-SO3H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes. Here, we review chemical proteomic strategies used to expand the landscape of thiol redox modifications. PMID:26518762

Oxidative Post-Translational Modifications of an Amyloidogenic Immunoglobulin Light Chain Protein.

PubMed

Lu, Yanyan; Jiang, Yan; Prokaeva, Tatiana; Connors, Lawreen H; Costello, Catherine E

2017-05-01

Immunoglobulin light chain amyloidosis (AL) is a plasma cell disorder characterized by overproduction and deposition of monoclonal immunoglobulin (Ig) light chains (LC) or variable region fragments as amyloid fibrils in various organs and tissues. Much clinical evidence indicates that patients with AL amyloidosis sustain cardiomyocyte impairment and suffer from oxidative stress. We seek to understand the underlying biochemical pathways whose disruption or amplification during sporadic or sustained disease states leads to harmful physiological consequences and to determine the detailed structures of intermediates and products that serve as signposts for the biochemical changes and represent potential biomarkers. In this study, matrix-assisted laser desorption/ionization mass spectrometry provided extensive evidence for oxidative post-translational modifications (PTMs) of an amyloidogenic Ig LC protein from a patient with AL amyloidosis. Some of the tyrosine residues were heavily mono- or di-chlorinated. In addition, a novel oxidative conversion to a nitrile moiety was observed for many of the terminal aminomethyl groups on lysine side chains. In vitro experiments using model peptides, in-solution oxidation, and click chemistry demonstrated that hypochlorous acid produced by the myeloperoxidase - hydrogen peroxide - chloride system could be responsible for these and other, more commonly observed modifications.

Basics and recent advances in peptide and protein drug delivery

PubMed Central

Bruno, Benjamin J; Miller, Geoffrey D; Lim, Carol S

2014-01-01

While the peptide and protein therapeutic market has developed significantly in the past decades, delivery has limited their use. Although oral delivery is preferred, most are currently delivered intravenously or subcutaneously due to degradation and limited absorption in the gastrointestinal tract. Therefore, absorption enhancers, enzyme inhibitors, carrier systems and stability enhancers are being studied to facilitate oral peptide delivery. Additionally, transdermal peptide delivery avoids the issues of the gastrointestinal tract, but also faces absorption limitations. Due to proteases, opsonization and agglutination, free peptides are not systemically stable without modifications. This review discusses oral and transdermal peptide drug delivery, focusing on barriers and solutions to absorption and stability issues. Methods to increase systemic stability and site-specific delivery are also discussed. PMID:24228993

Functions and substrates of NEDDylation during cell cycle in the silkworm, Bombyx mori.

PubMed

Li, Zhiqing; Cui, Qixin; Wang, Xiaoyan; Li, Bingqian; Zhao, Dongchao; Xia, Qingyou; Zhao, Ping

2017-11-01

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm. Copyright © 2017. Published by Elsevier Ltd.

Alpha-A crystallin: quantitation of C-terminal modification during lens aging

NASA Technical Reports Server (NTRS)

Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

1994-01-01

Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

Surface Modification of Biomaterials: A Quest for Blood Compatibility

PubMed Central

de Mel, Achala; Cousins, Brian G.; Seifalian, Alexander M.

2012-01-01

Cardiovascular implants must resist thrombosis and intimal hyperplasia to maintain patency. These implants when in contact with blood face a challenge to oppose the natural coagulation process that becomes activated. Surface protein adsorption and their relevant 3D confirmation greatly determine the degree of blood compatibility. A great deal of research efforts are attributed towards realising such a surface, which comprise of a range of methods on surface modification. Surface modification methods can be broadly categorized as physicochemical modifications and biological modifications. These modifications aim to modulate platelet responses directly through modulation of thrombogenic proteins or by inducing antithrombogenic biomolecules that can be biofunctionalised onto surfaces or through inducing an active endothelium. Nanotechnology is recognising a great role in such surface modification of cardiovascular implants through biofunctionalisation of polymers and peptides in nanocomposites and through nanofabrication of polymers which will pave the way for finding a closer blood match through haemostasis when developing cardiovascular implants with a greater degree of patency. PMID:22693509

Identification of O-linked β-d-N-acetylglucosamine-Modified Proteins from Arabidopsis

PubMed Central

Xu, Shou-Ling; Chalkley, Robert J.; Wang, Zhi-Yong; Burlingame, Alma L.

2013-01-01

The posttranslational modification of proteins with O-linked β-d-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

PubMed Central

McClure, Michelle; DeLucas, Lawrence J.; Wilson, Landon; Ray, Marjorie; Rowe, Steven M.; Wu, Xiaoyun; Dai, Qun; Hong, Jeong S.; Sorscher, Eric J.; Kappes, John C.; Barnes, Stephen

2012-01-01

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease. PMID:22119790

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

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry.

PubMed

Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J

2006-11-01

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.

Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts.

PubMed

Colzani, Mara; Aldini, Giancarlo; Carini, Marina

2013-10-30

Our current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

Chapter Seven - When Phosphorylation Encounters Ubiquitination: A Balanced Perspective on IGF-1R Signaling.

PubMed

Girnita, L; Takahashi, S-I; Crudden, C; Fukushima, T; Worrall, C; Furuta, H; Yoshihara, H; Hakuno, F; Girnita, A

2016-01-01

Cell-surface receptors govern the critical information passage from outside to inside the cell and hence control important cellular decisions such as survival, growth, and differentiation. These receptors, structurally grouped into different families, utilize common intracellular signaling-proteins and pathways, yet promote divergent biological consequences. In rapid processing of extracellular signals to biological outcomes, posttranslational modifications offer a repertoire of protein processing options. Protein ubiquitination was originally identified as a signal for protein degradation through the proteasome system. It is now becoming increasingly recognized that both ubiquitin and ubiquitin-like proteins, all evolved from a common ubiquitin structural superfold, are used extensively by the cell and encompass signal tags for many different cellular fates. In this chapter we examine the current understanding of the ubiquitin regulation surrounding the insulin-like growth factor and insulin signaling systems, major members of the larger family of receptor tyrosine kinases (RTKs) and key regulators of fundamental physiological and pathological states. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

NASA Astrophysics Data System (ADS)

Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

2016-03-01

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.

The etiology of human age-related cataract. Proteins don't last forever.

PubMed

Truscott, Roger J W; Friedrich, Michael G

2016-01-01

It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of Cryptococcus humicola response to aluminum stress.

PubMed

Zhang, Jingjing; Zhang, Lei; Qiu, Jinkui; Nian, Hongjuan

2015-10-01

Cryptococcus humicola is a highly aluminum (Al) tolerant yeast strain isolated from a tea field. Here the relative changes of protein expression in C. humicola undergoing aluminum stress were analyzed to understand the genetic basis of aluminum tolerance. In this work, iTRAQ-based (isobaric tags for relative and absolute quantification) quantitative proteomic technology was used to detect statistically significant proteins associated with the response to aluminum stress. A total of 625 proteins were identified and were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, energy production and conversion, and amino acid transport and metabolism. Of these proteins, 59 exhibited differential expression during aluminum stress. Twenty-nine proteins up-regulated by aluminum were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover and chaperones, and lipid transport and metabolism. Thirty proteins down-regulated by aluminum were mainly associated with energy transport and metabolism, translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, and lipid transport and metabolism. The potential functions of some proteins in aluminum tolerance are discussed. These functional changes may be beneficial for cells to protect themselves from aluminum toxic conditions. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

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

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

PubMed

Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B

1993-02-12

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

PubMed Central

Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

2014-01-01

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins. PMID:24865146

Preparation and Analysis of Positioned Mononucleosomes

PubMed Central

Kulaeva, Olga; Studitsky, Vasily M.

2016-01-01

Short DNA fragments containing single nucleosomes have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, covalent histone modifications, transcription, DNA repair and ATP-dependent chromatin remodeling. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes assembled on 200–350-bp DNA fragments. PMID:25827872

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

PubMed

Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

2016-08-01

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants

PubMed Central

Khodai-Kalaki, Maryam; Andrade, Angel; Fathy Mohamed, Yasmine

2015-01-01

ABSTRACT Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that “virulence” depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection. PMID:26045541

The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA

PubMed Central

Toh, Seok-Ming; Xiong, Liqun; Bae, Taeok; Mankin, Alexander S.

2008-01-01

A2503 in 23S rRNA of the Gram-negative bacterium Escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. In E. coli, and likely in other species, this adenosine residue is post-transcriptionally modified to m2A. The enzyme responsible for this modification was previously unknown. We identified E. coli protein YfgB, which belongs to the radical SAM enzyme superfamily, as the methyltransferase that modifies A2503 of 23S rRNA to m2A. Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA as revealed by primer extension analysis and thin layer chromatography. The A2503 modification was restored when YfgB protein was expressed in the yfgB knockout strain. A similar protein was shown to catalyze post-transcriptional modification of A2503 in 23S rRNA in Gram-positive Staphylococcus aureus. The yfgB knockout strain loses in competition with wild type in a co-growth experiment, indicating functional importance of A2503 modification. The location of A2503 in the exit tunnel suggests its possible involvement in interaction with the nascent peptide and raises the possibility that its post-transcriptional modification may influence such an interaction. PMID:18025251

Partial contribution of the Keap1–Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shinkai, Yasuhiro; Kimura, Tomoki; Itagaki, Ayaka

Cadmium is an environmental electrophile that modifies protein reactive thiols such as Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear factor-erythroid 2-related factor 2 (Nrf2). In the present study, we investigated a role of the Keap1–Nrf2 system in cellular response to cadmium in vascular endothelial cells. Exposure of bovine aortic endothelial cells to cadmium resulted in modification of Keap1 and Nrf2 activation, thereby up-regulating not only its typical downstream proteins but also metallothionein-1/2. Experiments with siRNA-mediated knockdown of Nrf2 or Keap1 supported participation of the Keap1–Nrf2 system in the modulation of metallothionein-1/2 expression. Furthermore, chromatin immunoprecipitation assay showedmore » that Nrf2 was recruited to the antioxidant response element of the promoter region of the bovine metallothionein-2 gene in the presence of cadmium. These results suggest that the transcription factor Nrf2 plays, at least in part, a role in the changes in metallothionein expression mediated by exposure to cadmium. - Highlights: • Role of the Keap1–Nrf2 system in cellular response to cadmium was examined. • We used bovine aortic endothelial cells as a model of the vascular endothelium. • Exposure of cells to cadmium resulted in modification of Keap1 and Nrf2 activation. • Keap1–Nrf2 system participated in the modulation of metallothionein-1/2 expression. • Nrf2 was recruited to the antioxidant response element of MT2 promoter region.« less

Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

PubMed

Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

2009-06-01

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a potential regulatory role. Through a wider analysis of other poorly characterized DNA-modifying enzymes we also show that the phage Mu Mom-like proteins, which catalyze the N6-carbamoylmethylation of adenines, are also linked to diverse families of bacterial transposases, suggesting that DNA modification by transposable elements might have a more general presence than previously appreciated. Among the other families of 2-oxoglutarate- and iron(II)-dependent dioxygenases identified in this study, one which is found in algae, is predicted to mainly comprise of RNA-modifying enzymes and shows a striking diversity in protein domain architectures suggesting the presence of RNA modifications with possibly unique adaptive roles. The results presented here are likely to provide the means for future investigation of unexpected epigenetic modifications, such as hydroxymethyl cytosine, that could profoundly impact our understanding of gene regulation and processes such as DNA demethylation.

Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory.

PubMed

Wilson, Karl A; Tan-Wilson, Anna

2013-01-01

Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed, requiring more time and expertise than instructors of large laboratory classes can devote. We have developed an experimental module for our Biochemistry Laboratory course that engages students in MS-based protein identification following protein separation by one-dimensional SDS-PAGE, a technique that is usually taught in this type of course. The module is based on soybean seed storage proteins, a relatively simple mixture of proteins present in high levels in the seed, allowing the identification of the main protein bands by MS/MS and in some cases, even by peptide mass fingerprinting. Students can identify their protein bands using software available on the Internet, and are challenged to deduce post-translational modifications that have occurred upon germination. A collection of mass spectral data and tutorials that can be used as a stand-alone computer-based laboratory module were also assembled. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function.

PubMed Central

Edge, Albert S B

2003-01-01

The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) [Edge, Faltynek, Hof, Reichert, and Weber, (1981) Anal. Biochem. 118, 131-137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function. PMID:12974674

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures.

PubMed

Bayart, Caroline; Peronin, Sébastien; Jean, Elisa; Paladino, Joseph; Talaga, Philippe; Borgne, Marc Le

2017-06-01

Aldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde. Copyright © 2017 Elsevier B.V. All rights reserved.

Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice

PubMed Central

Shearn, Colin T.; Fritz, Kristofer S.; Shearn, Alisabeth H.; Saba, Laura M.; Mercer, Kelly E.; Engi, Bridgette; Galligan, James J.; Zimniak, Piotr; Orlicky, David J.; Ronis, Martin J.; Petersen, Dennis R.

2015-01-01

Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4−/− mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4−/− mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4−/− mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4−/− mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4−/− PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins. PMID:26654979

Quantifying Ubiquitin Signaling

PubMed Central

Ordureau, Alban; Münch, Christian; Harper, J. Wade

2015-01-01

Ubiquitin (UB)-driven signaling systems permeate biology, and are often integrated with other types of post-translational modifications (PTMs), most notably phosphorylation. Flux through such pathways is typically dictated by the fractional stoichiometry of distinct regulatory modifications and protein assemblies as well as the spatial organization of pathway components. Yet, we rarely understand the dynamics and stoichiometry of rate-limiting intermediates along a reaction trajectory. Here, we review how quantitative proteomic tools and enrichment strategies are being used to quantify UB-dependent signaling systems, and to integrate UB signaling with regulatory phosphorylation events. A key regulatory feature of ubiquitylation is that the identity of UB chain linkage types can control downstream processes. We also describe how proteomic and enzymological tools can be used to identify and quantify UB chain synthesis and linkage preferences. The emergence of sophisticated quantitative proteomic approaches will set a new standard for elucidating biochemical mechanisms of UB-driven signaling systems. PMID:26000850

Toxicological evaluation of proteins introduced into food crops

PubMed Central

Kough, John; Herouet-Guicheney, Corinne; Jez, Joseph M.

2013-01-01

This manuscript focuses on the toxicological evaluation of proteins introduced into GM crops to impart desired traits. In many cases, introduced proteins can be shown to have a history of safe use. Where modifications have been made to proteins, experience has shown that it is highly unlikely that modification of amino acid sequences can make a non-toxic protein toxic. Moreover, if the modified protein still retains its biological function, and this function is found in related proteins that have a history of safe use (HOSU) in food, and the exposure level is similar to functionally related proteins, then the modified protein could also be considered to be “as-safe-as” those that have a HOSU. Within nature, there can be considerable evolutionary changes in the amino acid sequence of proteins within the same family, yet these proteins share the same biological function. In general, food crops such as maize, soy, rice, canola etc. are subjected to a variety of processing conditions to generate different food products. Processing conditions such as cooking, modification of pH conditions, and mechanical shearing can often denature proteins in these crops resulting in a loss of functional activity. These same processing conditions can also markedly lower human dietary exposure to (functionally active) proteins. Safety testing of an introduced protein could be indicated if its biological function was not adequately characterized and/or it was shown to be structurally/functionally related to proteins that are known to be toxic to mammals. PMID:24164515

C-reactive protein in degenerative aortic valve stenosis

PubMed Central

Sanchez, Pedro L; Mazzone, AnnaMaria

2006-01-01

Degenerative aortic valve stenosis includes a range of disorder severity from mild leaflet thickening without valve obstruction, "aortic sclerosis", to severe calcified aortic stenosis. It is a slowly progressive active process of valve modification similar to atherosclerosis for cardiovascular risk factors, lipoprotein deposition, chronic inflammation, and calcification. Systemic signs of inflammation, as wall and serum C-reactive protein, similar to those found in atherosclerosis, are present in patients with degenerative aortic valve stenosis and may be expression of a common disease, useful in monitoring of stenosis progression. PMID:16774687

Diversity of the Epsilonproteobacteria Dsb (disulfide bond) systems

PubMed Central

Bocian-Ostrzycka, Katarzyna M.; Grzeszczuk, Magdalena J.; Dziewit, Lukasz; Jagusztyn-Krynicka, Elżbieta K.

2015-01-01

The bacterial proteins of the Dsb family—important components of the post-translational protein modification system—catalyze the formation of disulfide bridges, a process that is crucial for protein structure stabilization and activity. Dsb systems play an essential role in the assembly of many virulence factors. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. While the Escherichia coli disulfide bond-forming system is quite well understood, the mechanisms of action of Dsb systems in other bacteria, including members of class Epsilonproteobacteria that contain pathogenic and non-pathogenic bacteria colonizing extremely diverse ecological niches, are poorly characterized. Here we present a review of current knowledge on Epsilonproteobacteria Dsb systems. We have focused on the Dsb systems of Campylobacter spp. and Helicobacter spp. because our knowledge about Dsb proteins of Wolinella and Arcobacter spp. is still scarce and comes mainly from bioinformatic studies. Helicobacter pylori is a common human pathogen that colonizes the gastric epithelium of humans with severe consequences. Campylobacter spp. is a leading cause of zoonotic enteric bacterial infections in most developed and developing nations. We focus on various aspects of the diversity of the Dsb systems and their influence on pathogenicity, particularly because Dsb proteins are considered as potential targets for a new class of anti-virulence drugs to treat human infections by Campylobacter or Helicobacter spp. PMID:26106374

A new class of animal collagen masquerading as an insect silk

PubMed Central

Sutherland, Tara D.; Peng, Yong Y.; Trueman, Holly E.; Weisman, Sarah; Okada, Shoko; Walker, Andrew A.; Sriskantha, Alagacone; White, Jacinta F.; Huson, Mickey G.; Werkmeister, Jerome A.; Glattauer, Veronica; Stoichevska, Violet; Mudie, Stephen T.; Haritos, Victoria S.; Ramshaw, John A. M.

2013-01-01

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. PMID:24091725

The Role of Protein-Mineral Interactions for Protein Adsorption or Fragmentation

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P.; Washton, N.; Kleber, M.

2014-12-01

Soil exo-enzymes (EE) are proteins with the capability to catalyze the depolymerization of soil organic matter (SOM). SOM must be disassembled by EEs in order to be transported through the microbial cell wall and become metabolized. One factor determining an EE's functionality is their affinity to mineral surfaces found in the soil. Our goal was to establish the range of protein modifications, either chemical or structural, as the protein becomes associated with mineral surfaces. We hypothesized that pedogenic oxides would generate more extensive chemical alterations to the protein structure than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We used 1H 15N Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance Spectroscopy (HSQC NMR) to observe structural modifications in the unadsorbed Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite and birnessite. Solid state NMR was used to observe the structural modifications of Gb1 while adsorbed onto kaolinite and montmorillonite. Preliminary results in the HSQC NMR spectra observed no changes in the native conformation of Gb1 when allowed to interact with goethite and kaolinite while birnessite induced strong structural modification of Gb1 at an acidic pH. Our results suggest that not all mineral surfaces in soil act as sorbents for EEs and changes in their catalytic activity upon adsorption to minerals surfaces may not just be an indication of conformational changes but of fragmentation of the protein itself.

Characterization of Surface Modification of Polyethersulfone Membrane

USDA-ARS?s Scientific Manuscript database

Surface modification of polyethersulfone (PES) membrane surface using UV/ozone-treated grafting and interfacial polymerization on membrane surface was investigated in order to improve the resistance of membrane surface to protein adsorption. These methods of surface modification were compared in te...

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells

PubMed Central

Tsuchiya, Yugo; Peak-Chew, Sew Yeu; Newell, Clare; Miller-Aidoo, Sheritta; Mangal, Sriyash; Zhyvoloup, Alexander; Bakovic´, Jovana; Malanchuk, Oksana; Pereira, Gonçalo C.; Kotiadis, Vassilios; Szabadkai, Gyorgy; Duchen, Michael R.; Campbell, Mark; Cuenca, Sergio Rodriguez; Vidal-Puig, Antonio; James, Andrew M.; Murphy, Michael P.; Filonenko, Valeriy; Skehel, Mark

2017-01-01

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions. PMID:28341808

Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

PubMed

Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

2017-10-07

The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeted gene disruption in Koji mold Aspergillus oryzae.

PubMed

Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2011-01-01

Filamentous fungi have received attentions as hosts for heterologous protein production because of their high secretion capability and eukaryotic post-translational modifications. One of the safest hosts for heterologous protein production is Koji mold Aspergillus oryzae since it has been used in the production of Japanese fermented foods for over 1,000 years. The production levels of proteins from higher eukaryotes are much lower than those of homologous (fungal) proteins. Bottlenecks in the heterologous protein production are suggested to be proteolytic degradation of the produced protein in the medium and the secretory pathway. For construction of excellent host strains, many genes causing the bottlenecks should be disrupted rapidly and efficiently. We developed a marker recycling system with the highly efficient gene-targeting background in A. oryzae. By employing this technique, we performed multiple gene disruption of the ten protease genes. The decuple protease gene disruptant showed fourfold production level of a heterologous protein compared with the wild-type strain.

The Modification of Tet1 in Male Germline Stem Cells and Interact with PCNA, HDAC1 to promote their Self-renewal and Proliferation

PubMed Central

Zheng, Liming; Zhai, Yuanxin; Li, Na; Ma, Fanglin; Zhu, Haijing; Du, Xiaomin; Li, Guangpeng; Hua, Jinlian

2016-01-01

Epigenetic modification plays key roles in spermatogenesis, especially DNA methylation dynamic is important in sustaining normal spermatogenesis. Ten-eleven translocation 1 (Tet1) is not only a key demethylase, which works in specific gene regions, but also crosstalks with partners to regulate epigenetic progress as protein complexes. Dairy goat is an important livestock in China, while the unstable culture system in vitro inhibits optimization of new dairy goat species. The study of epigenetic modification in male germline stem cells (mGSCs) is beneficial to the optimization of adult stem cell culture system in vitro, and the improvement of sperm quality and breeding of selected livestock. In our study, we not only analyzed the morphology, gene expression, DNA methylation and histone methylation dynamic in mouse Tet1 (mTet1) modified mGSCs, we also analyzed the stemness ability by in vivo transplantation and explored the functional mechanism of Tet1 in dairy goat mGSCs. The results showed mTet1 modified mGSCs had better self-renewal and proliferation ability than wild-type mGSCs, mTet1 could also up-regulate JMJD3 to decrease H3K27me3, which also showed to suppress the MEK-ERK pathway. Furthermore, Co-IP analysis demonstrated that TET1 interact with PCNA and HDAC1 by forming protein complexes to comprehensively regulate dairy goat mGSCs and spermatogenesis. PMID:27857213

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

NASA Astrophysics Data System (ADS)

Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

2015-11-01

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications

PubMed Central

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described. PMID:29849913

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

PubMed Central

Nuriel, Tal; Deeb, Ruba S.; Hajjar, David P.; Gross, Steven S.

2008-01-01

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues. PMID:18554526

Redox proteomics of tomato in response to Pseudomonas syringae infection

PubMed Central

Balmant, Kelly Mayrink; Parker, Jennifer; Yoo, Mi-Jeong; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2015-01-01

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation–reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses. PMID:26504582

Specificity and disease in the ubiquitin system

PubMed Central

Chaugule, Viduth K.; Walden, Helen

2016-01-01

Post-translational modification (PTM) of proteins by ubiquitination is an essential cellular regulatory process. Such regulation drives the cell cycle and cell division, signalling and secretory pathways, DNA replication and repair processes and protein quality control and degradation pathways. A huge range of ubiquitin signals can be generated depending on the specificity and catalytic activity of the enzymes required for attachment of ubiquitin to a given target. As a consequence of its importance to eukaryotic life, dysfunction in the ubiquitin system leads to many disease states, including cancers and neurodegeneration. This review takes a retrospective look at our progress in understanding the molecular mechanisms that govern the specificity of ubiquitin conjugation. PMID:26862208

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes.

PubMed

Nady, Norhan

2016-04-18

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled "green surface modification". This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers-ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)-is presented.

Greasy tactics in the plant-pathogen molecular arms race.

PubMed

Boyle, Patrick C; Martin, Gregory B

2015-03-01

The modification of proteins by the attachment of fatty acids is a targeting tactic involved in mechanisms of both plant immunity and bacterial pathogenesis. The plant plasma membrane (PM) is a key battleground in the war against disease-causing microbes. This membrane is armed with an array of sensor proteins that function as a surveillance system to detect invading pathogens. Several of these sensor proteins are directed to the plasma membrane through the covalent addition of fatty acids, a process termed fatty acylation. Phytopathogens secrete effector proteins into the plant cell to subvert these surveillance mechanisms, rendering the host susceptible to infection. The targeting of effectors to specific locales within plant cells, particularly the internal face of the host PM, is critical for their virulence function. Several bacterial effectors hijack the host fatty acylation machinery to be modified and directed to this contested locale. To find and fight these fatty acylated effectors the plant leverages lipid-modified intracellular sensors. This review provides examples featuring how fatty acylation is a battle tactic used by both combatants in the molecular arms race between plants and pathogens. Also highlighted is the exploitation of a specific form of host-mediated fatty acid modification, which appears to be exclusively employed by phytopathogenic effector proteins. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

PubMed Central

Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

2006-01-01

Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811

Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1

DOE PAGES

Leon, Deborah R.; Ytterberg, A. Jimmy; Boontheung, Pinmanee; ...

2015-03-05

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

A Robust Analytical Approach for the Identification of Specific Protein Carbonylation Sites: Metal-Catalyzed Oxidations of Human Serum Albumin

PubMed Central

Ugur, Zafer; Gronert, Scott

2017-01-01

The formation of protein carbonyls in the metal-catalyzed oxidation of human serum albumin (HSA) is characterized using a new analytical approach that involves tagging the modification site with multiple hydrazide reagents. Protein carbonyl formation at lysine and arginine residues was catalyzed with copper and iron ions, and the resulting oxidation patterns in HSA are contrasted. A total of 18 modification sites were identified with iron ion catalysis and 14 with copper ion catalysis. However, with the more stringent requirement of identification with at least two tagging reagents, the number of validated modification sites drops to 10 for iron and 9 for copper. Of the 14 total validated sites, there were only five in common for the two metal ions. The results illustrate the value of using multiple tagging agents and highlight the selective and specific nature of metal-catalyzed protein oxidations. PMID:28303033

Enrichment and separation techniques for large-scale proteomics analysis of the protein post-translational modifications.

PubMed

Huang, Junfeng; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

2014-11-06

Comprehensive analysis of the post-translational modifications (PTMs) on proteins at proteome level is crucial to elucidate the regulatory mechanisms of various biological processes. In the past decades, thanks to the development of specific PTM enrichment techniques and efficient multidimensional liquid chromatography (LC) separation strategy, the identification of protein PTMs have made tremendous progress. A huge number of modification sites for some major protein PTMs have been identified by proteomics analysis. In this review, we first introduced the recent progresses of PTM enrichment methods for the analysis of several major PTMs including phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and oxidation/reduction status. We then briefly summarized the challenges for PTM enrichment. Finally, we introduced the fractionation and separation techniques for efficient separation of PTM peptides in large-scale PTM analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Effects of Ionizing Radiation on Biological Molecules—Mechanisms of Damage and Emerging Methods of Detection

PubMed Central

Reisz, Julie A.; Bansal, Nidhi; Qian, Jiang; Zhao, Weiling

2014-01-01

Abstract Significance: The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR. Recent Advances: The development of high-throughput “omics” technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis. Critical Issues: In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and caveats for their use. Additional factors critical to the cellular response to radiation, including alterations in protein expression, metabolomics, and epigenetic factors, are also discussed. Future Directions: Throughout the review, the synergy of combined “omics” technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted. These are anticipated to lead to new hypotheses to understand IR effects on biological systems and improve IR-based therapies. Antioxid. Redox Signal. 21: 260–292. PMID:24382094

Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

PubMed

Korenori, Yoshimi; Tanigawa, Shunsuke; Kumamoto, Takuma; Qin, Si; Daikoku, Yosuke; Miyamori, Koji; Nagai, Masashi; Hou, De-Xing

2013-05-01

6-Methylthiohexyl isothiocyanate (6-MTITC), one of the major bioactive ingredients in Japanese Wasabi, has revealed cytoprotective and cancer chemopreventive effects. This study aims to clarify the molecular mechanisms how 6-MTITC modulates nuclear factor E2-related factor 2 (Nrf2)/Kelchlike ECH-associating protein 1 (Keap1) system in antioxidant-responsive element (ARE)-mediated nicotinamide adenine dinucleotide phosphate (NADP): quinone oxidoreductase 1 (NQO1) expression. HepG2 cells were treated with 6-MTITC with varying time and dose. NQO1, Nrf2, and Keap1 proteins were detected by Western blotting. ARE transactivation was detected by electrophilic mobility shift assay and reporter gene assay. Nuclear localization of Nrf2 was determined by immunocytochemistry assay. Ubiquitination of Nrf2 and Keap1 was detected using immunoprecipitation after treatment with MG132. Small interfering RNA was used to knockdown Nrf2 or Keap1. The results revealed that 6-MTITC modulated Nrf2/ARE pathway by stimulating Keap1 modification, and inhibiting Nrf2 ubiquitination and protein turnover. These actions finally increased nuclear Nrf2 accumulation and ARE-binding activity. Moreover, silencing Nrf2 markedly reduced ARE-driven activity induced by 6-MTITC. 6-MTITC modulated ARE-driven NQO1 expression by stabilizing Nrf2 with enhanced Keap1 modification and decreased Nrf2 degradation. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

PubMed Central

Ohga, Rie; Ota, Satoshi; Kawahara, Atsuo

2015-01-01

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish. PMID:26010089

EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

PubMed

Prasath, Thiruketheeswaran; Greven, Hartmut; D'Haese, Jochen

2012-06-01

Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships. © 2012 WILEY PERIODICALS, INC.

Potential for reduction of odorous compounds in swine manure through diet modification.

PubMed

Sutton, A L; Kephart, K B; Verstegen, M W; Canh, T T; Hobbs, P J

1999-02-01

Recent public concern about air pollution from pork production units has prompted more research to develop methods to reduce and control odors. Masking agents, enzymes and bacterial preparations, feed additives, chemicals, oxidation processes, air scrubbers, biofilters, and new ventilation systems have been studied. Research relating the effects of the swine diet on manure odors has been scarce. Introducing feed additives to bind ammonia, change digesta pH, affect specific enzyme activity, and mask odors has been either costly or not consistently successful. Recent research emphasis has focused on manipulating the diet 1) to increase the nutrient utilization of the diet to reduce excretion products, 2) to enhance microbial metabolism in the lower digestive tract to reduce excretion of odor-causing compounds, and 3) to change the physical characteristics of urine and feces to reduce odor emissions. Primary odor-causing compounds evolve from excess degradable proteins and lack of specific fermentable carbohydrates during microbial fermentation. Reductions in ammonia emissions by 28 to 79% through diet modifications have been reported. Limited research on reduction of other odorous volatile organic compounds through diet modifications is promising. Use of synthetic amino acids with reduced intact protein levels in diets significantly reduces nitrogen excretions and odor production. Addition of nonstarch polysaccharides and specific oligosaccharides further alters the pathway of nitrogen excretion and reduces odor emission. Continued nutritional and microbial research to incorporate protein degradation products, especially sulfur-containing organics, with fermentable carbohydrates in the lower gastrointestinal tract of pigs will further control odors from manure.

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

PubMed

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

2016-06-16

As one of the most common post-translational modifications in eukaryotic cells, lipid modification is an important mechanism for the regulation of variety aspects of protein function. Over the last decades, three classes of lipid modifications have been increasingly studied. The co-regulation of these different lipid modifications is beginning to be noticed. However, due to the lack of integrated bioinformatics resources, the studies of co-regulatory mechanisms are still very limited. In this work, we developed a tool called GPS-Lipid for the prediction of four classes of lipid modifications by integrating the Particle Swarm Optimization with an aging leader and challengers (ALC-PSO) algorithm. GPS-Lipid was proven to be evidently superior to other similar tools. To facilitate the research of lipid modification, we hosted a publicly available web server at http://lipid.biocuckoo.org with not only the implementation of GPS-Lipid, but also an integrative database and visualization tool. We performed a systematic analysis of the co-regulatory mechanism between different lipid modifications with GPS-Lipid. The results demonstrated that the proximal dual-lipid modifications among palmitoylation, myristoylation and prenylation are key mechanism for regulating various protein functions. In conclusion, GPS-lipid is expected to serve as useful resource for the research on lipid modifications, especially on their co-regulation.

Virus-like particles as a vaccine delivery system: myths and facts.

PubMed

Roy, Polly; Noad, Rob

2009-01-01

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this chapter we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus-like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the market place we argue that this technology has now 'come-of-age' and must be considered a viable vaccine strategy.

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

PubMed

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

2013-01-01

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

Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform

PubMed Central

Wagner, Tobias; Greschik, Holger; Burgahn, Teresa; Schmidtkunz, Karin; Schott, Anne-Kathrin; McMillan, Joel; Baranauskienė, Lina; Xiong, Yan; Fedorov, Oleg; Jin, Jian; Oppermann, Udo; Matulis, Daumantas; Schüle, Roland; Jung, Manfred

2016-01-01

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader. PMID:26893353

Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA

PubMed Central

Smith, Rachel M.; Marshall, Jacqueline J. T.; Jacklin, Alistair J.; Retter, Susan E.; Halford, Stephen E.; Sobott, Frank

2013-01-01

Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A2B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A2B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A2B units, each bound to a recognition site, with two more A2B units bridging the complexes by protein–protein interactions between the nuclease domains. PMID:23147005

Protein O-GlcNAcylation: A critical regulator of the cellular response to stress.

PubMed

Chatham, John C; Marchase, Richard B

2010-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203).

PubMed

Beninson, Lida A; Brown, Peter N; Loughridge, Alice B; Saludes, Jonel P; Maslanik, Thomas; Hills, Abigail K; Woodworth, Tyler; Craig, Wendy; Yin, Hang; Fleshner, Monika

2014-01-01

Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation.

Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

PubMed

Dewhurst, Henry M; Choudhury, Shilpa; Torres, Matthew P

2015-08-01

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)--a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits--conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of recombinant protein misfolding and aggregation on bacterial membranes.

PubMed

Ami, D; Natalello, A; Schultz, T; Gatti-Lafranconi, P; Lotti, M; Doglia, S M; de Marco, A

2009-02-01

The expression of recombinant proteins is known to induce a metabolic rearrangement in the host cell. We used aggregation-sensitive model systems to study the effects elicited in Escherichia coli cells by the aggregation of recombinant glutathione-S-transferase and its fusion with the green fluorescent protein that, according to the expression conditions, accumulate intracellularly as soluble protein, or soluble and insoluble aggregates. We show that the folding state of the recombinant protein and the complexity of the intracellular aggregates critically affect the cell response. Specifically, protein misfolding and aggregation induce changes in specific host proteins involved in lipid metabolism and oxidative stress, a reduction in the membrane permeability, as well as a rearrangement of its lipid composition. The temporal evolution of the host cell response and that of the aggregation process pointed out that the misfolded protein and soluble aggregates are responsible for the membrane modifications and the changes in the host protein levels. Interestingly, native recombinant protein and large insoluble aggregates do not seem to activate stress markers and membrane rearrangements.

Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

NASA Astrophysics Data System (ADS)

Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

2016-05-01

Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry.

PubMed

Mason, Alexander F; Thordarson, Pall

2016-07-20

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation. We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

PubMed

Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G; Lerouge, Patrice; Bardor, Muriel

2015-01-01

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

PubMed Central

Upadhyay, Srijana; Xu, Xinping

2016-01-01

ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

SUMO-Enriched Proteome for Drosophila Innate Immune Response

PubMed Central

Handu, Mithila; Kaduskar, Bhagyashree; Ravindranathan, Ramya; Soory, Amarendranath; Giri, Ritika; Elango, Vijay Barathi; Gowda, Harsha; Ratnaparkhi, Girish S.

2015-01-01

Small ubiquitin-like modifier (SUMO) modification modulates the expression of defense genes in Drosophila, activated by the Toll/nuclear factor-κB and immune-deficient/nuclear factor-κB signaling networks. We have, however, limited understanding of the SUMO-modulated regulation of the immune response and lack information on SUMO targets in the immune system. In this study, we measured the changes to the SUMO proteome in S2 cells in response to a lipopolysaccharide challenge and identified 1619 unique proteins in SUMO-enriched lysates. A confident set of 710 proteins represents the immune-induced SUMO proteome and analysis suggests that specific protein domains, cellular pathways, and protein complexes respond to immune stress. A small subset of the confident set was validated by in-bacto SUMOylation and shown to be bona-fide SUMO targets. These include components of immune signaling pathways such as Caspar, Jra, Kay, cdc42, p38b, 14-3-3ε, as well as cellular proteins with diverse functions, many being components of protein complexes, such as prosß4, Rps10b, SmD3, Tango7, and Aats-arg. Caspar, a human FAF1 ortholog that negatively regulates immune-deficient signaling, is SUMOylated at K551 and responds to treatment with lipopolysaccharide in cultured cells. Our study is one of the first to describe SUMO proteome for the Drosophila immune response. Our data and analysis provide a global framework for the understanding of SUMO modification in the host response to pathogens. PMID:26290570

SUMO-Enriched Proteome for Drosophila Innate Immune Response.

PubMed

Handu, Mithila; Kaduskar, Bhagyashree; Ravindranathan, Ramya; Soory, Amarendranath; Giri, Ritika; Elango, Vijay Barathi; Gowda, Harsha; Ratnaparkhi, Girish S

2015-08-18

Small ubiquitin-like modifier (SUMO) modification modulates the expression of defense genes in Drosophila, activated by the Toll/nuclear factor-κB and immune-deficient/nuclear factor-κB signaling networks. We have, however, limited understanding of the SUMO-modulated regulation of the immune response and lack information on SUMO targets in the immune system. In this study, we measured the changes to the SUMO proteome in S2 cells in response to a lipopolysaccharide challenge and identified 1619 unique proteins in SUMO-enriched lysates. A confident set of 710 proteins represents the immune-induced SUMO proteome and analysis suggests that specific protein domains, cellular pathways, and protein complexes respond to immune stress. A small subset of the confident set was validated by in-bacto SUMOylation and shown to be bona-fide SUMO targets. These include components of immune signaling pathways such as Caspar, Jra, Kay, cdc42, p38b, 14-3-3ε, as well as cellular proteins with diverse functions, many being components of protein complexes, such as prosß4, Rps10b, SmD3, Tango7, and Aats-arg. Caspar, a human FAF1 ortholog that negatively regulates immune-deficient signaling, is SUMOylated at K551 and responds to treatment with lipopolysaccharide in cultured cells. Our study is one of the first to describe SUMO proteome for the Drosophila immune response. Our data and analysis provide a global framework for the understanding of SUMO modification in the host response to pathogens. Copyright © 2015 Handu et al.

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease

PubMed Central

Ying, Peng; Serife, Ayaz-Guner; Deyang, Yu; Ying, Ge

2014-01-01

Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed. PMID:24945106

Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation

PubMed Central

Kovacs, Izabella; Lindermayr, Christian

2013-01-01

Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation. PMID:23717319

Alterations in the Ubiquitin Proteasome System in Persistent but Not Reversible Proteinuric Diseases

PubMed Central

Beeken, Maire; Lindenmeyer, Maja T.; Blattner, Simone M.; Radón, Victoria; Oh, Jun; Meyer, Tobias N.; Hildebrand, Diana; Schlüter, Hartmut; Reinicke, Anna T.; Knop, Jan-Hendrik; Vivekanandan-Giri, Anuradha; Münster, Silvia; Sachs, Marlies; Wiech, Thorsten; Pennathur, Subramaniam; Cohen, Clemens D.; Kretzler, Matthias; Stahl, Rolf A.K.

2014-01-01

Podocytes are the key cells affected in nephrotic glomerular kidney diseases, and they respond uniformly to injury with cytoskeletal rearrangement. In nephrotic diseases, such as membranous nephropathy and FSGS, persistent injury often leads to irreversible structural damage, whereas in minimal change disease, structural alterations are mostly transient. The factors leading to persistent podocyte injury are currently unknown. Proteolysis is an irreversible process and could trigger persistent podocyte injury through degradation of podocyte-specific proteins. We, therefore, analyzed the expression and functional consequence of the two most prominent proteolytic systems, the ubiquitin proteasome system (UPS) and the autophagosomal/lysosomal system, in persistent and transient podocyte injuries. We show that differential upregulation of both proteolytic systems occurs in persistent human and rodent podocyte injury. The expression of specific UPS proteins in podocytes differentiated children with minimal change disease from children with FSGS and correlated with poor clinical outcome. Degradation of the podocyte-specific protein α-actinin-4 by the UPS depended on oxidative modification in membranous nephropathy. Notably, the UPS was overwhelmed in podocytes during experimental glomerular disease, resulting in abnormal protein accumulation and compensatory upregulation of the autophagosomal/lysosomal system. Accordingly, inhibition of both proteolytic systems enhanced proteinuria in persistent nephrotic disease. This study identifies altered proteolysis as a feature of persistent podocyte injury. In the future, specific UPS proteins may serve as new biomarkers or therapeutic targets in persistent nephrotic syndrome. PMID:24722446

Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.

PubMed

Celedon, Jose M; Cline, Kenneth

2013-02-01

Protein trafficking and localization in plastids involve a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called 'conservative sorting'. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

Production of G protein-coupled receptors in an insect-based cell-free system.

PubMed

Sonnabend, Andrei; Spahn, Viola; Stech, Marlitt; Zemella, Anne; Stein, Christoph; Kubick, Stefan

2017-10-01

The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita

As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statisticalmore » inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.« less

Progress in Genome Editing Technology and Its Application in Plants

PubMed Central

Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

2017-01-01

Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. PMID:28261237

Epigenetic modifications: An important mechanism in diabetic disturbances.

PubMed

Rorbach-Dolata, Anna; Kubis, Adriana; Piwowar, Agnieszka

2017-11-29

In the search for explanations of diabetes pathomechanisms, especially the development of its vascular complications (micro- and macrovascular ), although current, good metabolic control of diabetes, attention was drawn to the role of epigenetic inheritance associated with epigenetic modifications of histone proteins and DNA in hyperglycemia conditions. This study showed the significant role of DNA methylation and histone epigenetic modifications (a different nature and a different degree) in the transmission of information that is not connected with gene inheritance but concerns the persistent changes induced by hyperglycemia..Attention was paid to the role of DNA methylation of pancreatic cells in the pathogenesis of type 1 diabetes, but also type 2. The important role of DNA methylation changes in a so-called intrauterine growth restriction (IUGR) as reason of subsequent development of diabetes was particularly emphasized. In the pathogenesis of type 2 diabetes and its complications, especially microvascular complications, the greatest share and importance of epigenetic modifications on mitochondrial DNA metylation are the most important. The multidirectionality Complicaand complexity of epigenetic modifications of histone proteins indicate their importance in the development of diabetic disturbances. An especially important role is attributed to methylation and acetylation of histone proteins, in particular on arginine and lysine, whose changes occur most frequently. Moreover, epigenetic modifications of the enzymes, especially methylases, responsible for these processes are the underlying. It has been indicated that the identification of epigenetic differences within the DNA or histone proteins may be a useful prognostic biomarker of susceptibility to the disease development in the future. Moreover, they may become a potential target for future therapeutic interventions for clinical disorders in diabetes.

Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.

PubMed

Stroylova, Yulia Y; Semenyuk, Pavel I; Asriyantz, Regina A; Gaillard, Cedric; Haertlé, Thomas; Muronetz, Vladimir I

2014-09-01

The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes. © 2014 Wiley Periodicals, Inc.

Strigolactone-nitric oxide interplay in plants: the story has just begun.

PubMed

Kolbert, Zsuzsanna

2018-02-26

Both strigolactones (SLs) and nitric oxide (NO) are regulatory signals with diverse roles during plant development and stress responses. This review aims to discuss the so far available data regarding SLs-NO interplay in plant systems. The majority of the few articles dealing with SL-NO interplay focuses on the root system and it seems that NO can be an upstream negative regulator of SL biosynthesis or an upstream positive regulator of SL signaling depending on the nutrient supply. From the so far published results it is clear that NO modifies the activity of target proteins involved in SL biosynthesis or signaling which may be a physiologically relevant interaction. Therefore, in silico analysis of NO-dependent posttranslational modifications in SL-related proteins was performed using computational prediction tools and putative NO-target proteins were specified. The picture is presumably more complicated, since also SL is able to modify NO levels. As a confirmation, author detected NO levels in different organs of max1-1 and max2-1 Arabidopsis and compared to the wild-type these mutants showed enhanced NO levels in their root tips indicating the negative effect of endogenous SLs on NO metabolism. Exogenous SL analogue-triggered NO production seems to contradict the results of the genetic study, which is an inconsistency should be taken into consideration in the future. In the coming years, the link between SL and NO signaling in further physiological processes should be examined and the possibilities of NO-dependent posttranslational modifications of SL biosynthetic and signaling proteins should be looked more closely. This article is protected by copyright. All rights reserved.

Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells

PubMed Central

King, Sarah L.; Joshi, Hiren J.; Schjoldager, Katrine T.; Halim, Adnan; Madsen, Thomas D.; Dziegiel, Morten H.; Woetmann, Anders; Vakhrushev, Sergey Y.

2017-01-01

The hemostatic system comprises platelet aggregation, coagulation, and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play important roles in the hemostatic system; however, most investigations have focused on N-glycans because of the complexity of O-glycan analysis. Here we performed the first systematic analysis of native-O-glycosylation using lectin affinity chromatography coupled to liquid chromatography mass spectrometry (LC-MS)/MS to determine the precise location of O-glycans in human plasma, platelets, and endothelial cells, which coordinately regulate hemostasis. We identified the hitherto largest O-glycoproteome from native tissue with a total of 649 glycoproteins and 1123 nonambiguous O-glycosites, demonstrating that O-glycosylation is a ubiquitous modification of extracellular proteins. Investigation of the general properties of O-glycosylation established that it is a heterogeneous modification, frequently occurring at low density within disordered regions in a cell-dependent manner. Using an unbiased screen to identify associations between O-glycosites and protein annotations we found that O-glycans were over-represented close (± 15 amino acids) to tandem repeat regions, protease cleavage sites, within propeptides, and located on a select group of protein domains. The importance of O-glycosites in proximity to proteolytic cleavage sites was further supported by in vitro peptide assays demonstrating that proteolysis of key hemostatic proteins can be inhibited by the presence of O-glycans. Collectively, these data illustrate the global properties of native O-glycosylation and provide the requisite roadmap for future biomarker and structure-function studies. PMID:29296958

Methods for evaluating the Caenorhabditis elegans dauer state: standard dauer-formation assay using synthetic daumones and proteomic analysis of O-GlcNAc modifications.

PubMed

Lee, Jeeyong; Kim, Kwang-Youl; Joo, Hyoe-Jin; Kim, Heekyeong; Jeong, Pan-Young; Paik, Young-Ki

2011-01-01

The dauer state is a non-feeding, alternative L3 state characterized by a number of distinctive metabolic and morphological changes. There are many naturally occurring dauer-inducing pheromones, termed daumones, that have been suggested by some to exhibit differences in dauer-inducing activity. Here, we have established a standard dauer-formation assay that uses synthetic daumones 1, 2, and 3, the three major daumones. To analyze the proteome of Caenorhabditis elegans in the dauer state, we focused on O-GlcNAc modification, a cytosolic modification of proteins that is known to interact either competitively or synergistically with protein phosphorylation. Protein O-GlcNAc modification is an important biological process in cells that can ensure the timely response to extracellular stimuli, such as daumone, and maintain cellular homeostasis. Establishing a standard method for assaying dauer formation using different synthetic daumones, and using differences in O-GlcNAcylated proteins during the dauer state to analyze the dauer proteome will lead to a better understanding of dauer biology of C. elegans in the context of animal longevity and adaptation under harsh environments. Copyright © 2011 Elsevier Inc. All rights reserved.

The story of protein arginine methylation: characterization, regulation, and function.

PubMed

Peng, Chao; Wong, Catherine Cl

2017-02-01

Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery. Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases. Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kou, Qiang; Zhu, Binhai; Wu, Si

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 comparedmore » 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.« less

Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis.

PubMed

Prause, J; Goswami, A; Katona, I; Roos, A; Schnizler, M; Bushuven, E; Dreier, A; Buchkremer, S; Johann, S; Beyer, C; Deschauer, M; Troost, D; Weis, J

2013-04-15

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patient's fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

Using an Engineered Protein Model to Constrain Protein-Mineral Interactions

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Washton, N.; Kleber, M.

2015-12-01

Exoenzymes are proteins that can catalyze the depolymerization of soil organic matter (SOM). Proteins can also be an important source of organic N for microorganisms, but must be fragmented into small peptides in order to be transported through their membranes. An exoenzyme's affinity to mineral surfaces found in soil affects their capacity to degrade SOM or other proteins. Our goal was to determine the range of modifications on proteins when they interact with a mineral surface. We hypothesized that pedogenic oxides would fragment or promote greater chemical modifications to a protein than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We then generated three engineered proxies of Gb1 by inserting either negatively charged, positively charged or aromatic amino acids into the second loop. We used liquid chromatography coupled with a mass spectrometer (LC-MS/MS) and solution-state Heteronuclear Single Quantum Coherence Spectroscopy Nuclear Magnetic Resonance (HSQC NMR) to observe modifications to Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite, birnessite, and montmorillonite. We also used Helium Ion Microscopy (HIM) to determine which surface archetypes Gb1 preferentially adsorbed to as a function of the mineral type. The three engineered proxies were used to determine how variation of the amino acid sequence affects a protein interaction with a mineral surface. Preliminary results in the LC-MS/MS indicate that birnessite hydrolytically fragments Gb1 into polypeptides. Our results suggest that not all mineral surfaces in soil may act as sorbents for EEs and that chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Our results also indicate an abiotic pathway for the turnover of proteins, although its relative contribution to protein turnover in soil must be further investigated.

Dynamic phosphorylation of RelA on Ser42 and Ser45 in response to TNFα stimulation regulates DNA binding and transcription.

PubMed

Lanucara, Francesco; Lam, Connie; Mann, Jelena; Monie, Tom P; Colombo, Stefano A P; Holman, Stephen W; Boyd, James; Dange, Manohar C; Mann, Derek A; White, Michael R H; Eyers, Claire E

2016-07-01

The NF-κB signalling module controls transcription through a network of protein kinases such as the IKKs, as well as inhibitory proteins (IκBs) and transcription factors including RelA/p65. Phosphorylation of the NF-κB subunits is critical for dictating system dynamics. Using both non-targeted discovery and quantitative selected reaction monitoring-targeted proteomics, we show that the cytokine TNFα induces dynamic multisite phosphorylation of RelA at a number of previously unidentified residues. Putative roles for many of these phosphorylation sites on RelA were predicted by modelling of various crystal structures. Stoichiometry of phosphorylation determination of Ser45 and Ser42 revealed preferential early phosphorylation of Ser45 in response to TNFα. Quantitative analyses subsequently confirmed differential roles for pSer42 and pSer45 in promoter-specific DNA binding and a role for both of these phosphosites in regulating transcription from the IL-6 promoter. These temporal dynamics suggest that RelA-mediated transcription is likely to be controlled by functionally distinct NF-κB proteoforms carrying different combinations of modifications, rather than a simple 'one modification, one effect' system. © 2016 The Authors.

Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

PubMed

Wanigasekara, Maheshika S K; Chowdhury, Saiful M

2016-09-07

Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. Copyright © 2016 Elsevier B.V. All rights reserved.

Tripping Up Trp: Modification of Protein Tryptophan Residues by Reactive Oxygen Species, Modes of Detection, and Biological Consequences

PubMed Central

Ehrenshaft, Marilyn; Deterding, Leesa J.; Mason, Ronald P.

2015-01-01

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes. PMID:26393422

Redox Proteomics: A Key Tool for New Insights into Protein Modification with Relevance to Disease.

PubMed

Butterfield, D Allan; Perluigi, Marzia

2017-03-01

Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the "triangle of death" composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277-279.

Mechanisms, biology and inhibitors of deubiquitinating enzymes.

PubMed

Love, Kerry Routenberg; Catic, André; Schlieker, Christian; Ploegh, Hidde L

2007-11-01

The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in eukaryotes, prokaryotes and viruses shows that these proteases constitute an essential class of enzymes. Here, we discuss how chemical tools, including activity-based probes and suicide inhibitors, have enabled (i) discovery of deubiquitinating enzymes, (ii) their functional profiling, crystallographic characterization and mechanistic classification and (iii) development of molecules for therapeutic purposes.

Crystal structure of the Tum1 protein from the yeast Saccharomyces cerevisiae.

PubMed

Qiu, Rui; Wang, Fengbin; Liu, Meiruo; Lou, Tiantian; Ji, Chaoneng

2012-11-01

Yeast tRNA-thiouridine modification protein 1 (Tum1) plays essential role in the sulfur transfer process of Urm1 system, which in turn is involved in many important cellular processes. In the rhodanese-like domain (RLD), conserved cysteine residue is proved to be the centre of active site of sulfurtransferases and crucial for the substrate recognition. In this report, we describe the crystal structure of Tum1 protein at 1.90 A resolution which, despite consisting of two RLDs, has only one conserved cysteine residue in the C-terminal RLD. An unaccounted electron density is found near the active site, which might point to the new cofactor in the sulfur transfer mechanism.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

PubMed Central

Naz, Anam; Obaid, Ayesha; Awan, Faryal M.; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells. PMID:28932213

Solid state, thermal synthesis of site-specific protein-boron cluster conjugates and their physicochemical and biochemical properties.

PubMed

Goszczyński, Tomasz M; Kowalski, Konrad; Leśnikowski, Zbigniew J; Boratyński, Janusz

2015-02-01

Boron clusters represent a vast family of boron-rich compounds with extraordinary properties that provide the opportunity of exploitation in different areas of chemistry and biology. In addition, boron clusters are clinically used in boron neutron capture therapy (BNCT) of tumors. In this paper, a novel, in solid state (solvent free), thermal method for protein modification with boron clusters has been proposed. The method is based on a cyclic ether ring opening in oxonium adduct of cyclic ether and a boron cluster with nucleophilic centers of the protein. Lysozyme was used as the model protein, and the physicochemical and biological properties of the obtained conjugates were characterized. The main residues of modification were identified as arginine-128 and threonine-51. No significant changes in the secondary or tertiary structures of the protein after tethering of the boron cluster were found using mass spectrometry and circular dichroism measurements. However, some changes in the intermolecular interactions and hydrodynamic and catalytic properties were observed. To the best of our knowledge, we have described the first example of an application of cyclic ether ring opening in the oxonium adducts of a boron cluster for protein modification. In addition, a distinctive feature of the proposed approach is performing the reaction in solid state and at elevated temperature. The proposed methodology provides a new route to protein modification with boron clusters and extends the range of innovative molecules available for biological and medical testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

PubMed

Naz, Anam; Obaid, Ayesha; Awan, Faryal M; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

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

PubMed

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

2009-01-01

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

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA.

PubMed

Kinsella, B T; Erdman, R A; Maltese, W A

1991-05-25

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

PubMed

Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

2017-03-02

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Controlled Cross-Linking with Glucose Oxidase for the Enhancement of Gelling Potential of Pork Myofibrillar Protein.

PubMed

Wang, Xu; Xiong, Youling L; Sato, Hiroaki; Kumazawa, Yoshiyuki

2016-12-21

Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO 4 versus a Fenton system (H 2 O 2 /FeSO 4 ) were investigated. Pork MP was modified at 4 °C and pH 6.25 with hydroxyl radicals produced from 1 mg/mL glucose in the presence of 80, 160, or 320 μg/mL GluOx and 10 μM FeSO 4 . Total sulfhydryl content, solubility, cross-linking pattern, and gelation properties of MP were measured. H 2 O 2 production proceeded linearly with the concentration of GluOx and increased with reaction time. GluOx- and H 2 O 2 -dose-dependent protein polymerization, evidenced by faded myosin heavy chain and actin in SDS-PAGE as well as significant decreases in sulfhydryls, coincided with protein solubility loss. Firmer and more elastic MP gels were produced by GluOx than by the Fenton system at comparable H 2 O 2 levels due to an altered radical reaction pathway.

In silico prediction of post-translational modifications.

PubMed

Liu, Chunmei; Li, Hui

2011-01-01

Methods for predicting protein post-translational modifications have been developed extensively. In this chapter, we review major post-translational modification prediction strategies, with a particular focus on statistical and machine learning approaches. We present the workflow of the methods and summarize the advantages and disadvantages of the methods.

Protein Arginine Methylation and Citrullination in Epigenetic Regulation

PubMed Central

2015-01-01

The post-translational modification of arginine residues represents a key mechanism for the epigenetic control of gene expression. Aberrant levels of histone arginine modifications have been linked to the development of several diseases including cancer. In recent years, great progress has been made in understanding the physiological role of individual arginine modifications and their effects on chromatin function. The present review aims to summarize the structural and functional aspects of histone arginine modifying enzymes and their impact on gene transcription. We will discuss the potential for targeting these proteins with small molecules in a variety of disease states. PMID:26686581

Integration of photoswitchable proteins, photosynthetic reaction centers and semiconductor/biomolecule hybrids with electrode supports for optobioelectronic applications.

PubMed

Wang, Fuan; Liu, Xiaoqing; Willner, Itamar

2013-01-18

Light-triggered biological processes provide the principles for the development of man-made optobioelectronic systems. This Review addresses three recently developed topics in the area of optobioelectronics, while addressing the potential applications of these systems. The topics discussed include: (i) the reversible photoswitching of the bioelectrocatalytic functions of redox proteins by the modification of proteins with photoisomerizable units or by the integration of proteins with photoisomerizable environments; (ii) the integration of natural photosynthetic reaction centers with electrodes and the construction of photobioelectrochemical cells and photobiofuel cells; and (iii) the synthesis of biomolecule/semiconductor quantum dots hybrid systems and their immobilization on electrodes to yield photobioelectrochemical and photobiofuel cell elements. The fundamental challenge in the tailoring of optobioelectronic systems is the development of means to electrically contact photoactive biomolecular assemblies with the electrode supports. Different methods to establish electrical communication between the photoactive biomolecular assemblies and electrodes are discussed. These include the nanoscale engineering of the biomolecular nanostructures on surfaces, the development of photoactive molecular wires and the coupling of photoinduced electron transfer reactions with the redox functions of proteins. The different possible applications of optobioelectronic systems are discussed, including their use as photosensors, the design of biosensors, and the construction of solar energy conversion and storage systems. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent trends in drug delivery system using protein nanoparticles.

PubMed

Sripriyalakshmi, S; Jose, Pinkybel; Ravindran, Aswathy; Anjali, C H

2014-09-01

Engineered nanoparticles that can facilitate drug formulation and passively target tumours have been under extensive research in recent years. These successes have driven a new wave of significant innovation in the generation of advanced particles. The fate and transport of diagnostic nanoparticles would significantly depend on nonselective drug delivery, and hence the use of high drug dosage is implemented. In this perspective, nanocarrier-based drug targeting strategies can be used which improve the selective delivery of drugs to the site of action, i.e. drug targeting. Pharmaceutical industries majorly focus on reducing the toxicity and side effects of drugs but only recently it has been realised that carrier systems themselves may pose risks to the patient. Proteins are compatible with biological systems and they are biodegradable. They offer a multitude of moieties for modifications to tailor drug binding, imaging or targeting entities. Thus, protein nanoparticles provide outstanding contributions as a carrier for drug delivery systems. This review summarises recent progress in particle-based therapeutic delivery and discusses important concepts in particle design and biological barriers for developing the next generation of particles drug delivery systems.

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

PubMed Central

Serfiotis-Mitsa, Dimitra; Herbert, Andrew P.; Roberts, Gareth A.; Soares, Dinesh C.; White, John H.; Blakely, Garry W.; Uhrín, Dušan; Dryden, David T. F.

2010-01-01

Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic. PMID:20007596

Modulation of BK channels by ethanol

PubMed Central

Dopico, Alex M.; Bukiya, Anna N.; Kuntamallappanavar, Guruprasad; Liu, Jianxi

2017-01-01

In alcohol-naïve systems, ethanol (<100 mM) exposure of calcium-gated BK channels perturbs physiology and behavior. Brief (several minutes) ethanol exposure usually leads to increased BK current, which results from ethanol interaction with a pocket mapped to the BK channel-forming slo1 protein cytosolic tail domain. The importance of this region in alcohol-induced intoxication has been addressed in Caenorhabditis elegans slo1 mutants. However, ethanol-induced BK activation is not universal as refractoriness and inhibition have been reported. The final effect depends on many factors, including intracellular calcium levels, slo1 isoform, BK beta subunit composition, post-translational modification of BK proteins, channel lipid microenvironment and type of ethanol administration. Studies in Drosophila melanogaster, Caenorhabditis elegans and rodents show that protracted/repeated ethanol administration leads to tolerance to alcohol-induced modification of BK-driven physiology and behavior. Unveiling the mechanisms underlying tolerance is of major importance, as tolerance to alcohol has been proposed as predictor of risk for alcoholism. PMID:27238266

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

NASA Astrophysics Data System (ADS)

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-11-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria--which models tuberculous granulomas--are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence

PubMed Central

Chionh, Yok Hian; McBee, Megan; Babu, I. Ramesh; Hia, Fabian; Lin, Wenwei; Zhao, Wei; Cao, Jianshu; Dziergowska, Agnieszka; Malkiewicz, Andrzej; Begley, Thomas J.; Alonso, Sylvie; Dedon, Peter C.

2016-01-01

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria. PMID:27834374

Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

NASA Astrophysics Data System (ADS)

Seaton, D. D.; Krishnan, J.

2012-08-01

Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.