multiple post-translational modifications: Topics by Science.gov

Sample records for multiple post-translational modifications

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

PubMed

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

2014-01-01

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

PubMed Central

Michelet, Laure; Zaffagnini, Mirko; Morisse, Samuel; Sparla, Francesca; Pérez-Pérez, María Esther; Francia, Francesco; Danon, Antoine; Marchand, Christophe H.; Fermani, Simona; Trost, Paolo; Lemaire, Stéphane D.

2013-01-01

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin–Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin–Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin–Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses. PMID:24324475
Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

DOE PAGES

Wu, Meiye; Singh, Anup K.

2014-07-15

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

PubMed

Matsubayashi, Yoshikatsu

2011-01-01

More than a dozen secreted peptides are now recognized as important hormones that coordinate and specify cellular functions in plants. Recent evidence has shown that secreted peptide hormones often undergo post-translational modification and proteolytic processing, which are critical for their function. Such 'small post-translationally modified peptide hormones' constitute one of the largest groups of peptide hormones in plants. This short review highlights recent progress in research on post-translationally modified peptide hormones, with particular emphasis on their structural characteristics and modification mechanisms.
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
Analysis of tau post-translational modifications in rTg4510 mice, a model of tau pathology.

PubMed

Song, Lixin; Lu, Sherry X; Ouyang, Xuesong; Melchor, Jerry; Lee, Julie; Terracina, Giuseppe; Wang, Xiaohai; Hyde, Lynn; Hess, J Fred; Parker, Eric M; Zhang, Lili

2015-03-26

Microtubule associated protein tau is the major component of the neurofibrillary tangles (NFTs) found in the brains of patients with Alzheimer's disease and several other neurodegenerative diseases. Tau mutations are associated with frontotemperal dementia with parkinsonism on chromosome 17 (FTDP-17). rTg4510 mice overexpress human tau carrying the P301L FTDP-17 mutation and develop robust NFT-like pathology at 4-5 months of age. The current study is aimed at characterizing the rTg4510 mice to better understand the genesis of tau pathology and to better enable the use of this model in drug discovery efforts targeting tau pathology. Using a panel of immunoassays, we analyzed the age-dependent formation of pathological tau in rTg4510 mice and our data revealed a steady age-dependent accumulation of pathological tau in the insoluble fraction of brain homogenates. The pathological tau was associated with multiple post-translational modifications including aggregation, phosphorylation at a wide variety of sites, acetylation, ubiquitination and nitration. The change of most tau species reached statistical significance at the age of 16 weeks. There was a strong correlation between the different post-translationally modified tau species in this heterogeneous pool of pathological tau. Total tau in the cerebrospinal fluid (CSF) displayed a multiphasic temporal profile distinct from the steady accumulation of pathological tau in the brain. Female rTg4510 mice displayed significantly more aggressive accumulation of pathological tau in the brain and elevation of total tau in CSF than their male littermates. The immunoassays described here were used to generate the most comprehensive description of the changes in various tau species across the lifespan of the rTg4510 mouse model. The data indicate that development of tauopathy in rTg4510 mice involves the accumulation of a pool of pathological tau that carries multiple post-translational modifications, a process that can be detected well before the histological detection of NFTs. Therapeutic treatment targeting tau should therefore aim to reduce all tau species associated with the pathological tau pool rather than reduce specific post-translational modifications. There is still much to learn about CSF tau in physiological and pathological processes in order to use it as a translational biomarker in drug discovery.
Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors

PubMed Central

Goo, Marisa S.; Scudder, Samantha L.; Patrick, Gentry N.

2015-01-01

Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses. PMID:26528125
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/
POSTMan (POST-translational modification analysis), a software application for PTM discovery.

PubMed

Arntzen, Magnus Ø; Osland, Christoffer Leif; Raa, Christopher Rasch-Olsen; Kopperud, Reidun; Døskeland, Stein-Ove; Lewis, Aurélia E; D'Santos, Clive S

2009-03-01

Post-translationally modified peptides present in low concentrations are often not selected for CID, resulting in no sequence information for these peptides. We have developed a software POSTMan (POST-translational Modification analysis) allowing post-translationally modified peptides to be targeted for fragmentation. The software aligns LC-MS runs (MS(1) data) between individual runs or within a single run and isolates pairs of peptides which differ by a user defined mass difference (post-translationally modified peptides). The method was validated for acetylated peptides and allowed an assessment of even the basal protein phosphorylation of phenylalanine hydroxylase (PHA) in intact cells.
Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis

PubMed Central

Emery, Samantha J; Baker, Louise; Ansell, Brendan R E; Mirzaei, Mehdi; Haynes, Paul A; McConville, Malcom J; Svärd, Staffan G; Jex, Aaron R

2018-01-01

Abstract Background Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein. Findings We performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity. Conclusions Our data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks. PMID:29688452
Differential protein expression and post-translational modifications in metronidazole-resistant Giardia duodenalis.

PubMed

Emery, Samantha J; Baker, Louise; Ansell, Brendan R E; Mirzaei, Mehdi; Haynes, Paul A; McConville, Malcom J; Svärd, Staffan G; Jex, Aaron R

2018-04-01

Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein. We performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity. Our data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Meiye; Singh, Anup K.

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

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

PubMed

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

2006-07-01

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

PubMed

Santos, Celio X C; Raza, Sadaf; Shah, Ajay M

2016-05-01

The specific effect of oxygen and reactive oxygen species (ROS) in mediating post-translational modification of protein targets has emerged as a key mechanism regulating signaling components, a process termed redox signaling. ROS act in the post-translational modification of multiple target proteins including receptors, kinases, phosphatases, ion channels and transcription factors. Both O2 and ROS are major source of electrons in redox reactions in aerobic organisms. Because the heart has the highest O2 consumption among body organs, it is not surprising that redox signaling is central to heart function and pathophysiology. In this article, we review some of the main cardiac redox signaling pathways and their roles in the cardiomyocyte and in heart failure, with particular focus on the specific molecular targets of ROS in the heart. Copyright © 2016 Elsevier Ltd. All rights reserved.
Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes

PubMed Central

MATSUBAYASHI, Yoshikatsu

2018-01-01

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone–receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation. PMID:29434080
Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes.

PubMed

Matsubayashi, Yoshikatsu

2018-01-01

The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone-receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation.
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
Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

PubMed Central

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

2015-01-01

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

PubMed Central

Shen, Siming; Casaccia-Bonnefil, Patrizia

2008-01-01

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

PubMed Central

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

2013-01-01

Gap junctions are comprised of connexins that form cell-to-cell channels which couple neighboring cells to accommodate the exchange of information. The need for communication does, however, change over time and therefore must be tightly controlled. Although the regulation of connexin protein expression by transcription and translation is of great importance, the trafficking, channel activity and degradation are also under tight control. The function of connexins can be regulated by several post translational modifications, which affect numerous parameters; including number of channels, open probability, single channel conductance or selectivity. The most extensively investigated post translational modifications are phosphorylations, which have been documented in all mammalian connexins. Besides phosphorylations, some connexins are known to be ubiquitinated, SUMOylated, nitrosylated, hydroxylated, acetylated, methylated, and γ-carboxyglutamated. The aim of the present review is to summarize our current knowledge of post translational regulation of the connexin family of proteins. PMID:24155720
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
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.
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/.
Post-translational modifications of linker histone H1 variants in mammals

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

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

Wu, Meiye; Singh, Anup K.

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.
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
KinView: A visual comparative sequence analysis tool for integrated kinome research

PubMed Central

McSkimming, Daniel Ian; Dastgheib, Shima; Baffi, Timothy R.; Byrne, Dominic P.; Ferries, Samantha; Scott, Steven Thomas; Newton, Alexandra C.; Eyers, Claire E.; Kochut, Krzysztof J.; Eyers, Patrick A.

2017-01-01

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats. PMID:27731453
Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry.

PubMed

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

2017-02-03

As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (PTMs) along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here we used state-of-the-art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as a search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

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

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473
RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

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

PubMed

Li, Ginny X H; Vogel, Christine; Choi, Hyungwon

2018-06-07

While tandem mass spectrometry can detect post-translational modifications (PTM) at the proteome scale, reported PTM sites are often incomplete and include false positives. Computational approaches can complement these datasets by additional predictions, but most available tools use prediction models pre-trained for single PTM type by the developers and it remains a difficult task to perform large-scale batch prediction for multiple PTMs with flexible user control, including the choice of training data. We developed an R package called PTMscape which predicts PTM sites across the proteome based on a unified and comprehensive set of descriptors of the physico-chemical microenvironment of modified sites, with additional downstream analysis modules to test enrichment of individual or pairs of PTMs in protein domains. PTMscape is flexible in the ability to process any major modifications, such as phosphorylation and ubiquitination, while achieving the sensitivity and specificity comparable to single-PTM methods and outperforming other multi-PTM tools. Applying this framework, we expanded proteome-wide coverage of five major PTMs affecting different residues by prediction, especially for lysine and arginine modifications. Using a combination of experimentally acquired sites (PSP) and newly predicted sites, we discovered that the crosstalk among multiple PTMs occur more frequently than by random chance in key protein domains such as histone, protein kinase, and RNA recognition motifs, spanning various biological processes such as RNA processing, DNA damage response, signal transduction, and regulation of cell cycle. These results provide a proteome-scale analysis of crosstalk among major PTMs and can be easily extended to other types of PTM.
S-Nitrosylation: Specificity, Occupancy, and Interaction with Other Post-Translational Modifications

PubMed Central

Kohr, Mark J.; Murphy, Elizabeth

2013-01-01

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

PubMed

Frye, Michaela; Blanco, Sandra

2016-11-01

Cells adapt to their environment by linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. Among these, mechanisms that couple environmental cues to the regulation of protein translation are not well understood. Chemical modifications of RNA allow rapid cellular responses to external stimuli by modulating a wide range of fundamental biochemical properties and processes, including the stability, splicing and translation of messenger RNA. In this Review, we focus on the occurrence of N 6 -methyladenosine (m 6 A), 5-methylcytosine (m 5 C) and pseudouridine (Ψ) in RNA, and describe how these RNA modifications are implicated in regulating pluripotency, stem cell self-renewal and fate specification. Both post-transcriptional modifications and the enzymes that catalyse them modulate stem cell differentiation pathways and are essential for normal development. © 2016. Published by The Company of Biologists Ltd.
Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications.

PubMed

Bunik, Victoria; Artiukhov, Artem; Aleshin, Vasily; Mkrtchyan, Garik

2016-12-14

Glutamate dehydrogenase (GDH) of animal cells is usually considered to be a mitochondrial enzyme. However, this enzyme has recently been reported to be also present in nucleus, endoplasmic reticulum and lysosomes. These extramitochondrial localizations are associated with moonlighting functions of GDH, which include acting as a serine protease or an ATP-dependent tubulin-binding protein. Here, we review the published data on kinetics and localization of multiple forms of animal GDH taking into account the splice variants, post-translational modifications and GDH isoenzymes, found in humans and apes. The kinetic properties of human GLUD1 and GLUD2 isoenzymes are shown to be similar to those published for GDH1 and GDH2 from bovine brain. Increased functional diversity and specific regulation of GDH isoforms due to alternative splicing and post-translational modifications are also considered. In particular, these structural differences may affect the well-known regulation of GDH by nucleotides which is related to recent identification of thiamine derivatives as novel GDH modulators. The thiamine-dependent regulation of GDH is in good agreement with the fact that the non-coenzyme forms of thiamine, i.e., thiamine triphosphate and its adenylated form are generated in response to amino acid and carbon starvation.
Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

PubMed

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

2018-05-23

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

PubMed Central

2018-01-01

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

PubMed

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

1996-01-01

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

PubMed Central

2017-01-01

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications. PMID:28135077
RNA-stabilization factors in chloroplasts of vascular plants.

PubMed

Manavski, Nikolay; Schmid, Lisa-Marie; Meurer, Jörg

2018-04-13

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants. © 2018 The Author(s).
Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

PubMed Central

Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

2015-01-01

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865
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.
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.
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
SIZ1-Dependent Post-Translational Modification by SUMO Modulates Sugar Signaling and Metabolism in Arabidopsis thaliana.

PubMed

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

2015-12-01

Post-translational modification mechanisms function as switches that mediate the balance between optimum growth and the response to environmental stimuli, by regulating the activity of key proteins. SUMO (small ubiquitin-like modifier) attachment, or sumoylation, is a post-translational modification that is essential for the plant stress response, also modulating hormonal circuits to co-ordinate developmental processes. The Arabidopsis SUMO E3 ligase SAP and Miz 1 (SIZ1) is the major SUMO conjugation enhancer in response to stress, and is implicated in several aspects of plant development. Here we report that known SUMO targets are over-represented in multiple carbohydrate-related proteins, suggesting a functional link between sumoylation and sugar metabolism and signaling in plants. We subsequently observed that SUMO-conjugated proteins accumulate in response to high doses of sugar in a SIZ1-dependent manner, and that the null siz1 mutant displays increased expression of sucrose and starch catabolic genes and shows reduced starch levels. We demonstrated that SIZ1 controls germination time and post-germination growth via osmotic and sugar-dependent signaling, respectively. Glucose was specifically linked to SUMO-sugar interplay, with high levels inducing root growth inhibition and aberrant root hair morphology in siz1. The use of sugar analogs and sugar marker gene expression analysis allowed us to implicate SIZ1 in a signaling pathway dependent on glucose metabolism, probably involving modulation of SNF1-related kinase 1 (SnRK1) activity. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
An improved ChIP-seq peak detection system for simultaneously identifying post-translational modified transcription factors by combinatorial fusion, using SUMOylation as an example.

PubMed

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

2014-01-01

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

PubMed

Martín-Campos, Trinidad; Mylonas, Roman; Masselot, Alexandre; Waridel, Patrice; Petricevic, Tanja; Xenarios, Ioannis; Quadroni, Manfredo

2017-08-04

Mass spectrometry (MS) has become the tool of choice for the large scale identification and quantitation of proteins and their post-translational modifications (PTMs). This development has been enabled by powerful software packages for the automated analysis of MS data. While data on PTMs of thousands of proteins can nowadays be readily obtained, fully deciphering the complexity and combinatorics of modification patterns even on a single protein often remains challenging. Moreover, functional investigation of PTMs on a protein of interest requires validation of the localization and the accurate quantitation of its changes across several conditions, tasks that often still require human evaluation. Software tools for large scale analyses are highly efficient but are rarely conceived for interactive, in-depth exploration of data on individual proteins. We here describe MsViz, a web-based and interactive software tool that supports manual validation of PTMs and their relative quantitation in small- and medium-size experiments. The tool displays sequence coverage information, peptide-spectrum matches, tandem MS spectra and extracted ion chromatograms through a single, highly intuitive interface. We found that MsViz greatly facilitates manual data inspection to validate PTM location and quantitate modified species across multiple samples.
Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs).

PubMed

Benjdia, Alhosna; Balty, Clémence; Berteau, Olivier

2017-01-01

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

PTMScout, a Web Resource for Analysis of High Throughput Post-translational Proteomics Studies*

PubMed Central

Naegle, Kristen M.; Gymrek, Melissa; Joughin, Brian A.; Wagner, Joel P.; Welsch, Roy E.; Yaffe, Michael B.; Lauffenburger, Douglas A.; White, Forest M.

2010-01-01

The rate of discovery of post-translational modification (PTM) sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. To help meet this challenge, we have created PTMScout, a web-based interface for viewing, manipulating, and analyzing high throughput experimental measurements of PTMs in an effort to facilitate biological understanding of protein modifications in signaling networks. PTMScout is constructed around a custom database of PTM experiments and contains information from external protein and post-translational resources, including gene ontology annotations, Pfam domains, and Scansite predictions of kinase and phosphopeptide binding domain interactions. PTMScout functionality comprises data set comparison tools, data set summary views, and tools for protein assignments of peptides identified by mass spectrometry. Analysis tools in PTMScout focus on informed subset selection via common criteria and on automated hypothesis generation through subset labeling derived from identification of statistically significant enrichment of other annotations in the experiment. Subset selection can be applied through the PTMScout flexible query interface available for quantitative data measurements and data annotations as well as an interface for importing data set groupings by external means, such as unsupervised learning. We exemplify the various functions of PTMScout in application to data sets that contain relative quantitative measurements as well as data sets lacking quantitative measurements, producing a set of interesting biological hypotheses. PTMScout is designed to be a widely accessible tool, enabling generation of multiple types of biological hypotheses from high throughput PTM experiments and advancing functional assignment of novel PTM sites. PTMScout is available at http://ptmscout.mit.edu. PMID:20631208
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.
UC/MALDI-MS analysis of HDL; evidence for density-dependent post-translational modifications

NASA Astrophysics Data System (ADS)

Johnson, Jeffery D.; Henriquez, Ronald R.; Tichy, Shane E.; Russell, David H.; McNeal, Catherine J.; Macfarlane, Ronald D.

2007-12-01

The purpose of this study is to determine whether the nature of the post-translational modifications of the major apolipoproteins of HDL is different for density-distinct subclasses. These subclasses were separated by ultracentrifugation using a novel density-forming solute to yield a high-resolution separation. The serum of two subjects, a control with a normolipidemic profile and a subject with diagnosed cardiovascular disease, was studied. Aliquots of three HDL subclasses were analyzed by MALDI and considerable differences were seen when comparing density-distinct subclasses and also when comparing the two subjects. A detailed analysis of the post-translational modification pattern of apoA-1 shows evidence of considerable protease activity, particularly in the more dense fractions. We conclude that part of the heterogeneity of the population of HDL particles is due to density-dependent protease activity.
Multiple Forms of Glutamate Dehydrogenase in Animals: Structural Determinants and Physiological Implications

PubMed Central

Bunik, Victoria; Artiukhov, Artem; Aleshin, Vasily; Mkrtchyan, Garik

2016-01-01

Glutamate dehydrogenase (GDH) of animal cells is usually considered to be a mitochondrial enzyme. However, this enzyme has recently been reported to be also present in nucleus, endoplasmic reticulum and lysosomes. These extramitochondrial localizations are associated with moonlighting functions of GDH, which include acting as a serine protease or an ATP-dependent tubulin-binding protein. Here, we review the published data on kinetics and localization of multiple forms of animal GDH taking into account the splice variants, post-translational modifications and GDH isoenzymes, found in humans and apes. The kinetic properties of human GLUD1 and GLUD2 isoenzymes are shown to be similar to those published for GDH1 and GDH2 from bovine brain. Increased functional diversity and specific regulation of GDH isoforms due to alternative splicing and post-translational modifications are also considered. In particular, these structural differences may affect the well-known regulation of GDH by nucleotides which is related to recent identification of thiamine derivatives as novel GDH modulators. The thiamine-dependent regulation of GDH is in good agreement with the fact that the non-coenzyme forms of thiamine, i.e., thiamine triphosphate and its adenylated form are generated in response to amino acid and carbon starvation. PMID:27983623
Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors.

PubMed

Olson, Michael F

2018-05-04

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

PubMed Central

Benjdia, Alhosna; Balty, Clémence; Berteau, Olivier

2017-01-01

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota. PMID:29167789
Glycation & Insulin Resistance: Novel Mechanisms and Unique Targets?

PubMed Central

Song, Fei; Schmidt, Ann Marie

2012-01-01

Objectives Multiple biochemical, metabolic and signal transduction pathways contribute to insulin resistance. In this review, we present the evidence that the post-translational process of protein glycation may play role in insulin resistance. The post-translational modifications, the advanced glycation endproducts (AGEs), are formed and accumulate by endogenous and exogenous mechanisms. Methods and Results AGEs may contribute to insulin resistance by a variety of mechanisms, including generation of tumor necrosis factor-alpha, direct modification of the insulin molecule thereby leading to its impaired action, generation of oxidative stress, and impairment of mitochondrial function, as examples. AGEs may stimulate signal transduction via engagement of cellular receptors, such as RAGE, or receptor for AGE. AGE-RAGE interaction perpetuates AGE formation and cellular stress via induction of inflammation, oxidative stress and reduction in the expression and activity of the enzyme, glyoxalase I that detoxifies the AGE precursor, methylglyoxal, or MG. Conclusions Once set in motion, glycation-promoting mechanisms may stimulate ongoing AGE production and target tissue stresses that reduce insulin responsiveness. Strategies to limit AGE accumulation and action may contribute to prevention of insulin resistance and its consequences. PMID:22815341
Functional advantages of dynamic protein disorder.

PubMed

Berlow, Rebecca B; Dyson, H Jane; Wright, Peter E

2015-09-14

Intrinsically disordered proteins participate in many important cellular regulatory processes. The absence of a well-defined structure in the free state of a disordered domain, and even on occasion when it is bound to physiological partners, is fundamental to its function. Disordered domains are frequently the location of multiple sites for post-translational modification, the key element of metabolic control in the cell. When a disordered domain folds upon binding to a partner, the resulting complex buries a far greater surface area than in an interaction of comparably-sized folded proteins, thus maximizing specificity at modest protein size. Disorder also maintains accessibility of sites for post-translational modification. Because of their inherent plasticity, disordered domains frequently adopt entirely different structures when bound to different partners, increasing the repertoire of available interactions without the necessity for expression of many different proteins. This feature also adds to the faithfulness of cellular regulation, as the availability of a given disordered domain depends on competition between various partners relevant to different cellular processes. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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.
Redox Aspects of Chaperones in Cardiac Function

PubMed Central

Penna, Claudia; Sorge, Matteo; Femminò, Saveria; Pagliaro, Pasquale; Brancaccio, Mara

2018-01-01

Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury. PMID:29615920
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
Hunting for unexpected post-translational modifications by spectral library searching with tier-wise scoring.

PubMed

Ma, Chun Wai Manson; Lam, Henry

2014-05-02

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

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

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

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

Mei, Yang; Glover, Karen; Su, Minfei

BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3-kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hubmore » for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled-coil domain (CCD); and a β-α-repeated autophagy-specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post-translational modifications. A better structure-based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post-translational modifications will be essential to elucidating the mechanism of its multiple biological roles.« less
Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry

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

Zhou, Mowei; Wu, Si; Stenoien, David L.

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

PubMed

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

2013-12-01

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

PubMed

Chakraborty, Joy; Basso, Valentina; Ziviani, Elena

2017-02-21

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin are associated to a rare form of familiar autosomal recessive Parkinsonism. Despite decades of research on the Parkin protein, whose structure has been recently solved, little is known about the specific signalling pathways that lead to Parkin activation. Parkin activity spans from mitochondria quality control to tumor suppression and stress protection; it is thus tempting to hypothesize that the broad impact of Parkin on cellular physiology might be the result of different post translational modifications that can be controlled by balanced opposing events. Sequence alignment of Parkin from different species indicates high homology between domains across Parkin orthologs and identifies highly conserved amino acid residues that, if modified, impinge on Parkin functions. In this review, we summarize findings on post translational modifications that have been shown to affect Parkin activity and stability. This article was reviewed by Prof. Dr. Konstanze F. Winklhofer and by Prof. Thomas Simmen. Both reviewers have been nominated by Professor Luca Pellegrini.
The role of proteomics in studies of protein moonlighting.

PubMed

Beynon, Robert J; Hammond, Dean; Harman, Victoria; Woolerton, Yvonne

2014-12-01

The increasing acceptance that proteins may exert multiple functions in the cell brings with it new analytical challenges that will have an impact on the field of proteomics. Many proteomics workflows begin by destroying information about the interactions between different proteins, and the reduction of a complex protein mixture to constituent peptides also scrambles information about the combinatorial potential of post-translational modifications. To bring the focus of proteomics on to the domain of protein moonlighting will require novel analytical and quantitative approaches.
Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer.

PubMed

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Mulderrig, Lee; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2009-05-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer

PubMed Central

van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O’Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

2010-01-01

Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029
Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals.

PubMed

Maynard, Jason C; Burlingame, Alma L; Medzihradszky, Katalin F

2016-11-01

Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Heterodimers of tyrosylprotein sulfotransferases suggest existence of a higher organization level of transferases in the membrane of the trans-Golgi apparatus.

PubMed

Hartmann-Fatu, Cristina; Trusch, Franziska; Moll, Carina N; Michin, Irina; Hassinen, Antti; Kellokumpu, Sakari; Bayer, Peter

2015-03-27

Tyrosine sulfation of proteins is an important post-translational modification shown to play a role in many membrane-associated or extracellular processes such as virus entry, blood clotting, antibody-mediated immune response, inflammation and egg fecundation. The sole two human enzymes that transfer sulfate moieties from 3'-phospho-adenosine-5'-phospho-sulfate onto tyrosine residues, TPST1 and TPST2, are anchored to the membranes of the trans-Golgi compartment with the catalytic domain oriented to the lumen. In contrast to the relatively well studied organization of medial Golgi enzymes, the organization of trans-Golgi transferases remains elusive. Although tyrosylprotein sulfotransferases are known to exist as homodimers in the Golgi membranes, this organization level may represent only a small piece of a puzzle that is linked to the entire picture. Here we report the formation of TPST1/TPST2 heterodimers and a novel interaction between either TPST1 or TPST2 and the α-2,6-sialyltransferase, indicating a higher organization level of tyrosylprotein sulfotransferases that may serve for substrate selectivity and/or effective organization of multiple post-translational modification of proteins. Copyright © 2015. Published by Elsevier Ltd.
Molecular farming on rescue of pharma industry for next generations.

PubMed

Moustafa, Khaled; Makhzoum, Abdullah; Trémouillaux-Guiller, Jocelyne

2016-10-01

Recombinant proteins expressed in plants have been emerged as a novel branch of the biopharmaceutical industry, offering practical and safety advantages over traditional approaches. Cultivable in various platforms (i.e. open field, greenhouses or bioreactors), plants hold great potential to produce different types of therapeutic proteins with reduced risks of contamination with human and animal pathogens. To maximize the yield and quality of plant-made pharmaceuticals, crucial factors should be taken into account, including host plants, expression cassettes, subcellular localization, post-translational modifications, and protein extraction and purification methods. DNA technology and genetic transformation methods have also contributed to great parts with substantial improvements. To play their proper function and stability, proteins require multiple post-translational modifications such as glycosylation. Intensive glycoengineering research has been performed to reduce the immunogenicity of recombinant proteins produced in plants. Important strategies have also been developed to minimize the proteolysis effects and enhance protein accumulation. With growing human population and new epidemic threats, the need for new medications will be paramount so that the traditional pharmaceutical industry will not be alone to answer medication demands for upcoming generations. Here, we review several aspects of plant molecular pharming and outline some important challenges that hamper these ambitious biotechnological developments.
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
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
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
Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin.

PubMed

Kluskens, Leon D; Kuipers, Anneke; Rink, Rick; de Boef, Esther; Fekken, Susan; Driessen, Arnold J M; Kuipers, Oscar P; Moll, Gert N

2005-09-27

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.
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
Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry.

PubMed

Zhou, Mowei; Wu, Si; Stenoien, David L; Zhang, Zhaorui; Connolly, Lanelle; Freitag, Michael; Paša-Tolić, Ljiljana

2017-01-01

Top-down mass spectrometry is a valuable tool for understanding gene expression through characterization of combinatorial histone post-translational modifications (i.e., histone code). In this protocol, we describe a top-down workflow that employs liquid chromatography (LC) coupled to mass spectrometry (MS), for fast global profiling of changes in histone proteoforms, and apply LCMS top-down approach for comparative analysis of a wild-type and a mutant fungal species. The proteoforms exhibiting differential abundances can be subjected to further targeted studies by other MS or orthogonal (e.g., biochemical) assays. This method can be generally adapted for screening of changes in histone modifications between samples such as wild type vs. mutant or healthy vs. diseased.
Role of novel histone modifications in cancer

PubMed Central

Shanmugam, Muthu K.; Arfuso, Frank; Arumugam, Surendar; Chinnathambi, Arunachalam; Jinsong, Bian; Warrier, Sudha; Wang, Ling Zhi; Kumar, Alan Prem; Ahn, Kwang Seok; Sethi, Gautam; Lakshmanan, Manikandan

2018-01-01

Oncogenesis is a multistep process mediated by a variety of factors including epigenetic modifications. Global epigenetic post-translational modifications have been detected in almost all cancers types. Epigenetic changes appear briefly and do not involve permanent changes to the primary DNA sequence. These epigenetic modifications occur in key oncogenes, tumor suppressor genes, and transcription factors, leading to cancer initiation and progression. The most commonly observed epigenetic changes include DNA methylation, histone lysine methylation and demethylation, histone lysine acetylation and deacetylation. However, there are several other novel post-translational modifications that have been observed in recent times such as neddylation, sumoylation, glycosylation, phosphorylation, poly-ADP ribosylation, ubiquitination as well as transcriptional regulation and these have been briefly discussed in this article. We have also highlighted the diverse epigenetic changes that occur during the process of tumorigenesis and described the role of histone modifications that can occur on tumor suppressor genes as well as oncogenes, which regulate tumorigenesis and can thus form the basis of novel strategies for cancer therapy. PMID:29541423
BSPS Program (ESI-Mass Spectrometry) Biological Sample Data Analysis; Disruption of Bacteria Spores

DTIC Science & Technology

2005-10-01

the original usage of the translational as a broad description of the entire process by which the polymer of the three-letter code in the mRNA is...translated. There is extensive review of post transnational modifications of proteins by Finn Wold(1981)24, given as in vivo chemical modifications... thiolation , biotin, bromination, carbamylation, deamidation, methylation, glu- cosylation, lipoyl, phosphorylation,, pyridoxal phosphate
Histone ubiquitination: a tagging tail unfolds?

PubMed

Jason, Laure J M; Moore, Susan C; Lewis, John D; Lindsey, George; Ausió, Juan

2002-02-01

Despite the fact that histone H2A ubiquitination affects about 10-15% of this histone in most eukaryotic cells, histone ubiquitination is among one of the less-well-characterized post-translational histone modifications. Nevertheless, some important observations have been made in recent years. Whilst several enzymes had been known to ubiquitinate histones in vitro, recent studies in yeast have led to the unequivocal identification of the enzyme responsible for this post-translational modification in this organism. A strong functional co-relation to meiosis and spermiogenesis has also now been well documented, although its participation in other functional aspects of chromatin metabolism, such as transcription or DNA repair, still remains rather speculative and controversial. Because of its nature, histone ubiquitination represents the most bulky structural change to histones and as such it would be expected to exert an important effect on chromatin structure. Past and recent structural studies, however, indicate a surprising lack of effect of (H2A/H2B) ubiquitination on nucleosome architecture and of uH2A on chromatin folding. These results suggest that this modification may serve as a signal for recognition by functionally relevant trans-acting factors and/or operate synergistically in conjunction with other post-translational modifications such as for instance acetylation. Copyright 2002 Wiley Periodicals, Inc.
Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure

PubMed Central

Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

2015-01-01

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm5U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. PMID:26438534
Profiling post-translational modifications of histones in human monocyte-derived macrophages.

PubMed

Olszowy, Pawel; Donnelly, Maire Rose; Lee, Chanho; Ciborowski, Pawel

2015-01-01

Histones and their post-translational modifications impact cellular function by acting as key regulators in the maintenance and remodeling of chromatin, thus affecting transcription regulation either positively (activation) or negatively (repression). In this study we describe a comprehensive, bottom-up proteomics approach to profiling post-translational modifications (acetylation, mono-, di- and tri-methylation, phosphorylation, biotinylation, ubiquitination, citrullination and ADP-ribosylation) in human macrophages, which are primary cells of the innate immune system. As our knowledge expands, it becomes more evident that macrophages are a heterogeneous population with potentially subtle differences in their responses to various stimuli driven by highly complex epigenetic regulatory mechanisms. To profile post-translational modifications (PTMs) of histones in macrophages we used two platforms of liquid chromatography and mass spectrometry. One platform was based on Sciex5600 TripleTof and the second one was based on VelosPro Orbitrap Elite ETD mass spectrometers. We provide side-by-side comparison of profiling using two mass spectrometric platforms, ion trap and qTOF, coupled with the application of collisional induced and electron transfer dissociation. We show for the first time methylation of a His residue in macrophages and demonstrate differences in histone PTMs between those currently reported for macrophage cell lines and what we identified in primary cells. We have found a relatively low level of histone PTMs in differentiated but resting human primary monocyte derived macrophages. This study is the first comprehensive profiling of histone PTMs in primary human MDM. Our study implies that epigenetic regulatory mechanisms operative in transformed cell lines and primary cells are overlapping to a limited extent. Our mass spectrometric approach provides groundwork for the investigation of how histone PTMs contribute to epigenetic regulation in primary human macrophages.
Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

DOE PAGES

Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; ...

2010-01-01

Promore » teasomes are composed of 20S core particles (CPs) of α - and β -type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α 1 and α 2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α 1 Thr147, α 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α 1 , thus, revealing a new type of proteasomal modification. bing the biological role of α 1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α 1 variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α 1 . The α 1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.« less
Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry.

PubMed

Sidoli, Simone; Cheng, Lei; Jensen, Ole N

2012-06-27

Histone proteins contribute to the maintenance and regulation of the dynamic chromatin structure, to gene activation, DNA repair and many other processes in the cell nucleus. Site-specific reversible and irreversible post-translational modifications of histone proteins mediate biological functions, including recruitment of transcription factors to specific DNA regions, assembly of epigenetic reader/writer/eraser complexes onto DNA, and modulation of DNA-protein interactions. Histones thereby regulate chromatin structure and function, propagate inheritance and provide memory functions in the cell. Dysfunctional chromatin structures and misregulation may lead to pathogenic states, including diabetes and cancer, and the mapping and quantification of multivalent post-translational modifications has therefore attracted significant interest. Mass spectrometry has quickly been accepted as a versatile tool to achieve insights into chromatin biology and epigenetics. High sensitivity and high mass accuracy and the ability to sequence post-translationally modified peptides and perform large-scale analyses make this technique very well suited for histone protein characterization. In this review we discuss a range of analytical methods and various mass spectrometry-based approaches for histone analysis, from sample preparation to data interpretation. Mass spectrometry-based proteomics is already an integrated and indispensable tool in modern chromatin biology, providing insights into the mechanisms and dynamics of nuclear and epigenetic processes. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry. Copyright © 2011 Elsevier B.V. All rights reserved.
Quantitative proteomics in Giardia duodenalis-Achievements and challenges.

PubMed

Emery, Samantha J; Lacey, Ernest; Haynes, Paul A

2016-08-01

Giardia duodenalis (syn. G. lamblia and G. intestinalis) is a protozoan parasite of vertebrates and a major contributor to the global burden of diarrheal diseases and gastroenteritis. The publication of multiple genome sequences in the G. duodenalis species complex has provided important insights into parasite biology, and made post-genomic technologies, including proteomics, significantly more accessible. The aims of proteomics are to identify and quantify proteins present in a cell, and assign functions to them within the context of dynamic biological systems. In Giardia, proteomics in the post-genomic era has transitioned from reliance on gel-based systems to utilisation of a diverse array of techniques based on bottom-up LC-MS/MS technologies. Together, these have generated crucial foundations for subcellular proteomes, elucidated intra- and inter-assemblage isolate variation, and identified pathways and markers in differentiation, host-parasite interactions and drug resistance. However, in Giardia, proteomics remains an emerging field, with considerable shortcomings evident from the published research. These include a bias towards assemblage A, a lack of emphasis on quantitative analytical techniques, and limited information on post-translational protein modifications. Additionally, there are multiple areas of research for which proteomic data is not available to add value to published transcriptomic data. The challenge of amalgamating data in the systems biology paradigm necessitates the further generation of large, high-quality quantitative datasets to accurately model parasite biology. This review surveys the current proteomic research available for Giardia and evaluates their technical and quantitative approaches, while contextualising their biological insights into parasite pathology, isolate variation and eukaryotic evolution. Finally, we propose areas of priority for the generation of future proteomic data to explore fundamental questions in Giardia, including the analysis of post-translational modifications, and the design of MS-based assays for validation of differentially expressed proteins in large datasets. Copyright © 2016 Elsevier B.V. All rights reserved.
Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly.

PubMed

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

2015-08-25

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

Protein O-GlcNAcylation: emerging mechanisms and functions

PubMed Central

Yang, Xiaoyong; Qian, Kevin

2017-01-01

O-GlcNAcylation—the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins—is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes—O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)—controls the dynamic cycling of this post-translational modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels ranging from structural and molecular biology to cell signalling and gene regulation to physiology and disease. Emerging from these recent developments are new mechanisms and functions of O-GlcNAcylation that enable us to begin constructing a unified conceptual framework through which to understand the significance of this modification in cellular and organismal physiology. PMID:28488703
Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.
Post-translational Modifications of Chicken Myelin Basic Protein Charge Components

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

Kim, Jeongkwon; Zhang, Rui; Strittmatter, Eric F.

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

PubMed

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

2015-01-01

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

PubMed Central

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

2012-01-01

Aim Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca2+-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination. Materials & methods Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated. Results The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets. Conclusion Nanoparticle exposure facilitated post-translational citrullination of proteins. PMID:22625207
Insights into molecular plasticity in protein complexes from Trm9-Trm112 tRNA modifying enzyme crystal structure.

PubMed

Létoquart, Juliette; van Tran, Nhan; Caroline, Vonny; Aleksandrov, Alexey; Lazar, Noureddine; van Tilbeurgh, Herman; Liger, Dominique; Graille, Marc

2015-12-15

Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm(5)U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Pathogens and Disease Play Havoc on the Host Epiproteome-The "First Line of Response" Role for Proteomic Changes Influenced by Disorder.

PubMed

Rikkerink, Erik H A

2018-03-08

Organisms face stress from multiple sources simultaneously and require mechanisms to respond to these scenarios if they are to survive in the long term. This overview focuses on a series of key points that illustrate how disorder and post-translational changes can combine to play a critical role in orchestrating the response of organisms to the stress of a changing environment. Increasingly, protein complexes are thought of as dynamic multi-component molecular machines able to adapt through compositional, conformational and/or post-translational modifications to control their largely metabolic outputs. These metabolites then feed into cellular physiological homeostasis or the production of secondary metabolites with novel anti-microbial properties. The control of adaptations to stress operates at multiple levels including the proteome and the dynamic nature of proteomic changes suggests a parallel with the equally dynamic epigenetic changes at the level of nucleic acids. Given their properties, I propose that some disordered protein platforms specifically enable organisms to sense and react rapidly as the first line of response to change. Using examples from the highly dynamic host-pathogen and host-stress response, I illustrate by example how disordered proteins are key to fulfilling the need for multiple levels of integration of response at different time scales to create robust control points.
Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

PubMed

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

2012-08-01

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

PubMed

Larance, Mark; Lamond, Angus I

2015-05-01

The proteome is a dynamic system in which each protein has interconnected properties - dimensions - that together contribute to the phenotype of a cell. Measuring these properties has proved challenging owing to their diversity and dynamic nature. Advances in mass spectrometry-based proteomics now enable the measurement of multiple properties for thousands of proteins, including their abundance, isoform expression, turnover rate, subcellular localization, post-translational modifications and interactions. Complementing these experimental developments are new data analysis, integration and visualization tools as well as data-sharing resources. Together, these advances in the multidimensional analysis of the proteome are transforming our understanding of various cellular and physiological processes.
Review of S100A9 Biology and its Role in Cancer

PubMed Central

Markowitz, Joseph; Carson, William E.

2013-01-01

S100A9 is a calcium binding protein with multiple ligands and post-translation modifications that is involved in inflammatory events and the initial development of the cancer cell through to the development of metastatic disease. This review has a threefold purpose: 1) describe S100A9 structural elements important for its biological activity, 2) describe S100A9 biology in the context of the immune system, and 3) illustrate the role of S100A9 in the development of malignancy via interactions with the immune system and other cellular processes. PMID:23123827
Multiple forms of statherin in human salivary secretions.

PubMed

Jensen, J L; Lamkin, M S; Troxler, R F; Oppenheim, F G

1991-01-01

Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SV1, statherin SV2, and statherin SV3. SV1 is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene.
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.
TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP)

PubMed Central

Lau, Zerlina; Cheung, Pamela; Schneider, William M.; Bozzacco, Leonia; Buehler, Eugen; Takaoka, Akinori; Rice, Charles M.; Felsenfeld, Dan P.; MacDonald, Margaret R.

2017-01-01

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP’s antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP’s ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity. PMID:28060952
TRIM25 Enhances the Antiviral Action of Zinc-Finger Antiviral Protein (ZAP).

PubMed

Li, Melody M H; Lau, Zerlina; Cheung, Pamela; Aguilar, Eduardo G; Schneider, William M; Bozzacco, Leonia; Molina, Henrik; Buehler, Eugen; Takaoka, Akinori; Rice, Charles M; Felsenfeld, Dan P; MacDonald, Margaret R

2017-01-01

The host factor and interferon (IFN)-stimulated gene (ISG) product, zinc-finger antiviral protein (ZAP), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. To elucidate its antiviral mechanism, we perform a loss-of-function genome-wide RNAi screen to identify cellular cofactors required for ZAP antiviral activity against the prototype alphavirus, Sindbis virus (SINV). In order to exclude off-target effects, we carry out stringent confirmatory assays to verify the top hits. Important ZAP-liaising partners identified include proteins involved in membrane ion permeability, type I IFN signaling, and post-translational protein modification. The factor contributing most to the antiviral function of ZAP is TRIM25, an E3 ubiquitin and ISG15 ligase. We demonstrate here that TRIM25 interacts with ZAP through the SPRY domain, and TRIM25 mutants lacking the RING or coiled coil domain fail to stimulate ZAP's antiviral activity, suggesting that both TRIM25 ligase activity and its ability to form oligomers are critical for its cofactor function. TRIM25 increases the modification of both the short and long ZAP isoforms by K48- and K63-linked polyubiquitin, although ubiquitination of ZAP does not directly affect its antiviral activity. However, TRIM25 is critical for ZAP's ability to inhibit translation of the incoming SINV genome. Taken together, these data uncover TRIM25 as a bona fide ZAP cofactor that leads to increased ZAP modification enhancing its translational inhibition activity.
Post-Translational Modification Biology of Glutamate Receptors and Drug Addiction

PubMed Central

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

2011-01-01

Post-translational covalent modifications of glutamate receptors remain a hot topic. Early studies have established that this family of receptors, including almost all ionotropic and metabotropic glutamate receptor subtypes, undergoes active phosphorylation at serine, threonine, or tyrosine residues in their intracellular domains. Recent evidence identifies several glutamate receptor subtypes to be direct substrates for palmitoylation at cysteine residues. Other modifications such as ubiquitination and sumoylation at lysine residues also occur to certain glutamate receptors. These modifications are dynamic and reversible in nature and are regulatable by changing synaptic inputs. The regulated modifications significantly impact the receptor in many ways, including interrelated changes in biochemistry (synthesis, subunit assembling, and protein–protein interactions), subcellular redistribution (trafficking, endocytosis, synaptic delivery, and clustering), and physiology, usually associated with changes in synaptic plasticity. Glutamate receptors are enriched in the striatum and cooperate closely with dopamine to regulate striatal signaling. Emerging evidence shows that modification processes of striatal glutamate receptors are sensitive to addictive drugs, such as psychostimulants (cocaine and amphetamine). Altered modifications are believed to be directly linked to enduring receptor/synaptic plasticity and drug-seeking. This review summarizes several major types of modifications of glutamate receptors and analyzes the role of these modifications in striatal signaling and in the pathogenesis of psychostimulant addiction. PMID:21441996
DAG1, no gene for RNA regulation?

PubMed

Brancaccio, Andrea

2012-04-10

DAG1 encodes for a precursor protein that liberates the two subunits featured by the dystroglycan (DG) adhesion complex that are involved in an increasing number of cellular functions in a wide variety of cells and tissues. Aside from the proteolytic events producing the α and β subunits, especially the former undergoes extensive "post-production" modifications taking place within the ER/Golgi where its core protein is both N- and O-decorated with sugars. These post-translational events, that are mainly orchestrated by a plethora of certified, or putative, glycosyltransferases, prelude to the excocytosis-mediated trafficking and targeting of the DG complex to the plasma membrane. Extensive genetic and biochemical evidences have been accumulated so far on α-DG glycosylation, while little is know on possible regulatory events underlying the chromatine activation, transcription or post-transcription (splicing and escape from the nucleus) of DAG1 or of its mRNA. A scenario is envisaged in which cells would use a sort of preferential, and scarcely regulated, route for DAG1 activation, that would imply fast mRNA transcription, maturation and export to the cytosol, and would prelude to the multiple time-consuming enzymatic post-translational activities needed for its glycosylation. Such a provocative view might be helpful to trigger future work aiming at disclosing the complete molecular mechanisms underlying DAG1 activation and at improving our knowledge of any pre-translational step that is involved in dystroglycan regulation. Copyright © 2012 Elsevier B.V. All rights reserved.
Doubling down on peptide phosphorylation as a variable mass modification

USDA-ARS?s Scientific Manuscript database

Some mass spectrometrists believe that searching for variable post-translational modifications like phosphorylation of serine or threonine when using database-search algorithms to interpret peptide tandem mass spectra will increase false positive rates. The basis for this is the premise that the al...
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
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.
Proteomic Responses of BEAS-2B Cells to Nontoxic and Toxic Chromium: Protein Indicators of Cytotoxicity Conversion

EPA Science Inventory

Hexavalent chromium (Cr (VI)) is an environmental human carcinogen which primarily targets lungs. Among a variety of toxic mechanisms, disruption of biological pathways via translational and post-translational modifications represents a key mechanism through which Cr (VI) induces...

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

PubMed

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

2016-06-15

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

NASA Astrophysics Data System (ADS)

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

2014-02-01

The review summarizes main trends in the development of methods for the prediction of protein post-translational modifications (PTMs) by considering the three most common types of PTMs — phosphorylation, acetylation and glycosylation. Considerable attention is given to general characteristics of regulatory interactions associated with PTMs. Different approaches to the prediction of PTMs are analyzed. Most of the methods are based only on the analysis of the neighbouring environment of modification sites. The related software is characterized by relatively low accuracy of PTM predictions, which may be due both to the incompleteness of training data and the features of PTM regulation. Advantages and limitations of the phylogenetic approach are considered. The prediction of PTMs using data on regulatory interactions, including the modular organization of interacting proteins, is a promising field, provided that a more carefully selected training data will be used. The bibliography includes 145 references.
Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover

PubMed Central

Swaney, Danielle L; Rodríguez-Mias, Ricard A; Villén, Judit

2015-01-01

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology. PMID:26142280
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
Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications

PubMed Central

2015-01-01

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

PubMed

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

2014-10-03

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

PubMed

Ruiz-Ballesta, Isabel; Baena, Guillermo; Gandullo, Jacinto; Wang, Liqun; She, Yi-Min; Plaxton, William Charles; Echevarría, Cristina

2016-05-01

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
MyelStones: the executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis.

PubMed

Vassall, Kenrick A; Bamm, Vladimir V; Harauz, George

2015-11-15

The classic isoforms of myelin basic protein (MBP, 14-21.5 kDa) are essential to formation of the multilamellar myelin sheath of the mammalian central nervous system (CNS). The predominant 18.5-kDa isoform links together the cytosolic surfaces of oligodendrocytes, but additionally participates in cytoskeletal turnover and membrane extension, Fyn-mediated signalling pathways, sequestration of phosphoinositides and maintenance of calcium homoeostasis. All MBP isoforms are intrinsically disordered proteins (IDPs) that interact via molecular recognition fragments (MoRFs), which thereby undergo local disorder-to-order transitions. Their conformations and associations are modulated by environment and by a dynamic barcode of post-translational modifications, particularly phosphorylation by mitogen-activated and other protein kinases and deimination [a hallmark of demyelination in multiple sclerosis (MS)]. The MBPs are thus to myelin what basic histones are to chromatin. Originally thought to be merely structural proteins forming an inert spool, histones are now known to be dynamic entities involved in epigenetic regulation and diseases such as cancer. Analogously, the MBPs are not mere adhesives of compact myelin, but active participants in oligodendrocyte proliferation and in membrane process extension and stabilization during myelinogenesis. A central segment of these proteins is pivotal in membrane-anchoring and SH3 domain (Src homology 3) interaction. We discuss in the present review advances in our understanding of conformational conversions of this classic basic protein upon membrane association, including new thermodynamic analyses of transitions into different structural ensembles and how a shift in the pattern of its post-translational modifications is associated with the pathogenesis and potentially onset of demyelination in MS. © 2015 Authors; published by Portland Press Limited.
The story so far: post-translational regulation of peroxisome proliferator-activated receptors by ubiquitination and SUMOylation

PubMed Central

Wadosky, Kristine M.

2012-01-01

Many studies have implicated the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptor transcription factors in regulating cardiac substrate metabolism and ATP generation. Recently, evidence from a variety of cell culture and organ systems has implicated ubiquitin and small ubiquitin-like modifier (SUMO) conjugation as post-translational modifications that regulate the activity of PPAR transcription factors and their coreceptors/coactivators. Here we introduce the ubiquitin and SUMO conjugation systems and extensively review how they have been shown to regulate all three PPAR isoforms (PPARα, PPARβ/δ, and PPARγ) in addition to the retinoid X receptor and PPARγ coactivator-1α subunits of the larger PPAR transcription factor complex. We then present how the specific ubiquitin (E3) ligases have been implicated and review emerging evidence that post-translational modifications of PPARs with ubiquitin and/or SUMO may play a role in cardiac disease. Because PPAR activity is perturbed in a variety of forms of heart disease and specific proteins regulate this process (E3 ligases), this may be a fruitful area of investigation with respect to finding new therapeutic targets. PMID:22037188
A Design Principle for an Autonomous Post-translational Pattern Formation.

PubMed

Sugai, Shuhei S; Ode, Koji L; Ueda, Hiroki R

2017-04-25

Previous autonomous pattern-formation models often assumed complex molecular and cellular networks. This theoretical study, however, shows that a system composed of one substrate with multisite phosphorylation and a pair of kinase and phosphatase can generate autonomous spatial information, including complex stripe patterns. All (de-)phosphorylation reactions are described with a generic Michaelis-Menten scheme, and all species freely diffuse without pre-existing gradients. Computational simulation upon >23,000,000 randomly generated parameter sets revealed the design motifs of cyclic reaction and enzyme sequestration by slow-diffusing substrates. These motifs constitute short-range positive and long-range negative feedback loops to induce Turing instability. The width and height of spatial patterns can be controlled independently by distinct reaction-diffusion processes. Therefore, multisite reversible post-translational modification can be a ubiquitous source for various patterns without requiring other complex regulations such as autocatalytic regulation of enzymes and is applicable to molecular mechanisms for inducing subcellular localization of proteins driven by post-translational modifications. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
PHOXTRACK-a tool for interpreting comprehensive datasets of post-translational modifications of proteins.

PubMed

Weidner, Christopher; Fischer, Cornelius; Sauer, Sascha

2014-12-01

We introduce PHOXTRACK (PHOsphosite-X-TRacing Analysis of Causal Kinases), a user-friendly freely available software tool for analyzing large datasets of post-translational modifications of proteins, such as phosphorylation, which are commonly gained by mass spectrometry detection. In contrast to other currently applied data analysis approaches, PHOXTRACK uses full sets of quantitative proteomics data and applies non-parametric statistics to calculate whether defined kinase-specific sets of phosphosite sequences indicate statistically significant concordant differences between various biological conditions. PHOXTRACK is an efficient tool for extracting post-translational information of comprehensive proteomics datasets to decipher key regulatory proteins and to infer biologically relevant molecular pathways. PHOXTRACK will be maintained over the next years and is freely available as an online tool for non-commercial use at http://phoxtrack.molgen.mpg.de. Users will also find a tutorial at this Web site and can additionally give feedback at https://groups.google.com/d/forum/phoxtrack-discuss. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Top-Down Analysis of Highly Post-Translationally Modified Peptides by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

NASA Astrophysics Data System (ADS)

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

2015-03-01

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

PubMed

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

2016-01-01

Several proteins interact either to activate or repress the expression of other genes during transcription. Based on the impact of these activities, the proteins can be classified into readers, modifier writers, and modifier erasers depending on whether histone marks are read, added, or removed, respectively, from a specific amino acid. Transcription is controlled by dynamic epigenetic marks with serious health implications in certain complex diseases, whose understanding may be useful in gene therapy. This work highlights traditional and current advances in post-translational modifications with relevance to gene therapy delivery. We report that enhanced understanding of epigenetic machinery provides clues to functional implication of certain genes/gene products and may facilitate transition toward revision of our clinical treatment procedure with effective fortification of gene therapy delivery.
Pseudouridine and N6-methyladenosine modifications weaken PUF protein/RNA interactions

PubMed Central

AlSadhan, Ishraq; Merriman, Dawn K.; Al-Hashimi, Hashim M.; Herschlag, Daniel

2017-01-01

RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications—pseudouridine (Ψ) and N6-methyladenosine (m6A)—affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure. Here, we test the hypothesis that RNA modifications directly affect interactions between RNA-binding proteins and target RNA. We show that Ψ and m6A weaken the binding of the human single-stranded RNA binding protein Pumilio 2 (hPUM2) to its consensus motif, with individual modifications having effects up to approximately threefold and multiple modifications giving larger effects. While there are likely to be some cases where RNA modifications essentially fully ablate protein binding, here we see modest responses that may be more common. Such modest effects could nevertheless profoundly alter the complex landscape of RNA:protein interactions, and the quantitative rather than qualitative nature of these effects underscores the need for quantitative, systems-level accounting of RNA:protein interactions to understand post-transcriptional regulation. PMID:28138061
Targeting allosteric disulphide bonds in cancer.

PubMed

Hogg, Philip J

2013-06-01

Protein action in nature is generally controlled by the amount of protein produced and by chemical modification of the protein, and both are often perturbed in cancer. The amino acid side chains and the peptide and disulphide bonds that bind the polypeptide backbone can be post-translationally modified. Post-translational cleavage or the formation of disulphide bonds are now being identified in cancer-related proteins and it is timely to consider how these allosteric bonds could be targeted for new therapies.
Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

PubMed

Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality. © PDA, Inc. 2016.
Epigenomic landscape modified by histone modification correlated with activation of IGF2 gene

USDA-ARS?s Scientific Manuscript database

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

PubMed Central

Hsin, Jing-Ping; Manley, James L.

2012-01-01

The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1–Ser2–Pro3–Thr4–Ser5–Pro6–Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity. PMID:23028141

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases.

PubMed

Gianazza, Erica; Tremoli, Elena; Banfi, Cristina

2014-12-01

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.
Information Commons for Rice (IC4R)

PubMed Central

2016-01-01

Rice is the most important staple food for a large part of the world's human population and also a key model organism for plant research. Here, we present Information Commons for Rice (IC4R; http://ic4r.org), a rice knowledgebase featuring adoption of an extensible and sustainable architecture that integrates multiple omics data through community-contributed modules. Each module is developed and maintained by different committed groups, deals with data collection, processing and visualization, and delivers data on-demand via web services. In the current version, IC4R incorporates a variety of rice data through multiple committed modules, including genome-wide expression profiles derived entirely from RNA-Seq data, resequencing-based genomic variations obtained from re-sequencing data of thousands of rice varieties, plant homologous genes covering multiple diverse plant species, post-translational modifications, rice-related literatures and gene annotations contributed by the rice research community. Unlike extant related databases, IC4R is designed for scalability and sustainability and thus also features collaborative integration of rice data and low costs for database update and maintenance. Future directions of IC4R include incorporation of other omics data and association of multiple omics data with agronomically important traits, dedicating to build IC4R into a valuable knowledgebase for both basic and translational researches in rice. PMID:26519466
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
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.
Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.
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.
Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

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

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

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

PubMed

de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

2016-06-03

Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.
O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks.

PubMed

Slawson, Chad; Housley, Michael P; Hart, Gerald W

2006-01-01

O-GlcNAc is an ubiquitous post-translational protein modification consisting of a single N-acetlyglucosamine moiety linked to serine or threonine residues on nuclear and cytoplasmic proteins. Recent work has begun to uncover the functional roles of O-GlcNAc in cellular processes. O-GlcNAc modified proteins are involved in sensing the nutrient status of the surrounding cellular environment and adjusting the activity of cellular proteins accordingly. O-GlcNAc regulates cellular responses to hormones such as insulin, initiates a protective response to stress, modulates a cell's capacity to grow and divide, and regulates gene transcription. This review will focus on recent work involving O-GlcNAc in sensing the environment and regulating signaling cascades. (c) 2005 Wiley-Liss, Inc.
Challenges ahead for mass spectrometry and proteomics applications in epigenetics.

PubMed

Kessler, Benedikt M

2010-02-01

Inheritance of biological information to future generations depends on the replication of DNA and the Mendelian principle of distribution of genes. In addition, external and environmental factors can influence traits that can be propagated to offspring, but the molecular details of this are only beginning to be understood. The discoveries of DNA methylation and post-translational modifications on chromatin and histones provided entry points for regulating gene expression, an area now defined as epigenetics and epigenomics. Mass spectrometry turned out to be instrumental in uncovering molecular details involved in these processes. The central role of histone post-translational modifications in epigenetics related biological processes has revitalized mass spectrometry based investigations. In this special report, current approaches and future challenges that lay ahead due to the enormous complexity are discussed.
The Application of Multiple Reaction Monitoring to Assess Apo A-I Methionine Oxidations in Diabetes and Cardiovascular Disease

PubMed Central

Yassine, Hussein N.; Jackson, Angela M.; Reaven, Peter D.; Nedelkov, Dobrin; Nelson, Randall W.; Lau, Serrine S.; Borchers, Christoph H.

2014-01-01

The oxidative modification of apolipoprotein A-I ‘s methionine148(M148) is associated with defective HDL function in vitro. Multiple Reaction Monitoring (MRM) is a mass spectrometric technique that can be used to quantitate post-translational modifications. In this study, we developed an MRM assay to monitor the abundance ratio of the peptide containing oxidized M148 to the native peptide in Apo A-I. Measurement of the oxidized-to-unoxidized-M148 ratio was reproducible (CV<5%). The extent of methionine M148 oxidation in the HDL of healthy controls, and type 2 diabetic participants with and without prior cardiovascular events (CVD) were then examined. The results suggest a significant increase in the relative ratio of the peptide containing oxidized M148 to the unmodified peptide in the HDL of participants with diabetes and CVD (p<0.001), compared to participants without CVD. Monitoring the abundance ratio of the peptides containing oxidized and unoxidized M148 by MRM provides a means of examining the relationship between M148 oxidation and vascular complications in CVD. PMID:25705587
Expansion of Protein Farnesyltransferase Specificity Using “Tunable” Active Site Interactions

PubMed Central

Hougland, James L.; Gangopadhyay, Soumyashree A.; Fierke, Carol A.

2012-01-01

Post-translational modifications play essential roles in regulating protein structure and function. Protein farnesyltransferase (FTase) catalyzes the biologically relevant lipidation of up to several hundred cellular proteins. Site-directed mutagenesis of FTase coupled with peptide selectivity measurements demonstrates that molecular recognition is determined by a combination of multiple interactions. Targeted randomization of these interactions yields FTase variants with altered and, in some cases, bio-orthogonal selectivity. We demonstrate that FTase specificity can be “tuned” using a small number of active site contacts that play essential roles in discriminating against non-substrates in the wild-type enzyme. This tunable selectivity extends in vivo, with FTase variants enabling the creation of bioengineered parallel prenylation pathways with altered substrate selectivity within a cell. Engineered FTase variants provide a novel avenue for probing both the selectivity of prenylation pathway enzymes and the effects of prenylation pathway modifications on the cellular function of a protein. PMID:22992747
The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference

PubMed Central

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

2014-01-01

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3. PMID:24658679
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
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.
Structural characterization of thioether-bridged bacteriocins.

PubMed

Lohans, Christopher T; Vederas, John C

2014-01-01

Bacteriocins are a group of ribosomally synthesized antimicrobial peptides produced by bacteria, some of which are extensively post-translationally modified. Some bacteriocins, namely the lantibiotics and sactibiotics, contain one or more thioether bridges. However, these modifications complicate the structural elucidation of these bacteriocins using conventional techniques. This review will discuss the techniques and strategies that have been applied to determine the primary structures of lantibiotics and sactibiotics. A major challenge is to identify the topology of thioether bridges in these peptides (i.e., which amino-acid residues are involved in which bridges). Edman degradation, NMR spectroscopy and tandem MS have all been commonly applied to characterize these bacteriocins, but can be incompatible with the post-translational modifications present. Chemical modifications to the modified residues, such as desulfurization and reduction, make the treated bacteriocins more compatible to analysis by these standard peptide analytical techniques. Despite their differences in structure, similar strategies have proved useful to study the structures of both lantibiotics and sactibiotics.
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.
Sumoylation Modulates the Activity of Spalt-like Proteins during Wing Development in Drosophila*

PubMed Central

Sánchez, Jonatan; Talamillo, Ana; Lopitz-Otsoa, Fernando; Pérez, Coralia; Hjerpe, Roland; Sutherland, James D.; Herboso, Leire; Rodríguez, Manuel S.; Barrio, Rosa

2010-01-01

The Spalt-like family of zinc finger transcription factors is conserved throughout evolution and is involved in fundamental processes during development and during embryonic stem cell maintenance. Although human SALL1 is modified by SUMO-1 in vitro, it is not known whether this post-translational modification plays a role in regulating the activity of this family of transcription factors. Here, we show that the Drosophila Spalt transcription factors are modified by sumoylation. This modification influences their nuclear localization and capacity to induce vein formation through the regulation of target genes during wing development. Furthermore, spalt genes interact genetically with the sumoylation machinery to repress vein formation in intervein regions and to attain the wing final size. Our results suggest a new level of regulation of Sall activity in vivo during animal development through post-translational modification by sumoylation. The evolutionary conservation of this family of transcription factors suggests a functional role for sumoylation in vertebrate Sall members. PMID:20562097
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.
Post-translational regulation of plant immunity.

PubMed

Withers, John; Dong, Xinnian

2017-08-01

Plants have evolved multi-layered molecular defense strategies to protect against pathogens. Plant immune signaling largely relies on post-translational modifications (PTMs) to induce rapid alterations of signaling pathways to achieve a response that is appropriate to the type of pathogen and infection pressure. In host cells, dynamic PTMs have emerged as powerful regulatory mechanisms that cells use to adjust their immune response. PTM is also a virulence strategy used by pathogens to subvert host immunity through the activities of effector proteins secreted into the host cell. Recent studies focusing on deciphering post-translational mechanisms underlying plant immunity have offered an in-depth view of how PTMs facilitate efficient immune responses and have provided a more dynamic and holistic view of plant immunity. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

The prediction of palmitoylation site locations using a multiple feature extraction method.

PubMed

Shi, Shao-Ping; Sun, Xing-Yu; Qiu, Jian-Ding; Suo, Sheng-Bao; Chen, Xiang; Huang, Shu-Yun; Liang, Ru-Ping

2013-03-01

As an extremely important and ubiquitous post-translational lipid modification, palmitoylation plays a significant role in a variety of biological and physiological processes. Unlike other lipid modifications, protein palmitoylation and depalmitoylation are highly dynamic and can regulate both protein function and localization. The dynamic nature of palmitoylation is poorly understood because of the limitations in current assay methods. The in vivo or in vitro experimental identification of palmitoylation sites is both time consuming and expensive. Due to the large volume of protein sequences generated in the post-genomic era, it is extraordinarily important in both basic research and drug discovery to rapidly identify the attributes of a new protein's palmitoylation sites. In this work, a new computational method, WAP-Palm, combining multiple feature extraction, has been developed to predict the palmitoylation sites of proteins. The performance of the WAP-Palm model is measured herein and was found to have a sensitivity of 81.53%, a specificity of 90.45%, an accuracy of 85.99% and a Matthews correlation coefficient of 72.26% in 10-fold cross-validation test. The results obtained from both the cross-validation and independent tests suggest that the WAP-Palm model might facilitate the identification and annotation of protein palmitoylation locations. The online service is available at http://bioinfo.ncu.edu.cn/WAP-Palm.aspx. Copyright © 2013 Elsevier Inc. All rights reserved.
Incorporation of unnatural sugars for the identification of glycoproteins.

PubMed

Zaro, Balyn W; Hang, Howard C; Pratt, Matthew R

2013-01-01

Glycosylation is an abundant post-translational modification that alters the fate and function of its substrate proteins. To aid in understanding the significance of protein glycosylation, identification of target proteins is key. As with all proteomics experiments, mass spectrometry has been established as the desired method for substrate identification. However, these approaches require selective enrichment and purification of modified proteins. Chemical reporters in combination with bioorthogonal reactions have emerged as robust tools for identifying post-translational modifications including glycosylation. We provide here a method for the use of bioorthogonal chemical reporters for isolation and identification of glycosylated proteins. More specifically, this protocol is a representative procedure from our own work using an alkyne-bearing O-GlcNAc chemical reporter (GlcNAlk) and a chemically cleavable azido-azo-biotin probe for the identification of O-GlcNAc-modified proteins.
SUMO and Nucleocytoplasmic Transport.

PubMed

Ptak, Christopher; Wozniak, Richard W

2017-01-01

The transport of proteins between the nucleus and cytoplasm occurs through nuclear pore complexes and is facilitated by numerous transport factors. These transport processes are often regulated by post-translational modification or, reciprocally, transport can function to control post-translational modifications through regulated transport of key modifying enzymes. This interplay extends to relationships between nucleocytoplasmic transport and SUMO-dependent pathways. Examples of protein sumoylation inhibiting or stimulating nucleocytoplasmic transport have been documented, both through its effects on the physical properties of cargo molecules and by directly regulating the functions of components of the nuclear transport machinery. Conversely, the nuclear transport machinery regulates the localization of target proteins and enzymes controlling dynamics of sumoylation and desumoylation thereby affecting the sumoylation state of target proteins. These inter-relationships between SUMO and the nucleocytoplasmic transport machinery, and the varied ways in which they occur, are discussed.
Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

PubMed

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

2017-11-03

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

PubMed

Upadhyay, Vaibhav; Singh, Anupam; Panda, Amulya K

2016-01-01

Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications. Copyright © 2015 Elsevier Inc. All rights reserved.
Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

PubMed

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

2017-06-16

Alteration of ryanodine receptor (RyR)-mediated calcium (Ca 2+ ) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca 2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca 2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca 2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca 2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca 2+ leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Tax-1 and Tax-2 similarities and differences: focus on post-translational modifications and NF-κB activation

PubMed Central

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

2013-01-01

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

PubMed

Kim, Ingu; Saito, Takeshi; Fujii, Norihiko; Kanamoto, Takashi; Fujii, Noriko

2016-12-01

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

ERIC Educational Resources Information Center

Gitlin, Laura N.; Chernett, Nancy L.; Harris, Lynn Fields; Palmer, Delores; Hopkins, Paul; Dennis, Marie P.

2008-01-01

Purpose: We describe the translation of K. R. Lorig and colleagues' Chronic Disease Self-Management Program (CDSMP) for delivery in a senior center and evaluate pre-post benefits for African American participants. Design and Methods: Modifications to the CDSMP included a name change; an additional introductory session; and course augmentations…
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
Industrial production of clotting factors: Challenges of expression, and choice of host cells.

PubMed

Kumar, Sampath R

2015-07-01

The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

PubMed Central

Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

2015-01-01

Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995
Proteomic patterns for classification of ovarian cancer and CTCL serum samples utilizing peak pairs indicative of post-translational modifications.

PubMed

Liu, Chenwei; Shea, Nancy; Rucker, Sally; Harvey, Linda; Russo, Paul; Saul, Richard; Lopez, Mary F; Mikulskis, Alvydas; Kuzdzal, Scott; Golenko, Eva; Fishman, David; Vonderheid, Eric; Booher, Susan; Cowen, Edward W; Hwang, Sam T; Whiteley, Gordon R

2007-11-01

Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOF mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process.
Distinguishing Sulfotyrosine Containing Peptides from their Phosphotyrosine Counterparts Using Mass Spectrometry

NASA Astrophysics Data System (ADS)

Chen, Guangming; Zhang, Yixiang; Trinidad, Jonathan C.; Dann, Charles

2018-03-01

Sulfotyrosine and phosphotyrosine are two post-translational modifications present in higher eukaryotes. A simple and direct mass spectrometry method to distinguish between these modifications is crucial to advance our understanding of the sulfoproteome. While sulfation and phosphorylation are nominally isobaric, the accurate mass of the sulfuryl moiety is 9.6 mDa less than the phosphoryl moiety. Based on this difference, we have used an Orbitrap Fusion Lumos mass spectrometer to characterize, resolve, and distinguish between sulfotyrosine and phosphotyrosine modifications using a set of model peptides. Multiple fragmentation techniques, namely HCD, CID, ETD, ETciD, and EThcD, have been used to compare the different fragmentation behaviors between peptides modified with these species. Sulfotyrosine undergoes neutral loss using HCD and CID, but the sulfuryl moiety is largely stable under ETD. In contrast, phosphotyrosine is stable during fragmentation using all these methods. This differential stability provides a mechanism to distinguish sulfopeptides from phosphopeptides. Based on the rigorous characterization presented herein, this work serves as a model for accurate identification of phosphotyrosine and, more challenging, sulfotyrosine, in complex proteomic samples. [Figure not available: see fulltext.
Sequence-Independent Cloning and Post-Translational Modification of Repetitive Protein Polymers through Sortase and Sfp-Mediated Enzymatic Ligation.

PubMed

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

2016-04-11

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

PubMed Central

Balaban, Robert S.

2009-01-01

The control of mitochondria energy conversion by cytosolic processes is reviewed. The nature of the cytosolic and mitochondrial potential energy homeostasis over wide ranges of energy utilization is reviewed and the consequences of this homeostasis in the control network are discussed. An analysis of the major candidate cytosolic signaling molecules ADP, Pi and Ca2+ are reviewed based on the magnitude and source of the cytosolic concentration changes as well as the potential targets of action within the mitochondrial energy conversion system. Based on this analysis, Ca2+ is the best candidate as a cytosolic signaling molecule for this process based on its ability to act as both a feed-forward and feed-back indicator of ATP hydrolysis and numerous targets within the matrix to provide a balanced activation of ATP production. These targets include numerous dehydrogenases and the F1-F0-ATPase. Pi is also a good candidate since it is an early signal of a mismatch between cytosolic ATP production and ATP synthesis in the presence of creatine kinase and has multiple targets within oxidative phosphorylation including NADH generation, electron flux in the cytochrome chain and a substrate for the F1-F0-ATPase. The mechanism of the coordinated activation of oxidative phosphorylation by these signaling molecules in discussed in light of the recent discoveries of extensive protein phosphorylation sites and other post-translational modifications. From this review it is clear that the control network associated with the maintenance of the cytosolic potential energy homeostasis is extremely complex with multiple pathways orchestrated to balance the sinks and sources in this system. New tools are needed to image and monitor metabolites within subcellular compartments to resolve many of these issues as well as the functional characterization of the numerous matrix post-translational events being discovered along with the enzymatic processes generating and removing these protein modifications. PMID:19265699
NOVEL METHODS FOR TARGET PROTEIN IDENTIFICATION USING IMMUNOPRECIPITATION - LC/MS/MS

EPA Science Inventory

Proteomics provides a powerful approach to screen and analyze responses to environmental exposures which induce alterations in protein expression, phosphorylation. ubiquitinylation, oxidation. and modulation of general proteome function. Post-translational modifications (PTM) of ...
Ubiquitinated Proteome: Ready for Global?*

PubMed Central

Shi, Yi; Xu, Ping; Qin, Jun

2011-01-01

Ubiquitin (Ub) is a small and highly conserved protein that can covalently modify protein substrates. Ubiquitination is one of the major post-translational modifications that regulate a broad spectrum of cellular functions. The advancement of mass spectrometers as well as the development of new affinity purification tools has greatly expedited proteome-wide analysis of several post-translational modifications (e.g. phosphorylation, glycosylation, and acetylation). In contrast, large-scale profiling of lysine ubiquitination remains a challenge. Most recently, new Ub affinity reagents such as Ub remnant antibody and tandem Ub binding domains have been developed, allowing for relatively large-scale detection of several hundreds of lysine ubiquitination events in human cells. Here we review different strategies for the identification of ubiquitination site and discuss several issues associated with data analysis. We suggest that careful interpretation and orthogonal confirmation of MS spectra is necessary to minimize false positive assignments by automatic searching algorithms. PMID:21339389

Post-Translational Modification Control of Innate Immunity.

PubMed

Liu, Juan; Qian, Cheng; Cao, Xuetao

2016-07-19

A coordinated balance between the positive and negative regulation of pattern-recognition receptor (PRR)-initiated innate inflammatory responses is required to ensure the most favorable outcome for the host. Post-translational modifications (PTMs) of innate sensors and downstream signaling molecules influence their activity and function by inducing their covalent linkage to new functional groups. PTMs including phosphorylation and polyubiquitination have been shown to potently regulate innate inflammatory responses through the activation, cellular translocation, and interaction of innate receptors, adaptors, and downstream signaling molecules in response to infectious and dangerous signals. Other PTMs such as methylation, acetylation, SUMOylation, and succinylation are increasingly implicated in the regulation of innate immunity and inflammation. In this review, we focus on the roles of PTMs in controlling PRR-triggered innate immunity and inflammatory responses. The emerging roles of PTMs in the pathogenesis and potential treatment of infectious and inflammatory immune diseases are also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.
Protein prenylation: a new mode of host-pathogen interaction.

PubMed

Amaya, Moushimi; Baranova, Ancha; van Hoek, Monique L

2011-12-09

Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction. Copyright © 2011 Elsevier Inc. All rights reserved.
Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

PubMed

Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.
Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins.

PubMed

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

2011-02-18

O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Uncovering Global SUMOylation Signaling Networks in a Site-Specific Manner

PubMed Central

Hendriks, Ivo A.; D’Souza, Rochelle C.J.; Yang, Bing; Verlaan-de Vries, Matty; Mann, Matthias; Vertegaal, Alfred C.O.

2014-01-01

SUMOylation is a reversible post-translational modification essential for genome stability. Using high-resolution mass spectrometry, we have studied global SUMOylation in human cells and in a site-specific manner, identifying a total of over 4,300 SUMOylation sites in over 1,600 proteins. Moreover, for the first time in excess of 1,000 SUMOylation sites were identified under standard growth conditions. SUMOylation dynamics were quantitatively studied in response to SUMO protease inhibition, proteasome inhibition and heat shock. A considerable amount of SUMOylated lysines have previously been reported to be ubiquitylated, acetylated or methylated, indicating crosstalk between SUMO and other post-translational modifications. We identified 70 phosphorylation and 4 acetylation events in close proximity to SUMOylation sites, and provide evidence for acetylation-dependent SUMOylation of endogenous histone H3. SUMOylation regulates target proteins involved in all nuclear processes including transcription, DNA repair, chromatin remodeling, pre-mRNA splicing and ribosome assembly. PMID:25218447
Nitric oxide signalling via cytoskeleton in plants.

PubMed

Yemets, Alla I; Krasylenko, Yuliya A; Lytvyn, Dmytro I; Sheremet, Yarina A; Blume, Yaroslav B

2011-11-01

Nitric oxide (NO) in plant cell mediates processes of growth and development starting from seed germination to pollination, as well as biotic and abiotic stress tolerance. However, proper understanding of the molecular mechanisms of NO signalling in plants has just begun to emerge. Accumulated evidence suggests that in eukaryotic cells NO regulates functions of proteins by their post-translational modifications, namely tyrosine nitration and S-nitrosylation. Among the candidates for NO-downstream effectors are cytoskeletal proteins because of their involvement in many processes regulated by NO. This review discusses new insights in plant NO signalling focused mainly on the involvement of cytoskeleton components into NO-cascades. Herein, examples of NO-related post-translational modifications of cytoskeletal proteins, and also indirect NO impact, are discussed. Special attention is paid to plant α-tubulin tyrosine nitration as an emerging topic in plant NO research. Copyright © 2011 Elsevier Ireland Ltd. 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.
Extracellular Matrix and Redox Signaling in Cellular Responses to Stress.

PubMed

Roberts, David D

2017-10-20

Cells in multicellular organisms communicate extensively with neighboring cells and distant organs using a variety of secreted proteins and small molecules. Cells also reside in a structural extracellular matrix (ECM), and changes in its composition, mechanical properties, and post-translational modifications provide additional layers of communication. This Forum addresses emerging mechanisms by which redox signaling controls and is controlled by changes in the ECM, focusing on the roles of matricellular proteins. These proteins engage specific cell surface signaling receptors, integrins, and proteoglycans to regulate the biosynthesis and catabolism of redox signaling molecules and the activation of their signal transducers. These signaling pathways, in turn, regulate the composition of ECM and its function. Covalent post-translational modifications of ECM by redox molecules further regulate its structure and function. Recent studies of acute injuries and chronic disease have identified important pathophysiological roles for this cross-talk and new therapeutic opportunities. Antioxid. Redox Signal. 27, 771-773.
Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias*

PubMed Central

Zhang, Xi

2016-01-01

Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins—at high efficiency and peptide reproducibility—poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. PMID:27073180
Post-translationally modified muscle-specific ubiquitin ligases as circulating biomarkers in experimental cancer cachexia

PubMed Central

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

2017-01-01

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Up to 80% of cancer patients experience cachexia, with 20-30% of cancer-related deaths directly linked to cachexia. Despite efforts to identify early cachexia and cancer relapse, clinically useful markers are lacking. Recently, we identified the role of muscle-specific ubiquitin ligases Atrogin-1 (MAFbx, FBXO32) and Muscle Ring Finger-1 in the pathogenesis of cardiac atrophy and hypertrophy. We hypothesized that during cachexia, the Atrogin-1 and MuRF1 ubiquitin ligases are released from muscle and migrate to the circulation where they could be detected and serve as a cachexia biomarker. To test this, we induced cachexia in mice using the C26 adenocarcinoma cells or vehicle (control). Body weight, tumor volume, and food consumption were measured from inoculation until ~day 14 to document cachexia. Western blot analysis of serum identified the presence of Atrogin-1 and MuRF1 with unique post-translational modifications consistent with mono- and poly- ubiquitination of Atrogin-1 and MuRF1 found only in cachectic serum. These findings suggest that both increased Atrogin-1 and the presence of unique post-translational modifications may serve as a surrogate marker specific for cachexia. PMID:28979816
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.
Redox signaling, Nox5 and vascular remodeling in hypertension.

PubMed

Montezano, Augusto C; Tsiropoulou, Sofia; Dulak-Lis, Maria; Harvey, Adam; Camargo, Livia De Lucca; Touyz, Rhian M

2015-09-01

Extensive data indicate a role for reactive oxygen species (ROS) and redox signaling in vascular damage in hypertension. However, molecular mechanisms underlying these processes remain unclear, but oxidative post-translational modification of vascular proteins is critical. This review discusses how proteins are oxidatively modified and how redox signaling influences vascular smooth muscle cell growth and vascular remodeling in hypertension. We also highlight Nox5 as a novel vascular ROS-generating oxidase. Oxidative stress in hypertension leads to oxidative imbalance that affects vascular cell function through redox signaling. Many Nox isoforms produce ROS in the vascular wall, and recent findings show that Nox5 may be important in humans. ROS regulate signaling by numerous processes including cysteine oxidative post-translational modification such as S-nitrosylation, S-glutathionylation and sulfydration. In vascular smooth muscle cells, this influences cellular responses to oxidative stimuli promoting changes from a contractile to a proliferative phenotype. In hypertension, Nox-induced ROS production is increased, leading to perturbed redox signaling through oxidative modifications of vascular proteins. This influences mitogenic signaling and cell cycle regulation, leading to altered cell growth and vascular remodeling in hypertension.
Emerging roles of protein kinase CK2 in abscisic acid signaling.

PubMed

Vilela, Belmiro; Pagès, Montserrat; Riera, Marta

2015-01-01

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth and development as well as responses to multiple stresses. Post-translational modifications such as phosphorylation or ubiquitination have pivotal roles in the regulation of ABA signaling. In addition to the positive regulator sucrose non-fermenting-1 related protein kinase 2 (SnRK2), the relevance of the role of other protein kinases, such as CK2, has been recently highlighted. We have recently established that CK2 phosphorylates the maize ortholog of open stomata 1 OST1, ZmOST1, suggesting a role of CK2 phosphorylation in the control of ZmOST1 protein degradation (Vilela et al., 2015). CK2 is a pleiotropic enzyme involved in multiple developmental and stress-responsive pathways. This review summarizes recent advances that taken together suggest a prominent role of protein kinase CK2 in ABA signaling and related processes.
Early Detection of Cancer by Affinity Mass Spectrometry-Set Aside funds — EDRN Public Portal

Cancer.gov

A. RATIONALE The recent introduction of multiple reaction monitoring capabilities offers unprecedented capability to the research arsenal available to protein based biomarker discovery. Specific to the discovery process this technology offers an ability to monitor specific protein changes in concentration and/or post-translational modification. The ability to accurately confirm specific biomarkers in a sensitive and reproducible manner is critical to the confirmation and pre-validation process. We are proposing two collaborative studies that promise to develop Multiple Reaction Monitoring (MRM) work flows for the biomarker scientific community and specifically for EDRN. B. GOALS The overall goal for this proposal is the identification of protein biomarkers that can be associated with prostate cancer detection. The underlying goal is the application of a novel technological approach aided by MRM toward biomarker discovery. An additional goal will be the dissemination of knowledge gained from these studies EDRN wide.
Regulation of gap junction channels and hemichannels by phosphorylation and redox changes: a revision.

PubMed

Pogoda, Kristin; Kameritsch, Petra; Retamal, Mauricio A; Vega, José L

2016-05-24

Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S.
Co- and/or post-translational modifications are critical for TCH4 XET activity

NASA Technical Reports Server (NTRS)

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

1998-01-01

TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.
SIRT1: new avenues of discovery for disorders of oxidative stress.

PubMed

Chong, Zhao Zhong; Shang, Yan Chen; Wang, Shaohui; Maiese, Kenneth

2012-02-01

The sirtuin SIRT1 is expressed throughout the body, has broad biological effects and can significantly affect both cellular survival and longevity during acute and long-term injuries, which involve both oxidative stress and cell metabolism. SIRT1 has an intricate role in the pathology, progression, and treatment of several disease entities, including neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, tumorigenesis, cardiovascular disease with myocardial injury and atherosclerosis, metabolic disease, and aging-related disease. New areas of study in these disciplines, with discussion of the cellular biology, are highlighted. Novel signaling pathways for SIRT1, which can be targeted to enhance cellular protection and potentially extend lifespan, continue to emerge. Investigations that can further determine the intracellular signaling, trafficking and post-translational modifications that occur with SIRT1 in a variety of cell systems and environments will allow us to further translate this knowledge into effective therapeutic strategies that will be applicable to multiple systems of the body.
Vitamin K

USDA-ARS?s Scientific Manuscript database

Vitamin K, a fat-soluble vitamin, is an enzyme cofactor for post-translation modification of specific glutamate residues that are converted into '-carboxyglutamic acid (Gla) residues by a vitamin K-dependent (VKD) carboxylase. Seven VKD coagulation proteins are synthesized in the liver. The extra-he...
Claim to FAME

NASA Astrophysics Data System (ADS)

Mata, Alvaro

2018-05-01

Proteins are attractive material building blocks, yet their intrinsic functionality has remained largely untapped. Now, a protein-based material that exhibits controllable self-assembling behaviour has been prepared in a one-pot synthesis by simultaneous use of recombinant expression and post-translational modification.
Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease

PubMed Central

Vidal-Brime, Laia; Lynn, K. Sabrina

2018-01-01

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease. PMID:29701678

Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community

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

Li, Zhou; Wang, Yingfeng; Yao, Qiuming

2014-01-01

Detailed characterization of posttranslational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further,more » Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.« less
Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

PubMed Central

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

2014-01-01

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

PubMed

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

2016-11-01

Although the number of sequenced prokaryotic genomes is growing rapidly, experimentally verified annotation of prokaryotic genome remains patchy and challenging. To facilitate genome annotation efforts for prokaryotes, we developed an open source software called GAPP for genome annotation and global profiling of post-translational modifications (PTMs) in prokaryotes. With a single command, it provides a standard workflow to validate and refine predicted genetic models and discover diverse PTM events. We demonstrated the utility of GAPP using proteomic data from Helicobacter pylori, one of the major human pathogens that is responsible for many gastric diseases. Our results confirmed 84.9% of the existing predicted H. pylori proteins, identified 20 novel protein coding genes, and corrected four existing gene models with regard to translation initiation sites. In particular, GAPP revealed a large repertoire of PTMs using the same proteomic data and provided a rich resource that can be used to examine the functions of reversible modifications in this human pathogen. This software is a powerful tool for genome annotation and global discovery of PTMs and is applicable to any sequenced prokaryotic organism; we expect that it will become an integral part of ongoing genome annotation efforts for prokaryotes. GAPP is freely available at https://sourceforge.net/projects/gappproteogenomic/. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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
A Type III Protein Arginine Methyltransferase from the Protozoan Parasite Trypanosoma brucei*

PubMed Central

Fisk, John C.; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G.; Read, Laurie K.

2009-01-01

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle. PMID:19254949
A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.

PubMed

Fisk, John C; Sayegh, Joyce; Zurita-Lopez, Cecilia; Menon, Sarita; Presnyak, Vladimir; Clarke, Steven G; Read, Laurie K

2009-04-24

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.
A homology-based pipeline for global prediction of post-translational modification sites

NASA Astrophysics Data System (ADS)

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

2016-05-01

The pathways of protein post-translational modifications (PTMs) have been shown to play particularly important roles for almost any biological process. Identification of PTM substrates along with information on the exact sites is fundamental for fully understanding or controlling biological processes. Alternative computational strategies would help to annotate PTMs in a high-throughput manner. Traditional algorithms are suited for identifying the common organisms and tissues that have a complete PTM atlas or extensive experimental data. While annotation of rare PTMs in most organisms is a clear challenge. In this work, to this end we have developed a novel homology-based pipeline named PTMProber that allows identification of potential modification sites for most of the proteomes lacking PTMs data. Cross-promotion E-value (CPE) as stringent benchmark has been used in our pipeline to evaluate homology to known modification sites. Independent-validation tests show that PTMProber achieves over 58.8% recall with high precision by CPE benchmark. Comparisons with other machine-learning tools show that PTMProber pipeline performs better on general predictions. In addition, we developed a web-based tool to integrate this pipeline at http://bioinfo.ncu.edu.cn/PTMProber/index.aspx. In addition to pre-constructed prediction models of PTM, the website provides an extensional functionality to allow users to customize models.
The ubiquitin conjugating enzyme UbcH7, controls cell migration

USDA-ARS?s Scientific Manuscript database

Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we ...
Post-translational Modification of LipL32 during Leptospira interrogans Infection

PubMed Central

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

2014-01-01

Background Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. Methodology/Principal Findings Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. Conclusions/Significance The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira. PMID:25356675
The i6A37 tRNA modification is essential for proper decoding of UUX-Leucine codons during rpoS and iraP translation

PubMed Central

Aubee, Joseph I.; Olu, Morenike

2016-01-01

The translation of rpoS (σS), the general stress/stationary phase sigma factor, is tightly regulated at the post-transcriptional level by several factors via mechanisms that are not clearly defined. One of these factors is MiaA, the enzyme necessary for the first step in the N6-isopentyl-2-thiomethyladenosinemethyladenosine 37 (ms2i6A37) tRNA modification. We tested the hypothesis that an elevated UUX-Leucine/total leucine codon ratio can be used to identify transcripts whose translation would be sensitive to loss of the MiaA-dependent modification. We identified iraP as another candidate MiaA-sensitive gene, based on the UUX-Leucine/total leucine codon ratio. An iraP-lacZ fusion was significantly decreased in the absence of MiaA, consistent with our predictive model. To determine the role of MiaA in UUX-Leucine decoding in rpoS and iraP, we measured β-galactosidase-specific activity of miaA− rpoS and iraP translational fusions upon overexpression of leucine tRNAs. We observed suppression of the MiaA effect on rpoS, and not iraP, via overexpression of tRNALeuX but not tRNALeuZ. We also tested the hypothesis that the MiaA requirement for rpoS and iraP translation is due to decoding of UUX-Leucine codons within the rpoS and iraP transcripts, respectively. We observed a partial suppression of the MiaA requirement for rpoS and iraP translational fusions containing one or both UUX-Leucine codons removed. Taken together, this suggests that MiaA is necessary for rpoS and iraP translation through proper decoding of UUX-Leucine codons and that rpoS and iraP mRNAs are both modification tunable transcripts (MoTTs) via the presence of the modification. PMID:26979278
Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

PubMed

Ghazi, Terisha; Nagiah, Savania; Tiloke, Charlette; Sheik Abdul, Naeem; Chuturgoon, Anil A

2017-11-01

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG 2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG 2 cells with FA (IC 50 : 580.32 μM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG 2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG 2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG 2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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.
The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.
UnPAKing RUNX3 functions-Both sides of the coin.

PubMed

Kumar, Arun; Sundaram, Sandhya; Rayala, Suresh K; Venkatraman, Ganesh

2017-06-19

Post translational modifications of RUNX3 have been shown to play an important role in directing RUNX3 functions. In this review we highlight the phosphorylation dependent functions of RUNX3 as regulated by PAK1 and its implications on tumorigenesis.
Post-translational quantitation by SRM/MRM: applications in cardiology.

PubMed

Gianazza, Erica; Banfi, Cristina

2018-06-04

Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization and interaction with other molecules. PTMs apply a dynamic control of proteins both in physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution MS/MS analysis provide attractive results on PTMs characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples. Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported and a specific section is focused on PTMs detection in proteins that are involved in cardiovascular system and heart diseases. Expert commentary: PTMs characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded the knowledge in the last years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTMs complexity. Promising studies confirm the great potentiality of SRM/MRM to study PTMs in cardiovascular field and PTMomics could be very useful in a clinical perspective.
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.
Induction of the neural crest state: Control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions

PubMed Central

Prasad, Maneeshi S.; Sauka-Spengler, Tatjana; LaBonne, Carole

2012-01-01

Neural crest cells are a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates, migrate extensively, and give rise to diverse derivatives such as melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The neural crest gene regulatory network (NC-GRN) includes a number of key factors that are used reiteratively to control multiple steps in the development of neural crest cells, including the acquisition of stem cell attributes. It is therefore essential to understand the mechanisms that control the distinct functions of such reiteratively used factors in different cellular contexts. The context-dependent control of neural crest specification is achieved through combinatorial interaction with other factors, post-transcriptional and post-translational modifications, and the epigenetic status and chromatin state of target genes. Here we review the current understanding of the NC-GRN, including the role of the neural crest specifiers, their links to the control of “stemness,” and their dynamic context-dependent regulation during the formation of neural crest progenitors. PMID:22583479
Biomolecular engineering of intracellular switches in eukaryotes

PubMed Central

Pastuszka, M.K.; Mackay, J.A.

2010-01-01

Tools to selectively and reversibly control gene expression are useful to study and model cellular functions. When optimized, these cellular switches can turn a protein's function “on” and “off” based on cues designated by the researcher. These cues include small molecules, drugs, hormones, and even temperature variations. Here we review three distinct areas in gene expression that are commonly targeted when designing cellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, with examples including the tetracycline gene induction system as well as nuclear receptors. Translational switches target the process of turning the mRNA signal into protein, with examples including riboswitches and RNA interference. Post-translational switches control how proteins interact with one another to attenuate or relay signals. Examples of post-translational modification include dimerization and intein splicing. In general, the delay times between switch and effect decreases from transcription to translation to post-translation; furthermore, the fastest switches may offer the most elegant opportunities to influence and study cell behavior. We discuss the pros and cons of these strategies, which directly influence their usefulness to study and implement drug targeting at the tissue and cellular level. PMID:21209849
Molecular dynamics simulation of phosphorylated KID post-translational modification.

PubMed

Chen, Hai-Feng

2009-08-05

Kinase-inducible domain (KID) as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX). NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood. To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD) for both bound and apo phosphorylated KID (pKID). Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding. Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B), folding of helix alpha(A), completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A) and alpha(B) are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.
Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

PubMed

Böhm, Ernst; Seyfried, Birgit K; Dockal, Michael; Graninger, Michael; Hasslacher, Meinhard; Neurath, Marianne; Konetschny, Christian; Matthiessen, Peter; Mitterer, Artur; Scheiflinger, Friedrich

2015-09-18

BACKGROUND & Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins.

PubMed

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-08-18

Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20-85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications.
A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins

PubMed Central

Burbelo, Peter D; Goldman, Radoslav; Mattson, Thomas L

2005-01-01

Background Assays detecting human antigen-specific antibodies are medically useful. However, the usefulness of existing simple immunoassay formats is limited by technical considerations such as sera antibodies to contaminants in insufficiently pure antigen, a problem likely exacerbated when antigen panels are screened to obtain clinically useful data. Results We developed a novel and simple immunoprecipitation technology for identifying clinical sera containing antigen-specific antibodies and for generating quantitative antibody response profiles. This method is based on fusing protein antigens to an enzyme reporter, Renilla luciferase (Ruc), and expressing these fusions in mammalian cells, where mammalian-specific post-translational modifications can be added. After mixing crude extracts, sera and protein A/G beads together and incubating, during which the Ruc-antigen fusion become immobilized on the A/G beads, antigen-specific antibody is quantitated by washing the beads and adding coelenterazine substrate and measuring light production. We have characterized this technology with sera from patients having three different types of cancers. We show that 20–85% of these sera contain significant titers of antibodies against at least one of five frequently mutated and/or overexpressed tumor-associated proteins. Five of six colon cancer sera tested gave responses that were statistically significantly greater than the average plus three standard deviations of 10 control sera. The results of competition experiments, preincubating positive sera with unmodified E. coli-produced antigens, varied dramatically. Conclusion This technology has several advantages over current quantitative immunoassays including its relative simplicity, its avoidance of problems associated with E. coli-produced antigens and its use of antigens that can carry mammalian or disease-specific post-translational modifications. This assay should be generally useful for analyzing sera for antibodies recognizing any protein or its post-translational modifications. PMID:16109166
Molecular Characterization of Tick Salivary Gland Glutaminyl Cyclase

PubMed Central

Adamson, Steven W.; Browning, Rebecca E.; Chao, Chien-Chung; Bateman, Robert C.; Ching, Wei-Mei; Karim, Shahid

2013-01-01

Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood-feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc-binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the blood-meal of adult female ticks prior to fast feeding phases in both I. scapularis and A. maculatum suggesting a functional link with blood meal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC-transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496
Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

PubMed Central

Schultz, A M; Rabin, E H; Oroszlan, S

1979-01-01

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag. Images PMID:480454
Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

PubMed

Stinemetz, Emily K; Gao, Peng; Pinkston, Kenneth L; Montealegre, Maria Camila; Murray, Barbara E; Harvey, Barrett R

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.
Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation

PubMed Central

Pinkston, Kenneth L.; Montealegre, Maria Camila; Murray, Barbara E.

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation. PMID:29049345
Crosstalk between phosphorylation and O-GlcNAcylation: friend or foe.

PubMed

van der Laarse, Saar A M; Leney, Aneika C; Heck, Albert J R

2018-05-02

A wide variety of protein post-translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co-occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O-GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O-GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O-GlcNAc transferase), on the erasers (i.e., phosphatases and O-GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O-GlcNAcylation crosstalk. © 2018 Federation of European Biochemical Societies.
Genomic and Epigenomic Alterations in Cancer.

PubMed

Chakravarthi, Balabhadrapatruni V S K; Nepal, Saroj; Varambally, Sooryanarayana

2016-07-01

Multiple genetic and epigenetic events characterize tumor progression and define the identity of the tumors. Advances in high-throughput technologies, like gene expression profiling, next-generation sequencing, proteomics, and metabolomics, have enabled detailed molecular characterization of various tumors. The integration and analyses of these high-throughput data have unraveled many novel molecular aberrations and network alterations in tumors. These molecular alterations include multiple cancer-driving mutations, gene fusions, amplification, deletion, and post-translational modifications, among others. Many of these genomic events are being used in cancer diagnosis, whereas others are therapeutically targeted with small-molecule inhibitors. Multiple genes/enzymes that play a role in DNA and histone modifications are also altered in various cancers, changing the epigenomic landscape during cancer initiation and progression. Apart from protein-coding genes, studies are uncovering the critical regulatory roles played by noncoding RNAs and noncoding regions of the genome during cancer progression. Many of these genomic and epigenetic events function in tandem to drive tumor development and metastasis. Concurrent advances in genome-modulating technologies, like gene silencing and genome editing, are providing ability to understand in detail the process of cancer initiation, progression, and signaling as well as opening up avenues for therapeutic targeting. In this review, we discuss some of the recent advances in cancer genomic and epigenomic research. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
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.
Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability

PubMed Central

Rossin, A; Durivault, J; Chakhtoura-Feghali, T; Lounnas, N; Gagnoux-Palacios, L; Hueber, A-O

2015-01-01

The death receptor Fas undergoes a variety of post-translational modifications including S-palmitoylation. This protein acylation has been reported essential for an optimal cell death signaling by allowing both a proper Fas localization in cholesterol and sphingolipid-enriched membrane nanodomains, as well as Fas high-molecular weight complexes. In human, S-palmitoylation is controlled by 23 members of the DHHC family through their palmitoyl acyltransferase activity. In order to better understand the role of this post-translational modification in the regulation of the Fas-mediated apoptosis pathway, we performed a screen that allowed the identification of DHHC7 as a Fas-palmitoylating enzyme. Indeed, modifying DHHC7 expression by specific silencing or overexpression, respectively, reduces or enhances Fas palmitoylation and DHHC7 co-immunoprecipitates with Fas. At a functional level, DHHC7-mediated palmitoylation of Fas allows a proper Fas expression level by preventing its degradation through the lysosomes. Indeed, the decrease of Fas expression obtained upon loss of Fas palmitoylation can be restored by inhibiting the lysosomal degradation pathway. We describe the modification of Fas by palmitoylation as a novel mechanism for the regulation of Fas expression through its ability to circumvent its degradation by lysosomal proteolysis. PMID:25301068
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
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
Dietary fat and fiber interact to uniquely modify global histone post-translational epigenetic programming in a rat colon cancer progression model.

PubMed

Triff, Karen; McLean, Mathew W; Callaway, Evelyn; Goldsby, Jennifer; Ivanov, Ivan; Chapkin, Robert S

2018-04-16

Dietary fermentable fiber generates short-chain fatty acids (SCFA), e.g., butyrate, in the colonic lumen which serves as a chemoprotective histone deacetylase inhibitor and/or as an acetylation substrate for histone acetylases. In addition, n-3 polyunsaturated fatty acids (n-3 PUFA) in fish oil can affect the chromatin landscape by acting as ligands for tumor suppressive nuclear receptors. In an effort to gain insight into the global dimension of post-translational modification of histones (including H3K4me3 and H3K9ac) and clarify the chemoprotective impact of dietary bioactive compounds on transcriptional control in a preclinical model of colon cancer, we generated high-resolution genome-wide RNA (RNA-Seq) and "chromatin-state" (H3K4me3-seq and H3K9ac-seq) maps for intestinal (epithelial colonocytes) crypts in rats treated with a colon carcinogen and fed diets containing bioactive (i) fish oil, (ii) fermentable fiber (a rich source of SCFA), (iii) a combination of fish oil plus pectin or (iv) control, devoid of fish oil or pectin. In general, poor correlation was observed between differentially transcribed (DE) and enriched genes (DERs) at multiple epigenetic levels. The combinatorial diet (fish oil + pectin) uniquely affected transcriptional profiles in the intestinal epithelium, e.g., upregulating lipid catabolism and beta-oxidation associated genes. These genes were linked to activated ligand-dependent nuclear receptors associated with n-3 PUFA and were also correlated with the mitochondrial L-carnitine shuttle and the inhibition of lipogenesis. These findings demonstrate that the chemoprotective fish oil + pectin combination diet uniquely induces global histone state modifications linked to the expression of chemoprotective genes. This article is protected by copyright. All rights reserved. © 2018 UICC.
Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias.

PubMed

Zhang, Xi

2016-12-01

Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

PubMed

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

2015-08-19

Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites in all our example data sets by more than double when compared to those recovered using existing resources such as PhosphoSitePlus. PhosphOrtholog is the first tool that enables mapping of thousands of novel and known protein phosphorylation sites across species, accessible through an easy-to-use web interface. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of functional PTM sites. Moreover, PhosphOrtholog is generic being applicable to other PTM datasets such as acetylation, ubiquitination and methylation.
Vitamin K dependent protein activity and incident ischemic cardiovascular disease: The multi ethnic study of atherosclerosis

USDA-ARS?s Scientific Manuscript database

OBJECTIVE: Vitamin K-dependent proteins (VKDPs), which require post-translational modification to achieve biological activity, seem to contribute to thrombus formation, vascular calcification, and vessel stiffness. Whether VKDP activity is prospectively associated with incident cardiovascular diseas...
Post-translational modification of LipL32 during Leptospira interrogans infection

USDA-ARS?s Scientific Manuscript database

Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world’s most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize ...
Synthetic and semi-synthetic strategies to study ubiquitin signaling.

PubMed

van Tilburg, Gabriëlle Ba; Elhebieshy, Angela F; Ovaa, Huib

2016-06-01

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself. Copyright © 2016 Elsevier Ltd. All rights reserved.
Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

PubMed

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

2016-04-01

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

DOE PAGES

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

2013-01-01

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

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

PubMed Central

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

2016-01-01

Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance. PMID:26909095
Oligomerization of the protein tau in the Alzheimer's disease

NASA Astrophysics Data System (ADS)

Larini, Luca

The Alzheimer's disease is characterized by the formation of protein aggregates both within and outside of the brain's cells, the neurons. Within the neurons, the aggregation of the microtubule associated protein tau leads to the destruction of the microtubules in the axon of the neuron. Tau is extremely flexible and is classified as an intrinsically disordered protein due to its low propensity to form secondary structure. Tau promotes tubulin assembly into microtubules, which are an essential component of the cytoskeleton of the axon. The microtubule binding region of tau consists of 4 pseudo-repeats that are critical for aggregation as well. In this study, we focus on the aggregation propensity of different segments of the microtubule binding region as well as post-translational modifications that can alter tau dynamics and structure. We have performed replica exchange molecular dynamics simulations to characterize the ensemble of conformations of the monomer and small oligomers as well as how these structures are stabilized or destabilized by mutations and post-translational modifications.
Insight in the multilevel regulation of NER

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

Dijk, Madelon; Typas, Dimitris; Mullenders, Leon, E-mail: l.mullenders@lumc.nl

2014-11-15

Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of themore » core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.« less
Implication of SUMO E3 ligases in nucleotide excision repair.

PubMed

Tsuge, Maasa; Kaneoka, Hidenori; Masuda, Yusuke; Ito, Hiroki; Miyake, Katsuhide; Iijima, Shinji

2015-08-01

Post-translational modifications alter protein function to mediate complex hierarchical regulatory processes that are crucial to eukaryotic cellular function. The small ubiquitin-like modifier (SUMO) is an important post-translational modification that affects transcriptional regulation, nuclear localization, and the maintenance of genome stability. Nucleotide excision repair (NER) is a very versatile DNA repair system that is essential for protection against ultraviolet (UV) irradiation. The deficiencies in NER function remarkably increase the risk of skin cancer. Recent studies have shown that several NER factors are SUMOylated, which influences repair efficiency. However, how SUMOylation modulates NER has not yet been elucidated. In the present study, we performed RNAi knockdown of SUMO E3 ligases and found that, in addition to PIASy, the polycomb protein Pc2 affected the repair of cyclobutane pyrimidine dimers. PIAS1 affected both the removal of 6-4 pyrimidine pyrimidone photoproducts and cyclobutane pyrimidine dimers, whereas other SUMO E3 ligases did not affect the removal of either UV lesion.
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.
Protein biomarker validation via proximity ligation assays.

PubMed

Blokzijl, A; Nong, R; Darmanis, S; Hertz, E; Landegren, U; Kamali-Moghaddam, M

2014-05-01

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. © 2013.
Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3

PubMed Central

Tang, Liu-Ya; Yamashita, Motozo; Coussens, Nathan P; Tang, Yi; Wang, Xiangchun; Li, Cuiling; Deng, Chu-Xia; Cheng, Steven Y; Zhang, Ying E

2011-01-01

TGF-β signalling is regulated by post-translational modifications of Smad proteins to translate quantitative difference in ligand concentration into proportional transcriptional output. Previous studies in cell culture systems suggested that Smad ubiquitination regulatory factors (Smurfs) act in this regulation by targeting Smads for proteasomal degradation, but whether this mechanism operates under physiological conditions is not clear. Here, we generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, Smad-dependent transcriptional responses are elevated in the absence of Smurf2. Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data support a model in which Smurf2 negatively regulates TGF-β signalling by attenuating the activity of Smad3 rather than promoting its degradation. PMID:22045334
Impairment of Human Immunodeficiency Virus Type-1 Integrase SUMOylation Correlates with an Early Replication Defect*

PubMed Central

Zamborlini, Alessia; Coiffic, Audrey; Beauclair, Guillaume; Delelis, Olivier; Paris, Joris; Koh, Yashuiro; Magne, Fabian; Giron, Marie-Lou; Tobaly-Tapiero, Joelle; Deprez, Eric; Emiliani, Stephane; Engelman, Alan; de Thé, Hugues; Saïb, Ali

2011-01-01

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. PMID:21454548
Bacillus cereus Certhrax ADP-ribosylates Vinculin to Disrupt Focal Adhesion Complexes and Cell Adhesion*

PubMed Central

Simon, Nathan C.; Barbieri, Joseph T.

2014-01-01

Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton. PMID:24573681
Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids

DOE PAGES

Zhang, Xuebin; Liu, Chang-Jun

2014-12-11

Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway; it diverts the central flux of carbon from primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels control the transcription and the enzymatic activity of PALs. In this review, we present a current overview on our understanding of the complicated regulatory mechanisms governing PAL's activity; we particularlymore » highlight recent progresses in unraveling its post-translational modifications, its metabolite feedback regulation, and its enzyme organization.« less
Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoids

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

Zhang, Xuebin; Liu, Chang-Jun

Phenylpropanoid biosynthesis in plants engenders a vast variety of aromatic metabolites critically important for their growth, development, and environmental adaptation. Some of these aromatic compounds have high economic value. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway; it diverts the central flux of carbon from primary metabolism to the synthesis of myriad phenolics. Over the decades, many studies have shown that exquisite regulatory mechanisms at multiple levels control the transcription and the enzymatic activity of PALs. In this review, we present a current overview on our understanding of the complicated regulatory mechanisms governing PAL's activity; we particularlymore » highlight recent progresses in unraveling its post-translational modifications, its metabolite feedback regulation, and its enzyme organization.« less
Cortactin Branches Out: Roles in Regulating Protrusive Actin Dynamics

PubMed Central

Ammer, Amanda Gatesman; Weed, Scott A.

2008-01-01

Since its discovery in the early 1990’s, cortactin has emerged as a key signaling protein in many cellular processes, including cell adhesion, migration, endocytosis, and tumor invasion. While the list of cellular functions influenced by cortactin grows, the ability of cortactin to interact with and alter the cortical actin network is central to its role in regulating these processes. Recently, several advances have been made in our understanding of the interaction between actin and cortactin, providing insight into how these two proteins work together to provide a framework for normal and altered cellular function. This review examines how regulation of cortactin through post-translational modifications and interactions with multiple binding partners elicits changes in cortical actin cytoskeletal organization, impacting the regulation and formation of actin-rich motility structures. PMID:18615630
Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

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

PubMed

Tarasova, Irina A; Chumakov, Peter M; Moshkovskii, Sergei A; Gorshkov, Mikhail V

2018-05-17

Peptide mass shifts were profiled using ultra-tolerant database search strategy for shotgun proteomics data sets of human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The main objective of this profiling was revealing the cell response to IFN treatment at the level of protein modifications. To achieve this objective, statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples were analyzed. Detailed analysis of MS/MS spectra allowed further interpretation of the observed mass shifts and differentiation between post-translational and artifact modifications. Malignant cells typically acquire increased sensitivity to viruses due to the deregulated antiviral mechanisms. Therefore, a viral therapy is considered as one of the promising approaches to treat cancer. However, recent studies have demonstrated that malignant cells can preserve intact antiviral mechanisms, e.g. interferon signaling, and develop resistance to virus infection in response to interferon treatment. Post translational modifications, e.g. tyrosine phosphorylation, are the interferon signaling drivers. Thus, comprehensive characterization of modifications is crucially important, yet, most challenging problem in cancer proteomics. Here, we report on the application of the recently introduced ultra-tolerant search strategy for profiling peptide modifications in the human glioblastoma cell lines demonstrating strong response to the type I interferon (IFNα-2b) treatment. The specific aim of the study was identification of statistically significant changes in peptide mass shift profiles between IFN treated and untreated glioblastoma samples, as well as determination of whether these shifts represent the biologically relevant modification. Copyright © 2018 Elsevier B.V. All rights reserved.
Histones and their modifications in ovarian cancer - drivers of disease and therapeutic targets.

PubMed

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

2014-01-01

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

ERIC Educational Resources Information Center

Vernon, Jeffrey; Irvine, Elaine E.; Peters, Marco; Jeyabalan, Jeshmi; Giese, K. Peter

2016-01-01

Phosphorylation is a ubiquitous post-translational modification of proteins, and a known physiological regulator of K[superscript +] channel function. Phosphorylation of K[superscript +] channels by kinases has long been presumed to regulate neuronal processing and behavior. Although circumstantial evidence has accumulated from behavioral studies…
Biosynthesis of the Polycyclic Antimicrobial Peptides Lacticin 481, Haloduracin, and Cinnamycin

ERIC Educational Resources Information Center

Cooper, Lisa E.

2009-01-01

Lantibiotics are bacterial-derived polycyclic antimicrobial peptides. They are genetically encoded and ribosomally synthesized as precursor peptides containing a structural region that undergoes post-translational modification and a leader sequence that is not modified. Specific serine and threonine residues in the pre-lantibiotic structural…
Global regulation of post-translational modifications on core histones.

PubMed

Galasinski, Scott C; Louie, Donna F; Gloor, Kristen K; Resing, Katheryn A; Ahn, Natalie G

2002-01-25

Full-length masses of histones were analyzed by mass spectrometry to characterize post-translational modifications of bulk histones and their changes induced by cell stimulation. By matching masses of unique peptides with full-length masses, H4 and the variants H2A.1, H2B.1, and H3.1 were identified as the main histone forms in K562 cells. Mass changes caused by covalent modifications were measured in a dose- and time-dependent manner following inhibition of phosphatases by okadaic acid. Histones H2A, H3, and H4 underwent changes in mass consistent with altered acetylation and phosphorylation, whereas H2B mass was largely unchanged. Unexpectedly, histone H4 became almost completely deacetylated in a dose-dependent manner that occurred independently of phosphorylation. Okadaic acid also partially blocked H4 hyperacetylation induced by trichostatin-A, suggesting that the mechanism of deacetylation involves inhibition of H4 acetyltransferase activity, following perturbation of cellular phosphatases. In addition, mass changes in H3 in response to okadaic acid were consistent with phosphorylation of methylated, acetylated, and phosphorylated forms. Finally, kinetic differences were observed with respect to the rate of phosphorylation of H2A versus H4, suggesting differential regulation of phosphorylation at sites on these proteins, which are highly related by sequence. These results provide novel evidence that global covalent modifications of chromatin-bound histones are regulated through phosphorylation-dependent mechanisms.
REDOX REGULATION OF SIRT1 IN INFLAMMATION AND CELLULAR SENESCENCE

PubMed Central

Hwang, Jae-woong; Yao, Hongwei; Caito, Samuel; Sundar, Isaac K.; Rahman, Irfan

2013-01-01

Sirtuin1 (SIRT1) regulates inflammation, aging (lifespan and healthspan), calorie restriction/energetics, mitochondrial biogenesis, stress resistance, cellular senescence, endothelial functions, apoptosis/autophagy, and circadian rhythms through deacetylation of transcription factors and histones. SIRT1 level and activity are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. SIRT1 is regulated by a NAD+-dependent DNA repair enzyme poly(ADP-ribose)-polymerase-1 (PARP-1), and subsequent NAD+ depletion by oxidative stresses may have consequent effects on inflammatory and stress responses as well as cellular senescence. SIRT1 has been shown to undergo covalent oxidative modifications by cigarette smoke-derived oxidants/aldehydes, leading to post-translational modifications, inactivation, and protein degradation. Furthermore, oxidant/carbonyl stress-mediated reduction of SIRT1 leads to the loss of its control on acetylation of target proteins including p53, RelA/p65 and FOXO3, thereby enhancing the inflammatory, pro-senescent and apoptotic responses, as well as endothelial dysfunction. In this review, the mechanisms of cigarette smoke/oxidant-mediated redox post-translational modifications of SIRT1 and its role in PARP1, NF-κB activation, FOXO3 and eNOS regulation, as well as chromatin remodeling/histone modifications during inflammaging are discussed. Furthermore, we also discussed various novel ways to activate SIRT1 either directly or indirectly, which may have therapeutic potential in attenuating inflammation and premature senescence involved in chronic lung diseases. PMID:23542362

Interleukin-33, friend and foe in type-2 immune responses.

PubMed

Hardman, Clare; Ogg, Graham

2016-10-01

IL-33 is the most recent addition to the IL-1 cytokine family, identified in 2005 as the ligand of T1/ST2 and inducer of type-2 immune responses. IL-33 has been implicated in a wide range of disease settings, in anti-inflammatory responses and homeostasis, and thus signalling must be strictly regulated. Altered gene expression, post-translational modification, decoy receptor, and receptor signalling are all modulatory mechanisms used to control the IL-33 pathway. Understanding both the genetic and post-translational factors influencing IL-33 activity will be critical for provision of safe effective treatment of type-2 disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.
PML nuclear bodies: from architecture to function.

PubMed

Lallemand-Breitenbach, Valérie; de Thé, Hugues

2018-06-01

PML nuclear bodies are nucleated by the PML protein, which polymerizes into spherical shells where it concentrates many unrelated partner proteins. Emerging data has connected PML bodies to post-translational control, notably conjugation by SUMOs. High concentrations of SUMO-bound proteins were proposed to condense into liquid-like droplets and such phase transition may occur within NBs. Many stress pathways modulate NB formation and recent findings have directly implicated PML in oxidative stress response in vivo. PML may also undergo SUMO-dependent ubiquitination/degradation. We highlight recent advances linking PML to partner degradation and other adaptative post-translational modifications in the context of chromatin remodeling, telomere biology, senescence or viral infections. Copyright © 2018. Published by Elsevier Ltd.
Principles and methodology for translation and cross-cultural adaptation of the Nordic Occupational Skin Questionnaire (NOSQ-2002) to Spanish and Catalan.

PubMed

Sala-Sastre, Nohemi; Herdman, Mike; Navarro, Lidia; de la Prada, Miriam; Pujol, Ramón M; Serra, Consol; Alonso, Jordi; Flyvholm, Mari-Ann; Giménez-Arnau, Ana M

2009-08-01

Occupational skin diseases are among the most frequent work-related diseases in industrialized countries. The Nordic Occupational Skin Questionnaire (NOSQ-2002), developed in English, is a useful tool for screening of occupational skin diseases. To culturally adapt the NOSQ-2002 to Spanish and Catalan and to assess the clarity, comprehension, cultural relevance and appropriateness of the translated versions. The International Society for Pharmacoeconomics and Outcomes Research (ISPOR) principles of good practice for the translation and cultural adaptation of patient-reported outcomes were followed. After translation into the target language, a first consensus version of the questionnaire was evaluated in multiple cognitive debriefing interviews. The expert panel introduced some modifications in 39 (68%) and 27 (47%) items in the Spanish and Catalan version, respectively (e.g. addition of examples and definitions, reformulation of instructions and use of direct question format). This version was back translated and submitted to the original authors, who suggested a further seven and two modifications in the Spanish and Catalan versions, respectively. A second set of cognitive interviews were performed. A consensus version of both questionnaires was obtained after final modifications based on comments by the patients. The final versions of the Spanish and Catalan NOSQ-2002 questionnaires are now available at www.NRCWE.dk/NOSQ.
Dynamic m(6)A mRNA methylation directs translational control of heat shock response.

PubMed

Zhou, Jun; Wan, Ji; Gao, Xiangwei; Zhang, Xingqian; Jaffrey, Samie R; Qian, Shu-Bing

2015-10-22

The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.
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.
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
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
A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs

PubMed Central

Taoka, Masato; Nobe, Yuko; Hori, Masayuki; Takeuchi, Aiko; Masaki, Shunpei; Yamauchi, Yoshio; Nakayama, Hiroshi; Takahashi, Nobuhiro; Isobe, Toshiaki

2015-01-01

We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA. PMID:26013808
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
IA channels: diverse regulatory mechanisms.

PubMed

Carrasquillo, Yarimar; Nerbonne, Jeanne M

2014-04-01

In many peripheral and central neurons, A-type K(+) currents, IA, have been identified and shown to be key determinants in shaping action potential waveforms and repetitive firing properties, as well as in the regulation of synaptic transmission and synaptic plasticity. The functional properties and physiological roles of native neuronal IA, however, have been shown to be quite diverse in different types of neurons. Accumulating evidence suggests that this functional diversity is generated by multiple mechanisms, including the expression and subcellular distributions of IA channels encoded by different voltage-gated K(+) (Kv) channel pore-forming (α) subunits, interactions of Kv α subunits with cytosolic and/or transmembrane accessory subunits and regulatory proteins and post-translational modifications of channel subunits. Several recent reports further suggest that local protein translation in the dendrites of neurons and interactions between IA channels with other types of voltage-gated ion channels further expands the functional diversity of native neuronal IA channels. Here, we review the diverse molecular mechanisms that have been shown or proposed to underlie the functional diversity of native neuronal IA channels.
Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

USDA-ARS?s Scientific Manuscript database

Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...
Protein Glycosylation in Archaea: A Post-Translational Modification to Enhance Extremophilic Protein Stability

DTIC Science & Technology

2010-01-15

Analysis of the chemical composition of the Asn-linked polysaccharides decorating many archaeal proteins has revealed the use of a wider variety of sugar...reminiscent of the eukaryal glycan-charged lipid, linked to a variety of monosaccharides , including glucose, mannose, and N-acetylglucosamine (GlcNAc
Drug development and manufacturing

DOEpatents

Warner, Benjamin P.; McCleskey, T. Mark; Burrell, Anthony K.

2015-10-13

X-ray fluorescence (XRF) spectrometry has been used for detecting binding events and measuring binding selectivities between chemicals and receptors. XRF may also be used for estimating the therapeutic index of a chemical, for estimating the binding selectivity of a chemical versus chemical analogs, for measuring post-translational modifications of proteins, and for drug manufacturing.
Pathogenic leptospires modulate protein expression and post-translational modifications in response to mammalian host signals

USDA-ARS?s Scientific Manuscript database

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs and cattle, exhibit little to no signs of disease but shed large numbers of organisms in...
Understanding and Targeting Epigenetic Alterations in Acquired Bone Marrow Failure

DTIC Science & Technology

2016-07-01

should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation. REPORT...modifiers may specifically impact DNA methylation and/or histone post -translational modifications in a manner that is therapeutically targetable, and (c) if...2016 Tisch Cancer Institute Seminar Series, Icahn School of Medicine at Mount Sinai, New York, NY 2016 11th CML & MPN Post -ASH Workshop, La Jolla
S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

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

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

Hu, Hao; Gu, Xi; Xue, Liang-Jiao

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less
Tubulin C-terminal Post-translational Modifications Do Not Occur in Wood Forming Tissue of Populus

DOE PAGES

Hu, Hao; Gu, Xi; Xue, Liang-Jiao; ...

2016-10-13

Cortical microtubules (MTs) are evolutionarily conserved cytoskeletal components with specialized roles in plants, including regulation of cell wall biogenesis. MT functions and dynamics are dictated by the composition of their monomeric subunits, α- (TUA) and β-tubulins (TUB), which in animals and protists are subject to both transcriptional regulation and post-translational modifications (PTM). While spatiotemporal regulation of tubulin gene expression has been reported in plants, whether and to what extent tubulin PTMs occur in these species remain poorly understood. We chose the woody perennial Populus for investigation of tubulin PTMs in this study, with a particular focus on developing xylem wheremore » high tubulin transcript levels support MT-dependent secondary cell wall deposition. Mass spectrometry and immunodetection concurred that detyrosination, non-tyrosination and glutamylation were essentially absent in tubulins isolated from wood-forming tissues of P. deltoides and P. tremula ×alba. Label-free quantification of tubulin isotypes and RNA-Seq estimation of tubulin transcript abundance were largely consistent with transcriptional regulation. However, two TUB isotypes were detected at noticeably lower levels than expected based on RNA-Seq transcript abundance in both Populus species. These findings led us to conclude that MT composition during wood formation depends exclusively on transcriptional and, to a lesser extent, translational regulation of tubulin isotypes.« less
The GAGA protein of Drosophila is phosphorylated by CK2.

PubMed

Bonet, Carles; Fernández, Irene; Aran, Xavier; Bernués, Jordi; Giralt, Ernest; Azorín, Fernando

2005-08-19

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.

Commercial processed soy-based food product contains glycated and glycoxidated lunasin proteoforms.

PubMed

Serra, Aida; Gallart-Palau, Xavier; See-Toh, Rachel Su-En; Hemu, Xinya; Tam, James P; Sze, Siu Kwan

2016-05-18

Nutraceuticals have been proposed to exert positive effects on human health and confer protection against many chronic diseases. A major bioactive component of soy-based foods is lunasin peptide, which has potential to exert a major impact on the health of human consumers worldwide, but the biochemical features of dietary lunasin still remain poorly characterized. In this study, lunasin was purified from a soy-based food product via strong anion exchange solid phase extraction and then subjected to top-down mass spectrometry analysis that revealed in detail the molecular diversity of lunasin in processed soybean foods. We detected multiple glycated proteoforms together with potentially toxic advanced glycation end products (AGEs) derived from lunasin. In both cases, modification sites were Lys24 and Lys29 located at the helical region that shows structural homology with a conserved region of chromatin-binding proteins. The identified post-translational modifications may have an important repercussion on lunasin epigenetic regulatory capacity. Taking together, our results demonstrate the importance of proper chemical characterization of commercial processed food products to assess their impact on consumer's health and risk of chronic diseases.
A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

PubMed

Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

2018-02-01

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. 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.
Regulation of mRNA Trafficking by Nuclear Pore Complexes

PubMed Central

Bonnet, Amandine; Palancade, Benoit

2014-01-01

Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs). mRNPs are then exported through nuclear pore complexes (NPCs), which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed. PMID:25184662
Disulfide bonds in ER protein folding and homeostasis

PubMed Central

Feige, Matthias J.; Hendershot, Linda M.

2010-01-01

Proteins that are expressed outside the cell must be synthesized, folded and assembled in a way that ensures they can function in their designate location. Accordingly these proteins are primarily synthesized in the endoplasmic reticulum (ER), which has developed a chemical environment more similar to that outside the cell. This organelle is equipped with a variety of molecular chaperones and folding enzymes that both assist the folding process, while at the same time exerting tight quality control measures that are largely absent outside the cell. A major post-translational modification of ER-synthesized proteins is disulfide bridge formation, which is catalyzed by the family of protein disulfide isomerases. As this covalent modification provides unique structural advantages to extracellular proteins, multiple pathways to their formation have evolved. However, the advantages that disulfide bonds impart to these proteins come at a high cost to the cell. Very recent reports have shed light on how the cell can deal with or even exploit the side reactions of disulfide bond formation to maintain homeostasis of the ER and its folding machinery. PMID:21144725
Commercial processed soy-based food product contains glycated and glycoxidated lunasin proteoforms

PubMed Central

Serra, Aida; Gallart-Palau, Xavier; See-Toh, Rachel Su-En; Hemu, Xinya; Tam, James P.; Sze, Siu Kwan

2016-01-01

Nutraceuticals have been proposed to exert positive effects on human health and confer protection against many chronic diseases. A major bioactive component of soy-based foods is lunasin peptide, which has potential to exert a major impact on the health of human consumers worldwide, but the biochemical features of dietary lunasin still remain poorly characterized. In this study, lunasin was purified from a soy-based food product via strong anion exchange solid phase extraction and then subjected to top-down mass spectrometry analysis that revealed in detail the molecular diversity of lunasin in processed soybean foods. We detected multiple glycated proteoforms together with potentially toxic advanced glycation end products (AGEs) derived from lunasin. In both cases, modification sites were Lys24 and Lys29 located at the helical region that shows structural homology with a conserved region of chromatin-binding proteins. The identified post-translational modifications may have an important repercussion on lunasin epigenetic regulatory capacity. Taking together, our results demonstrate the importance of proper chemical characterization of commercial processed food products to assess their impact on consumer’s health and risk of chronic diseases. PMID:27189269
Post-translational modifications of transthyretin affect the triiodonine-binding potential

PubMed Central

Henze, Andrea; Homann, Thomas; Serteser, Mustafa; Can, Ozge; Sezgin, Ozlem; Coskun, Abdurrahman; Unsal, Ibrahim; Schweigert, Florian J; Ozpinar, Aysel

2015-01-01

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys10), which is frequently affected by oxidative post-translational modifications. As Cys10 is part of the thyroid hormone-binding channel within the TTR molecule, PTM of Cys10 may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys10 modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype- and site-specific manner with S-glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function-specific patterns of TTR with a substantial decrease in S-sulphonated, S-cysteinylglycinated and S-glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys10 seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability. PMID:25311081
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
The CpG island encompassing the promoter and first exon of human DNMT3L gene is a PcG/TrX response element (PRE).

PubMed

Basu, Amitava; Dasari, Vasanthi; Mishra, Rakesh K; Khosla, Sanjeev

2014-01-01

DNMT3L, a member of DNA methyltransferases family, is present only in mammals. As it provides specificity to the action of de novo methyltransferases, DNMT3A and DNMT3B and interacts with histone H3, DNMT3L has been invoked as the molecule that can read the histone code and translate it into DNA methylation. It plays an important role in the initiation of genomic imprints during gametogenesis and in nuclear reprogramming. With important functions attributed to it, it is imperative that the DNMT3L expression is tightly controlled. Previously, we had identified a CpG island within the human DNMT3L promoter and first exon that showed loss of DNA methylation in cancer samples. Here we show that this Differentially Methylated CpG island within DNMT3L (DNMT3L DMC) acts to repress transcription, is a Polycomb/Trithorax Response Element (PRE) and interacts with both PRC1 and PRC2 Polycomb repressive complexes. In addition, it adopts inactive chromatin conformation and is associated with other inactive chromatin-specific proteins like SUV39H1 and HP1. The presence of DNMT3L DMC also influences the adjacent promoter to adopt repressive histone post-translational modifications. Due to its association with multiple layers of repressive epigenetic modifications, we believe that PRE within the DNMT3L DMC is responsible for the tight regulation of DNMT3L expression and the aberrant epigenetic modifications of this region leading to DNMT3L overexpression could be the reason of nuclear programming during carcinogenesis.
Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed.

PubMed

He, Dongli; Wang, Qiong; Li, Ming; Damaris, Rebecca Njeri; Yi, Xingling; Cheng, Zhongyi; Yang, Pingfang

2016-03-04

Regulation of rice seed germination has been shown to mainly occur at post-transcriptional levels, of which the changes on proteome status is a major one. Lysine acetylation and succinylation are two prevalent protein post-translational modifications (PTMs) involved in multiple biological processes, especially for metabolism regulation. To investigate the potential mechanism controlling metabolism regulation in rice seed germination, we performed the lysine acetylation and succinylation analyses simultaneously. Using high-accuracy nano-LC-MS/MS in combination with the enrichment of lysine acetylated or succinylated peptides from digested embryonic proteins of 24 h after imbibition (HAI) rice seed, a total of 699 acetylated sites from 389 proteins and 665 succinylated sites from 261 proteins were identified. Among these modified lysine sites, 133 sites on 78 proteins were commonly modified by two PTMs. The overlapped PTM sites were more likely to be in polar acidic/basic amino acid regions and exposed on the protein surface. Both of the acetylated and succinylated proteins cover nearly all aspects of cellular functions. Ribosome complex and glycolysis/gluconeogenesis-related proteins were significantly enriched in both acetylated and succinylated protein profiles through KEGG enrichment and protein-protein interaction network analyses. The acetyl-CoA and succinyl-CoA metabolism-related enzymes were found to be extensively modified by both modifications, implying the functional interaction between the two PTMs. This study provides a rich resource to examine the modulation of the two PTMs on the metabolism pathway and other biological processes in germinating rice seed.
Modification site localization scoring integrated into a search engine.

PubMed

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

2011-07-01

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

PubMed Central

van Niekerk, Erna A.; Tuszynski, Mark H.; Lu, Paul; Dulin, Jennifer N.

2016-01-01

Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. PMID:26695766
Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

DOE PAGES

Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

2015-09-21

The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less
A workflow for large-scale empirical identification of cell wall N-linked glycoproteins of tomato (Solanum lycopersicum) fruit by tandem mass spectrometry

USDA-ARS?s Scientific Manuscript database

Glycosylation is a common post-translational modification of plant proteins that impacts a large number of important biological processes. Nevertheless, the impacts of differential site occupancy and the nature of specific glycoforms are obscure. Historically, characterization of glycoproteins has b...
A race-specific interaction between vitamin K status and statin use during warfarin therapy initiation

USDA-ARS?s Scientific Manuscript database

Vitamin K (VK) is required for the post-translational modification of several clotting factors. Warfarin is a vitamin K antagonist and anticoagulant. The most common dietary and circulating form of VK is phylloquinone (PK). PK is lipid soluble, carried by triglyceride-rich lipoproteins, and shares a...
Glutaredoxin GrxC2 catalyzes the glutathionylation and inactivation of Arabidopsis BRI1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) in vitro

USDA-ARS?s Scientific Manuscript database

Reversible protein phosphorylation, catalyzed by protein kinases, is the most widely studied post-translational modification (PTM) both in terms of its occurrence and the regulatory consequences of phosphorylation events on phosphorylated proteins. In addition to reversible phosphorylation, many pro...
Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

ERIC Educational Resources Information Center

Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel

2013-01-01

Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…
Detection of the ubiquitinome in cells undergoing oncogene-induced senescence

PubMed Central

Zhu, Hengrui; Le, Linh; Tang, Hsin-Yao; Speicher, David W.; Zhang, Rugang

2017-01-01

Summary Senescent cells exhibit dramatic changes in protein post-translational modifications. Here, we describe a method, stable isotope labeling with amino acids in cell culture (SILAC) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS), to identify changes in the ubiquitinome in cells that have undergone oncogene-induced senescence. PMID:27812874
Mass spectrometry: Raw protein from the top down

NASA Astrophysics Data System (ADS)

Breuker, Kathrin

2018-02-01

Mass spectrometry is a powerful technique for analysing proteins, yet linking higher-order protein structure to amino acid sequence and post-translational modifications is far from simple. Now, a native top-down method has been developed that can provide information on higher-order protein structure and different proteoforms at the same time.
Characterization and expression analysis of genes involved in SUMOylation during embryogenesis in rainbow trout (Oncorhynchus mykiss)

USDA-ARS?s Scientific Manuscript database

SUMOylation is the post-translational modification of proteins by the addition of the small ubiquitin-like modifier (SUMO), which plays an important role in various cellular processes. It has been reported that SUMO and its related proteins are important in diverse reproductive functions such as ovu...

Congenital Disorders of Glycosylation and Intellectual Disability

ERIC Educational Resources Information Center

Wolfe, Lynne A.; Krasnewich, Donna

2013-01-01

The congenital disorders of glycosylation (CDG) are a rapidly growing group of inborn errors of metabolism that result from defects in the synthesis of glycans. Glycosylation is a major post-translational protein modification and an estimated 2% of the human genome encodes proteins for glycosylation. The molecular bases for the current 60…
O-mannosylation of the Mycobacterium tuberculosis Adhesin Apa Is Crucial for T Cell Antigenicity during Infection but Is Expendable for Protection

PubMed Central

Dobos, Karen M.; Lucas, Megan; Spencer, John S.; Fang, Sunan; McDonald, Melissa A.; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V.; Plikaytis, Bonnie B.; Posey, James E.; Amara, Rama Rao

2013-01-01

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis. PMID:24130497
O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

PubMed

Nandakumar, Subhadra; Kannanganat, Sunil; Dobos, Karen M; Lucas, Megan; Spencer, John S; Fang, Sunan; McDonald, Melissa A; Pohl, Jan; Birkness, Kristin; Chamcha, Venkateswarlu; Ramirez, Melissa V; Plikaytis, Bonnie B; Posey, James E; Amara, Rama Rao; Sable, Suraj B

2013-01-01

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.
Phosphorylation of glutaminase by PKCε is essential for its enzymatic activity and critically contributes to tumorigenesis.

PubMed

Han, Tianyu; Zhan, Weihua; Gan, Mingxi; Liu, Fanrong; Yu, Bentong; Chin, Y Eugene; Wang, Jian-Bin

2018-06-01

Glutamine metabolism plays an important role in cancer development and progression. Glutaminase C (GAC), the first enzyme in glutaminolysis, has emerged as an important target for cancer therapy and many studies have focused on the mechanism of enhanced GAC expression in cancer cells. However, little is known about the post-translational modification of GAC. Here, we report that phosphorylation is a crucial post-translational modification of GAC, which is responsible for the higher glutaminase activity in lung tumor tissues and cancer cells. We identify the key Ser314 phosphorylation site on GAC that is regulated by the NF-κB-PKCε axis. Blocking Ser314 phosphorylation by the S314A mutation in lung cancer cells inhibits the glutaminase activity, triggers genetic reprogramming, and alleviates tumor malignancy. Furthermore, we find that a high level of GAC phosphorylation correlates with poor survival rate of lung cancer patients. These findings highlight a previously unappreciated mechanism for activation of GAC by phosphorylation and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.
Evolution of Src Homology 2 (SH2) Domain to Recognize Sulfotyrosine.

PubMed

Ju, Tong; Niu, Wei; Guo, Jiantao

2016-09-16

Protein tyrosine O-sulfation is considered as the most common type of post-translational tyrosine modification in nature and plays important roles in extracellular biomolecular interactions. To facilitate the mapping, biological study, and medicinal application of this type of post-translational modification, we seek to evolve a small protein scaffold that recognizes sulfotyrosine with high affinity. We focused our efforts on the engineering of the Src Homology 2 (SH2) domain, which represents the largest class of known phosphotyrosine-recognition domain in nature and has a highly evolvable binding pocket. By using phage display, we successfully engineered the SH2 domain to recognize sulfotyrosine with high affinity. The best mutant, SH2-60.1, displayed more than 1700 fold higher sulfotyrosine-binding affinity than that of the wild-type SH2 domain. We also demonstrated that the evolved SH2 domain mutants could be used to detect sulfoprotein levels on the cell surface. These evolved SH2 domain mutants can be potentially applied to the study of protein tyrosine O-sulfation with proper experimental designs.
Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin

NASA Astrophysics Data System (ADS)

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

2016-11-01

The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.
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
The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity.

PubMed

Zhang, Yiguo; Hayes, John D

2013-01-01

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

PubMed Central

Mantena, Sudheer K; King, Adrienne L; Andringa, Kelly K; Landar, Aimee; Darley-Usmar, Victor; Bailey, Shannon M

2007-01-01

Mitochondrial dysfunction is known to be a contributing factor to a number of diseases including chronic alcohol induced liver injury. While there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known regarding how changes in the mitochondrial proteome may contribute to the development of hepatic pathologies. Emerging evidence indicates that reactive oxygen and nitrogen species disrupt mitochondrial function through post-translational modifications to the mitochondrial proteome. Indeed, various new affinity labeling reagents are available to test the hypothesis that post-translational modification of proteins by reactive species contributes to mitochondrial dysfunction and alcoholic fatty liver disease. Specialized proteomic techniques are also now available, which allow for identification of defects in the assembly of multi-protein complexes in mitochondria and the resolution of the highly hydrophobic proteins of the inner membrane. In this review knowledge gained from the study of changes to the mitochondrial proteome in alcoholic hepatotoxicity will be described and placed into a mechanistic framework to increase understanding of the role of mitochondrial dysfunction in liver disease. PMID:17854139
Simultaneous monitoring of oxidation, deamidation, isomerization, and glycosylation of monoclonal antibodies by liquid chromatography-mass spectrometry method with ultrafast tryptic digestion.

PubMed

Wang, Yi; Li, Xiaojuan; Liu, Yan-Hui; Richardson, Daisy; Li, Huijuan; Shameem, Mohammed; Yang, Xiaoyu

Monoclonal antibodies are subjected to a wide variety of post-translational modifications (PTMs) that cause structural heterogeneity. Characterization and control of these modifications or quality attributes are critical to ensure antibody quality and to define any potential effects on the ultimate safety and potency of antibody therapeutics. The biopharmaceutical industry currently uses numerous tools to analyze these quality attributes individually, which requires substantial time and resources. Here, we report a simple and ultrafast bottom-up liquid chromatography-mass spectrometry (uLC-MS) method with 5 min tryptic digestion to simultaneously analyze multiple modifications, including oxidation, deamidation, isomerization, glycation, glycosylation, and N-terminal pyro-glutamate formation, which can occur during antibody production in mammalian cell culture, during purification and/or on storage. Compared to commonly used preparation procedures, this uLC-MS method eliminates assay artifacts of falsely-increased Met oxidation, Asp isomerization, and Asn deamidation, a problem associated with long digestion times in conventional LC-MS methods. This simple, low artifact multi-attribute uLC-MS method can be used to quickly and accurately analyze samples at any stage of antibody drug development, in particular for clone and media selection during cell culture development.
Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa).

PubMed

Tan, Feng; Zhang, Kangling; Mujahid, Hana; Verma, Desh Pal S; Peng, Zhaohua

2011-02-04

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.
Direct targeting of human plasma for matrix-assisted laser desorption/ionization and analysis of plasma proteins by time of flight-mass spectrometry.

PubMed

Jin, Ya; Manabe, Takashi

2005-07-01

A method to analyze human plasma proteins without fractionation, directly applying a plasma-matrix mixture on the target plate of a matrix-assisted laser desorption/ionization-time of flight-mass spectrometer (MALDI-TOF-MS), has been described. Peaks of ionized plasma proteins could not be detected applying a mixture of an undiluted plasma sample and a matrix solution, but they appeared when the plasma was diluted before mixing with the matrix. Tenfold diluted plasma provided well-resolved protein peaks in the m/z range from 4000 to 30,000. The addition of a simple post-crystallization washing procedure performed on the target plate further improved the quality of mass spectra. We numbered 58 peaks in the range of 4-160 kDa and 32 out of which were assigned to the plasma protein species which have been reported. Especially high sensitivity and resolution were obtained in the region < 30 kDa, where multiple isoforms of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, and transthyretin could be assigned. Various post-translational modifications are involved in the isoforms, e.g., proteolytic cleavage, glycosylation and chemical modifications. This method will become complementary with the present electrophoretic techniques, especially for the analysis of low-molecular-mass proteins.
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
Identification of Protein Succination as a Novel Modification of Tubulin

PubMed Central

Piroli, Gerardo G.; Manuel, Allison M.; Walla, Michael D.; Jepson, Matthew J.; Brock, Jonathan W.C.; Rajesh, Mathur P.; Tanis, Ross M.; Cotham, William E.; Frizzell, Norma

2015-01-01

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). We demonstrate that both alpha (α) and beta (β) tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound dimethylfumarate (DMF, 500 μM) inhibited polymerization up to 35% and 59%, respectively. Using mass spectrometry we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteines increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose vs. normal glucose also had reduced reactivity with the anti-αtubulin antibody; suggesting that succination may interfere with tubulin:protein interactions. DMF reacted rapidly with 11 of the 20 cysteines in the αβ tubulin dimer, decreased the number of free sulfhydryls and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggests that inhibition of tubulin polymerization is an important, undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. PMID:24909641
Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications.

PubMed

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling.
Regulation of T cell receptor complex-mediated signaling by ubiquitin and ubiquitin-like modifications

PubMed Central

Friend, Samantha F; Deason-Towne, Francina; Peterson, Lisa K; Berger, Allison J; Dragone, Leonard L

2014-01-01

Post-translational protein modifications are a dynamic method of regulating protein function in response to environmental signals. As with any cellular process, T cell receptor (TCR) complex-mediated signaling is highly regulated, since the strength and duration of TCR-generated signals governs T cell development and activation. While regulation of TCR complex-mediated signaling by phosphorylation has been well studied, regulation by ubiquitin and ubiquitin-like modifiers is still an emerging area of investigation. This review will examine how ubiquitin, E3 ubiquitin ligases, and other ubiquitin-like modifications such as SUMO and NEDD8 regulate TCR complex-mediated signaling. PMID:25628960
Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

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

Nayak, Dipti D.; Mahanta, Nilkamal; Mitchell, Douglas A.

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather thanmore » thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.« less
Post-translational thioamidation of methyl-coenzyme M reductase, a key enzyme in methanogenic and methanotrophic Archaea

DOE PAGES

Nayak, Dipti D.; Mahanta, Nilkamal; Mitchell, Douglas A.; ...

2017-09-07

Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and/or ycaO revealed the presence of glycine, rather thanmore » thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.« less
Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy

PubMed Central

Barhoumi, Aoune; Halas, Naomi J.

2013-01-01

Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics. PMID:24427449
Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy.

PubMed

Barhoumi, Aoune; Halas, Naomi J

2011-12-15

Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.

Protein S-Nitrosylation Regulates Xylem Vessel Cell Differentiation in Arabidopsis.

PubMed

Kawabe, Harunori; Ohtani, Misato; Kurata, Tetsuya; Sakamoto, Tomoaki; Demura, Taku

2018-01-01

Post-translational modifications of proteins have important roles in the regulation of protein activity. One such modification, S-nitrosylation, involves the covalent binding of nitric oxide (NO)-related species to a cysteine residue. Recent work showed that protein S-nitrosylation has crucial functions in plant development and environmental responses. In the present study, we investigated the importance of protein S-nitrosylation for xylem vessel cell differentiation using a forward genetics approach. We performed ethyl methanesulfonate mutagenesis of a transgenic Arabidopsis 35S::VND7-VP16-GR line in which the activity of VASCULAR-RELATED NAC-DOMAIN7 (VND7), a key transcription factor involved in xylem vessel cell differentiation, can be induced post-translationally by glucocorticoid treatment, with the goal of obtaining suppressor mutants that failed to differentiate ectopic xylem vessel cells; we named these mutants suppressor of ectopic vessel cell differentiation induced by VND7 (seiv) mutants. We found the seiv1 mutant to be a recessive mutant in which ectopic xylem cell differentiation was inhibited, especially in aboveground organs. In seiv1 mutants, a single nucleic acid substitution (G to A) leading to an amino acid substitution (E36K) was present in the gene encoding S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which regulates the turnover of the natural NO donor, S-nitrosoglutathione. An in vitro S-nitrosylation assay revealed that VND7 can be S-nitrosylated at Cys264 and Cys320 located near the transactivation activity-related domains, which were shown to be important for transactivation activity of VND7 by transient reporter assay. Our results suggest crucial roles for GSNOR1-regulated protein S-nitrosylation in xylem vessel cell differentiation, partly through the post-translational modification of VND7. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Widespread occurrence of lysine methylation in Plasmodium falciparum proteins at asexual blood stages.

PubMed

Kaur, Inderjeet; Zeeshan, Mohammad; Saini, Ekta; Kaushik, Abhinav; Mohmmed, Asif; Gupta, Dinesh; Malhotra, Pawan

2016-10-20

Post-transcriptional and post-translational modifications play a major role in Plasmodium life cycle regulation. Lysine methylation of histone proteins is well documented in several organisms, however in recent years lysine methylation of proteins outside histone code is emerging out as an important post-translational modification (PTM). In the present study we have performed global analysis of lysine methylation of proteins in asexual blood stages of Plasmodium falciparum development. We immunoprecipitated stage specific Plasmodium lysates using anti-methyl lysine specific antibodies that immunostained the asexual blood stage parasites. Using liquid chromatography and tandem mass spectrometry analysis, 570 lysine methylated proteins at three different blood stages were identified. Analysis of the peptide sequences identified 605 methylated sites within 422 proteins. Functional classification of the methylated proteins revealed that the proteins are mainly involved in nucleotide metabolic processes, chromatin organization, transport, homeostatic processes and protein folding. The motif analysis of the methylated lysine peptides reveals novel motifs. Many of the identified lysine methylated proteins are also interacting partners/substrates of PfSET domain proteins as revealed by STRING database analysis. Our findings suggest that the protein methylation at lysine residues is widespread in Plasmodium and plays an important regulatory role in diverse set of the parasite pathways.
Arginine Methylation: The Coming of Age.

PubMed

Blanc, Roméo S; Richard, Stéphane

2017-01-05

Arginine methylation is a common post-translational modification functioning as an epigenetic regulator of transcription and playing key roles in pre-mRNA splicing, DNA damage signaling, mRNA translation, cell signaling, and cell fate decision. Recently, a wealth of studies using transgenic mouse models and selective PRMT inhibitors helped define physiological roles for protein arginine methyltransferases (PRMTs) linking them to diseases such as cancer and metabolic, neurodegenerative, and muscular disorders. This review describes the recent molecular advances that have been uncovered in normal and diseased mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.
Quantification of Treatment Effect Modification on Both an Additive and Multiplicative Scale

PubMed Central

Girerd, Nicolas; Rabilloud, Muriel; Pibarot, Philippe; Mathieu, Patrick; Roy, Pascal

2016-01-01

Background In both observational and randomized studies, associations with overall survival are by and large assessed on a multiplicative scale using the Cox model. However, clinicians and clinical researchers have an ardent interest in assessing absolute benefit associated with treatments. In older patients, some studies have reported lower relative treatment effect, which might translate into similar or even greater absolute treatment effect given their high baseline hazard for clinical events. Methods The effect of treatment and the effect modification of treatment were respectively assessed using a multiplicative and an additive hazard model in an analysis adjusted for propensity score in the context of coronary surgery. Results The multiplicative model yielded a lower relative hazard reduction with bilateral internal thoracic artery grafting in older patients (Hazard ratio for interaction/year = 1.03, 95%CI: 1.00 to 1.06, p = 0.05) whereas the additive model reported a similar absolute hazard reduction with increasing age (Delta for interaction/year = 0.10, 95%CI: -0.27 to 0.46, p = 0.61). The number needed to treat derived from the propensity score-adjusted multiplicative model was remarkably similar at the end of the follow-up in patients aged < = 60 and in patients >70. Conclusions The present example demonstrates that a lower treatment effect in older patients on a relative scale can conversely translate into a similar treatment effect on an additive scale due to large baseline hazard differences. Importantly, absolute risk reduction, either crude or adjusted, can be calculated from multiplicative survival models. We advocate for a wider use of the absolute scale, especially using additive hazard models, to assess treatment effect and treatment effect modification. PMID:27045168
Identification of Phosphorylated Proteins on a Global Scale.

PubMed

Iliuk, Anton

2018-05-31

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.
Characterization of wood decay enzymes by MALDI-MS for post-translational modification and gene identification.

Treesearch

Theodorus H. de Koker; Philip J. Kersten

2002-01-01

The recent sequencing of the Phanerochaete chrysosporium genome presents many opportunities, including the possibility of rapidly correlating specific wood decay proteins of the fungus with the corresponding gene sequences. Here we compare mass fragments of trypsin digests, determined by MALDI-MS (Matrix Assisted Laser Desorption Ionization-Mass Spectrometry), with...
Synthesis of Globulins in Maize Embryos 1

PubMed Central

Kriz, Alan L.; Schwartz, Drew

1986-01-01

The two major components of the globulin fraction in Zea mays embryos are specified by the Prot gene. Pulse-chase analysis of protein synthesis in cultured, immature embryos indicates that the smaller Prot-specific polypeptide, PROT, is derived from the larger polypeptide, PROT'. These experiments also demonstrate that PROT' is derived from a short-lived precursor polypeptide, prePROT'. The primary Prot-specific translation product, as detected by in vitro translation of immature embryo RNA, is of a lower apparent molecular weight than pre-PROT', suggesting the involvement of co- and/or post-translational modification in the production of prePROT'. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16665136
Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

PubMed

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

2002-01-01

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

PubMed Central

Krassowski, Michal; Paczkowska, Marta; Cullion, Kim; Huang, Tina; Dzneladze, Irakli; Ouellette, B F Francis; Yamada, Joseph T; Fradet-Turcotte, Amelie

2018-01-01

Abstract Interpretation of genetic variation is needed for deciphering genotype-phenotype associations, mechanisms of inherited disease, and cancer driver mutations. Millions of single nucleotide variants (SNVs) in human genomes are known and thousands are associated with disease. An estimated 21% of disease-associated amino acid substitutions corresponding to missense SNVs are located in protein sites of post-translational modifications (PTMs), chemical modifications of amino acids that extend protein function. ActiveDriverDB is a comprehensive human proteo-genomics database that annotates disease mutations and population variants through the lens of PTMs. We integrated >385,000 published PTM sites with ∼3.6 million substitutions from The Cancer Genome Atlas (TCGA), the ClinVar database of disease genes, and human genome sequencing projects. The database includes site-specific interaction networks of proteins, upstream enzymes such as kinases, and drugs targeting these enzymes. We also predicted network-rewiring impact of mutations by analyzing gains and losses of kinase-bound sequence motifs. ActiveDriverDB provides detailed visualization, filtering, browsing and searching options for studying PTM-associated mutations. Users can upload mutation datasets interactively and use our application programming interface in pipelines. Integrative analysis of mutations and PTMs may help decipher molecular mechanisms of phenotypes and disease, as exemplified by case studies of TP53, BRCA2 and VHL. The open-source database is available at https://www.ActiveDriverDB.org. PMID:29126202
Redox biology and the interface between bioenergetics, autophagy and circadian control of metabolism.

PubMed

Wende, Adam R; Young, Martin E; Chatham, John; Zhang, Jianhua; Rajasekaran, Namakkal S; Darley-Usmar, Victor M

2016-11-01

Understanding molecular mechanisms that underlie the recent emergence of metabolic diseases such as diabetes and heart failure has revealed the need for a multi-disciplinary research integrating the key metabolic pathways which change the susceptibility to environmental or pathologic stress. At the physiological level these include the circadian control of metabolism which aligns metabolism with temporal demand. The mitochondria play an important role in integrating the redox signals and metabolic flux in response to the changing activities associated with chronobiology, exercise and diet. At the molecular level this involves dynamic post-translational modifications regulating transcription, metabolism and autophagy. In this review we will discuss different examples of mechanisms which link these processes together. An important pathway capable of linking signaling to metabolism is the post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc). This is a nutrient regulated protein modification that plays an important role in impaired cellular stress responses. Circadian clocks have also emerged as critical regulators of numerous cardiometabolic processes, including glucose/lipid homeostasis, hormone secretion, redox status and cardiovascular function. Central to these pathways are the response of autophagy, bioenergetics to oxidative stress, regulated by Keap1/Nrf2 and mechanisms of metabolic control. The extension of these ideas to the emerging concept of bioenergetic health will be discussed. Copyright © 2016 Elsevier Inc. All rights reserved.
Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638
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.
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.
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
Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation*

PubMed Central

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

2016-01-01

Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12−/−), PRC1 (Ring1A/B−/−) and (Dnmt1/3a/3b−/−) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk. PMID:27302890
Accumulation of carboxymethyl-lysine (CML) in human cortical bone.

PubMed

Thomas, Corinne J; Cleland, Timothy P; Sroga, Grazyna E; Vashishth, Deepak

2018-05-01

Advanced glycation end-products (AGEs) are a category of post translational modification associated with the degradation of the structural properties of multiple different types of tissues. Typically, AGEs are the result of a series of post-translational modification reactions between sugars and proteins through a process known as non-enzymatic glycation (NEG). Increases in the rate of NEG of bone tissue are associated with type 2 diabetes and skeletal fragility. Current methods of assessing NEG and its impact on bone fracture risk involve measurement of pentosidine or total fluorescent AGEs (fAGEs). However, pentosidine represents only a small fraction of possible fAGEs present in bone, and neither pentosidine nor total fAGE measurement accounts for non-fluorescent AGEs, which are known to form in significant amounts in skin and other collagenous tissues. Carboxymethyl-lysine (CML) is a non-fluorescent AGE that is often measured and has been shown to accumulate in tissues such as skin, heart, arteries, and intervertebral disks, but is currently not assessed in bone. Here we show the localization of CML to collagen I using mass spectrometry for the first time in human bone. We then present a new method using demineralization followed by heating and trypsin digestion to measure CML content in human bone and demonstrate that CML in bone is 40-100 times greater than pentosidine (the current most commonly used marker of AGEs in bone). We then establish the viability of CML as a measurable AGE in bone by showing that levels of CML, obtained from bone using this technique, increase with age (p<0.05) and are correlated with previously reported measures of bone toughness. Thus, CML is a viable non-fluorescent AGE target to assess AGE accumulation and fragility in bone. The method developed here to extract and measure CML from human bone could facilitate the development of a new diagnostic assay to evaluate fracture risk and potentially lead to new therapeutic approaches to address bone fragility. Copyright © 2018 Elsevier Inc. All rights reserved.
The cyanogenic syndrome in rubber tree Hevea brasiliensis: tissue-damage-dependent activation of linamarase and hydroxynitrile lyase accelerates hydrogen cyanide release

PubMed Central

Kadow, Daniel; Voß, Karsten; Selmar, Dirk; Lieberei, Reinhard

2012-01-01

Background and Aims The release of hydrogen cyanide (HCN) from injured plant tissue affects multiple ecological interactions. Plant-derived HCN can act as a defence against herbivores and also plays an important role in plant–pathogen interactions. Crucial for activity as a feeding deterrent is the amount of HCN generated per unit time, referred to as cyanogenic capacity (HCNc). Strong intraspecific variation in HCNc has been observed among cyanogenic plants. This variation, in addition to genotypic variability (e.g. in Trifolium repens), can result from modifications in the expression level of the enzymes involved in either cyanogenic precursor formation or HCN release (as seen in Sorghum bicolor and Phaseolus lunatus). Thus, a modification or modulation of HCNc in reaction to the environment can only be achieved from one to the next generation when under genetic control and within days or hours when transcriptional regulations are involved. In the present study, it is shown that in rubber tree (Hevea brasiliensis) HCNc is modulated by post-translational activity regulation of the key enzymes for cyanide release. Methods Linamarase (LIN) and hydroxynitrile lyase (HNL) activity was determined by colorimetric assays utilizing dissociation of the substrates p-nitrophenyl-β-d-glucopyranoside and acetone cyanohydrin, respectively. Key Results In rubber tree leaves, LIN and HNL show up to ten-fold increased activity in response to tissue damage. This enzyme activation occurs within seconds and results in accelerated HCN formation. It is restricted to the damaged leaf area and depends on the severity of tissue damage. Conclusions LIN and HNL activation (in contrast to genetic and transcriptional regulations) allows an immediate, local and damage type-dependent modulation of the cyanogenic response. Accordingly, this post-translational activation plays a decisive role in the defence of H. brasiliensis against herbivores as well as pathogens and may allow more flexible reactions in response to these different antagonists. PMID:22451599
Post-transcriptional modifications in the small subunit ribosomal RNA from Thermotoga maritima, including presence of a novel modified cytidine

PubMed Central

Guymon, Rebecca; Pomerantz, Steven C.; Ison, J. Nicholas; Crain, Pamela F.; McCloskey, James A.

2007-01-01

Post-transcriptional modifications of RNA are nearly ubiquitous in the principal RNAs involved in translation. However, in the case of rRNA the functional roles of modification are far less established than for tRNA, and are subject to less knowledge in terms of specific nucleoside identities and their sequence locations. Post-transcriptional modifications have been studied in the SSU rRNA from Thermotoga maritima (optimal growth 80°C), one of the most deeply branched organisms in the Eubacterial phylogenetic tree. A total of 10 different modified nucleosides were found, the greatest number reported for bacterial SSU rRNA, occupying a net of ∼14 sequence sites, compared with a similar number of sites recently reported for Thermus thermophilus and 11 for Escherichia coli. The relatively large number of modifications in Thermotoga offers modest support for the notion that thermophile rRNAs are more extensively modified than those from mesophiles. Seven of the Thermotoga modified sites are identical (location and identity) to those in E. coli. An unusual derivative of cytidine was found, designated N-330 (M r 330.117), and was sequenced to position 1404 in the decoding region of the rRNA. It was unexpectedly found to be identical to an earlier reported nucleoside of unknown structure at the same location in the SSU RNA of the archaeal mesophile Haloferax volcanii. PMID:17255199
Signaling coupled epigenomic regulation of gene expression.

PubMed

Kumar, R; Deivendran, S; Santhoshkumar, T R; Pillai, M R

2017-10-26

Inheritance of genomic information independent of the DNA sequence, the epigenetics, as well as gene transcription are profoundly shaped by serine/threonine and tyrosine signaling kinases and components of the chromatin remodeling complexes. To precisely respond to a changing external milieu, human cells efficiently translate upstream signals into post-translational modifications (PTMs) on histones and coregulators such as corepressors, coactivators, DNA-binding factors and PTM modifying enzymes. Because a protein with multiple residues for putative PTMs is expected to undergo more than one PTM in cells stimulated with growth factors, the outcome of combinational PTM codes on histones and coregulators is profoundly shaped by regulatory interplays between PTMs. The genomic functions of signaling kinases in cancer cells are manifested by the downstream effectors of cytoplasmic signaling cascades as well as translocation of the cytoplasmic signaling kinases to the nucleus. Signaling-mediated phosphorylation of histones serves as a regulatory switch for other PTMs, and connects chromatin remodeling complexes into gene transcription and gene activity. Here, we will discuss the recent advances in signaling-dependent epigenomic regulation of gene transcription using a few representative cancer-relevant serine/threonine and tyrosine kinases and their interplay with chromatin remodeling factors in cancer cells.
Emerging roles of post-translational modifications in signal transduction and angiogenesis.

PubMed

Rahimi, Nader; Costello, Catherine E

2015-01-01

The vascular endothelial growth factor receptor-2 (VEGFR-2) belongs to the family of receptor tyrosine kinases and is a key player in vasculogenesis and pathological angiogenesis. An emerging picture of PTMs of VEGFR-2 suggests that they play central roles in generating a highly dynamic and complex signaling system that regulates key angiogenic responses ranging from endothelial cell differentiation, proliferation, migration to permeability. Recent MS analysis of VEGFR-2 uncovered previously unrecognized PTMs on VEGFR-2 with a distinct function. The ligand binding extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains highly decorated with N-glycosylation, while its cytoplasmic domain is subject to multiple PTMs including Tyr, Ser/Thr phosphorylation, Arg and Lys methylation, acetylation and ubiquitination. Here we review the PTMs on VEGFR-2, their importance in angiogenic signaling relays and possible novel therapeutic potentials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Proteome-wide Domain-centric Perspective on Protein Phosphorylation *

PubMed Central

Palmeri, Antonio; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela; Gherardini, Pier Federico

2014-01-01

Phosphorylation is a widespread post-translational modification that modulates the function of a large number of proteins. Here we show that a significant proportion of all the domains in the human proteome is significantly enriched or depleted in phosphorylation events. A substantial improvement in phosphosites prediction is achieved by leveraging this observation, which has not been tapped by existing methods. Phosphorylation sites are often not shared between multiple occurrences of the same domain in the proteome, even when the phosphoacceptor residue is conserved. This is partly because of different functional constraints acting on the same domain in different protein contexts. Moreover, by augmenting domain alignments with structural information, we were able to provide direct evidence that phosphosites in protein-protein interfaces need not be positionally conserved, likely because they can modulate interactions simply by sitting in the same general surface area. PMID:24830415
Alga-PrAS (Algal Protein Annotation Suite): A Database of Comprehensive Annotation in Algal Proteomes

PubMed Central

Kurotani, Atsushi; Yamada, Yutaka

2017-01-01

Algae are smaller organisms than land plants and offer clear advantages in research over terrestrial species in terms of rapid production, short generation time and varied commercial applications. Thus, studies investigating the practical development of effective algal production are important and will improve our understanding of both aquatic and terrestrial plants. In this study we estimated multiple physicochemical and secondary structural properties of protein sequences, the predicted presence of post-translational modification (PTM) sites, and subcellular localization using a total of 510,123 protein sequences from the proteomes of 31 algal and three plant species. Algal species were broadly selected from green and red algae, glaucophytes, oomycetes, diatoms and other microalgal groups. The results were deposited in the Algal Protein Annotation Suite database (Alga-PrAS; http://alga-pras.riken.jp/), which can be freely accessed online. PMID:28069893
Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury.

PubMed

van Niekerk, Erna A; Tuszynski, Mark H; Lu, Paul; Dulin, Jennifer N

2016-02-01

Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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.
Epigenetics of oropharyngeal squamous cell carcinoma: opportunities for novel chemotherapeutic targets.

PubMed

Lindsay, Cameron; Seikaly, Hadi; Biron, Vincent L

2017-01-31

Epigenetic modifications are heritable changes in gene expression that do not directly alter DNA sequence. These modifications include DNA methylation, histone post-translational modifications, small and non-coding RNAs. Alterations in epigenetic profiles cause deregulation of fundamental gene expression pathways associated with carcinogenesis. The role of epigenetics in oropharyngeal squamous cell carcinoma (OPSCC) has recently been recognized, with implications for novel biomarkers, molecular diagnostics and chemotherapeutics. In this review, important epigenetic pathways in human papillomavirus (HPV) positive and negative OPSCC are summarized, as well as the potential clinical utility of this knowledge.This material has never been published and is not currently under evaluation in any other peer-reviewed publication.
Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies

PubMed Central

Dashivets, Tetyana; Stracke, Jan; Dengl, Stefan; Knaupp, Alexander; Pollmann, Jan; Buchner, Johannes; Schlothauer, Tilman

2016-01-01

ABSTRACT Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the VL domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation. PMID:27612038
Actin filaments-A target for redox regulation.

PubMed

Wilson, Carlos; Terman, Jonathan R; González-Billault, Christian; Ahmed, Giasuddin

2016-10-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates-the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL-and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Actin filaments – a target for redox regulation

PubMed Central

Wilson, Carlos; Terman, Jonathan R.; González-Billault, Christian; Ahmed, Giasuddin

2016-01-01

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through non-covalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation-reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post-translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates – the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL – and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease. PMID:27309342
Dealing with an Unconventional Genetic Code in Mitochondria: The Biogenesis and Pathogenic Defects of the 5-Formylcytosine Modification in Mitochondrial tRNAMet.

PubMed

Van Haute, Lindsey; Powell, Christopher A; Minczuk, Michal

2017-03-02

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt-tRNAMet) also recognises AUA during translation initiation and elongation. Post-transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5-formylcytosine (f5C) modification of position 34 in mt-tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.
Enhancement of antibody functions through Fc multiplications

PubMed Central

Wang, Qun; Cvitkovic, Romana; Bonnell, Jessica; Chang, Chien-Ying; Koksal, Adem C.; O'Connor, Ellen; Gao, Xizhe; Yu, Xiang-Qing; Wu, Herren; Stover, C. Kendall; Dall'Acqua, William F.; Xiao, Xiaodong

2017-01-01

ABSTRACT Antibodies carry out a plethora of functions through their crystallizable fragment (Fc) regions, which can be naturally tuned by the adoption of several isotypes and post-translational modifications. Protein engineering enables further Fc function modulations through modifications of the interactions between the Fc and its functional partners, including FcγR, FcRn, complement complex, and additions of auxiliary functional units. Due to the many functions embedded within the confinement of an Fc, a suitable balance must be maintained for a therapeutic antibody to be effective and safe. The outcome of any Fc engineering depends on the interplay among all the effector molecules involved. In this report, we assessed the effects of Fc multiplication (or tandem Fc) on antibody functions. Using IgG1 as a test case, we found that, depending on the specifically designed linker, Fc multiplication led to differentially folded, stable molecules with unique pharmacokinetic profiles. Interestingly, the variants with 3 copies of Fc improved in vitro opsonophagocytic killing activity and displayed significantly improved protective efficacies in a Klebsiella pneumoniae mouse therapeutic model despite faster clearance compared with its IgG1 counterpart. There was no adverse effect observed or pro-inflammatory cytokine release when the Fc variants were administered to animals. We further elucidated that enhanced binding to various effector molecules by IgG-3Fc created a “sink” leading to the rapid clearance of the 3Fc variants, and identified the increased FcRn binding as one strategy to facilitate “sink” escape. These findings reveal new opportunities for novel Fc engineering to further expand our abilities to manipulate and improve antibody therapeutics. PMID:28102754
Post-translational regulation of nitrogen transporters in plants and microorganisms.

PubMed

Jacquot, Aurore; Li, Zhi; Gojon, Alain; Schulze, Waltraud; Lejay, Laurence

2017-05-01

For microorganisms and plants, nitrate and ammonium are the main nitrogen sources and they are also important signaling molecules controlling several aspects of metabolism and development. Over the past decade, numerous studies revealed that nitrogen transporters are strongly regulated at the transcriptional level. However, more and more reports are now showing that nitrate and ammonium transporters are also subjected to post-translational regulations in response to nitrogen availability. Phosphorylation is so far the most well studied post-translational modification for these transporters and it affects both the regulation of nitrogen uptake and nitrogen sensing. For example, in Arabidopsis thaliana, phosphorylation was shown to activate the sensing function of the root nitrate transporter NRT1.1 and to switch the transport affinity. Also, for ammonium transporters, a phosphorylation-dependent activation/inactivation mechanism was elucidated in recent years in both plants and microorganisms. However, despite the fact that these regulatory mechanisms are starting to be thoroughly described, the signaling pathways involved and their action on nitrogen transporters remain largely unknown. In this review, we highlight the inorganic nitrogen transporters regulated at the post-translational level and we compare the known mechanisms in plants and microorganisms. We then discuss how these mechanisms could contribute to the regulation of nitrogen uptake and/or nitrogen sensing. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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.
Synergistic Action of FOXP3 and TSC1 Pathways During Tumor Progression

DTIC Science & Technology

2016-10-01

Tumor progression, Gene therapy, Transcriptional regulation, Post -translational modification ACCOMPLISHMENTS Our preliminary studies provide evidence...Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for...10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S REPORT
RESEARCH INVESTIGATIONS ON THE PROTEOME: 1. MECHANISMS OF REGULATING PROTEIN SYNTHESIS, AND 2. GLOBAL CHARACTERIZATION OF PROTEOMIC RESPONSES TO ARSENIC EXPOSURES

EPA Science Inventory

Eukaryotic Elongation Factor 2 (eEF2) mediates translocation in protein synthesis. eEF2 is modified by two post-translational modifications: the phosphorylation of Thr57 in the G domain and a unique conversion of His699 to diphthamide at the tip of domain IV. Diphthamide is the t...
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.
Proteomic analysis of post translational modifications in cyanobacteria.

PubMed

Xiong, Qian; Chen, Zhuo; Ge, Feng

2016-02-16

Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive in various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. The systematic investigation of PTMs could contribute to the comprehensive description of protein species and to elucidate potential biological roles of each protein species in cyanobacteria. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This review aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed. Copyright © 2015 Elsevier B.V. All rights reserved.
Mitochondrial Proteome Studies in Seeds during Germination

PubMed Central

Czarna, Malgorzata; Kolodziejczak, Marta; Janska, Hanna

2016-01-01

Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination is progressive development of structurally simple and metabolically quiescent promitochondria into fully active and cristae-containing mitochondria, known as mitochondrial biogenesis. This is a complex and tightly regulated process, which is accompanied by sequential and dynamic gene expression, protein synthesis, and post-translational modifications. The aim of this review is to give a comprehensive summary of seed mitochondrial proteome studies during germination of various plant model organisms. We describe different gel-based and gel-free proteomic approaches used to characterize mitochondrial proteomes of germinating seeds as well as challenges and limitations of these proteomic studies. Furthermore, the dynamic changes in the abundance of the mitochondrial proteomes of germinating seeds are illustrated, highlighting numerous mitochondrial proteins involved in respiration, tricarboxycylic acid (TCA) cycle, metabolism, import, and stress response as potentially important for seed germination. We then review seed mitochondrial protein carbonylation, phosphorylation, and S-nitrosylation as well as discuss the possible link between these post-translational modifications (PTMs) and the regulation of seed germination. PMID:28248229
Treatments and compositions targeting α-synuclein: a patent review (2010-2016).

PubMed

Jęśko, Henryk; Lenkiewicz, Anna M; Adamczyk, Agata

2017-04-01

Abnormal deposition of α-synuclein (ASN) is a hallmark and possible central mechanism of Parkinson's disease and other synucleinopathies. Their therapy is currently hampered by the lack of early, screening-compatible diagnostic methods and efficient treatments. Areas covered: Patent applications related to synucleinopathies obtained from Patentscope and Espacenet databases are described against the background of current knowledge regarding the regulatory mechanisms of ASN behavior including alternative splicing, post-translational modifications, molecular interactions, aggregation, degradation, and changes in localization. Expert opinion: As the central pathological feature and possibly one of root causes in a number of neurodegenerative diseases, deregulation of ASN is a potentially optimal diagnostic and therapeutic target. Changes in total ASN may have diagnostic value, especially if non-invasive /peripheral tissue tests can be developed. Targeting the whole ASN pool for therapeutic purposes may be problematic, however. ASN mutations, truncation, and post-translational modifications have great potential value; therapeutic approaches selective towards aggregated or aggregation-prone ASN forms may lead to more successful and safe treatments. Numerous ASN interactions with signaling pathways, protein degradation and stress mechanisms widen its potential therapeutic significance dramatically. However, significant improvement in the basic knowledge on ASN is necessary to fully exploit these opportunities.
Galectin-3: A novel substrate for c-Abl kinase.

PubMed

Balan, Vitaly; Nangia-Makker, Pratima; Jung, Young Suk; Wang, Yi; Raz, Avraham

2010-10-01

Galectin-3, a beta-galactoside-binding lectin, is found in cellular and extracellular location of the cell and has pleiotropic biological functions such as cell growth, cell adhesion and cell-cell interaction. It may exhibit anti- or pro-apoptotic activity depending on its localization and post-translational modifications. Two important post-translational modifications of galectin-3 have been reported: its cleavage and phosphorylation. Cleavage of galectin-3 was reported to be involved with angiogenic potential and apoptotic resistance. Phosphorylation of galectin-3 regulates its sugar-binding ability. In this report we have identified novel tyrosine phosphorylation sites in galectin-3 as well as the kinase responsible for its phosphorylation. Our results demonstrate that tyrosines at positions 79, 107 and 118 can be phosphorylated in vitro and in vivo by c-Abl kinase. Tyrosine 107 is the main target of c-Abl. Expression of galectin-3 Y107F mutant in galectin-3 null SK-Br-3 cells leads to morphological changes and increased motility compared to wild type galectin-3. Further investigation is needed to better understand the functional significance of the novel tyrosine phosphorylated sites of galectin-3. Copyright © 2010 Elsevier B.V. All rights reserved.
Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

PubMed Central

Eyre, David R.; Ann Weis, Mary

2013-01-01

Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

Mechanisms of CaMKII Activation in the Heart.

PubMed

Erickson, Jeffrey R

2014-01-01

Calcium/calmodulin (Ca(2+)/CaM) dependent protein kinase II (CaMKII) has emerged as a key nodal protein in the regulation of cardiac physiology and pathology. Due to the particularly elegant relationship between the structure and function of the kinase, CaMKII is able to translate a diverse set of signaling events into downstream physiological effects. While CaMKII is typically autoinhibited at basal conditions, prolonged rapid Ca(2+) cycling can activate the kinase and allow post-translational modifications that depend critically on the biochemical environment of the heart. These modifications result in sustained, autonomous CaMKII activation and have been associated with pathological cardiac signaling. Indeed, improved understanding of CaMKII activation mechanisms could potentially lead to new clinical therapies for the treatment or prevention of cardiovascular disease. Here we review the known mechanisms of CaMKII activation and discuss some of the pathological signaling pathways in which they play a role.
Modulation of farnesoid X receptor results in post-translational modification of poly (ADP-ribose) polymerase 1 in the liver

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

Zhu, Yan; Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS; Li, Guodong

2013-01-15

The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR deficiency in mice results in cholestasis, metabolic disorders, and tumorigenesis in liver and intestine. FXR is known to contribute to pathogenesis by regulating gene transcription; however, changes in the post-transcriptional modification of proteins associated with FXR modulation have not been determined. In the current study, proteomic analysis of the livers of wild-type (WT) and FXR knockout (FXR-KO) mice treated with a FXR synthetic ligand or vehicle was performed. The results identified five proteins as novel FXR targets. Since FXR deficiency in micemore » leads to liver tumorigenesis, poly (ADP-ribose) polymerase family, member 1 (Parp1) that is important for DNA repair, was validated in the current study by quantitative real-time PCR, and 1- and 2-dimensional gel electrophoresis/western blot. The results showed that Parp1 mRNA levels were not altered by FXR genetic status or by agonist treatment. However, total Parp1 protein levels were increased in FXR-KO mice as early as 3 month old. Interestingly, total Parp1 protein levels were increased in WT mice in an age-dependent manner (from 3 to 18 months), but not in FXR-KO mice. Finally, activation of FXR in WT mice resulted in reduction of phosporylated Parp1 protein in the liver without affecting total Parp1 protein levels. In conclusion, this study reveals that FXR genetic status and agonist treatment affects basal levels and phosphorylation state of Parp1, respectively. These alterations, in turn, may be associated with the hepatobiliary alterations observed in FXR-KO mice and participate in FXR agonist-induced protection in the liver. -- Highlights: ► Proteomic analysis identified novel FXR targets. ► FXR modification altered post-translational modification of the Parp1 protein. ► Altered Parp1 function may contribute to mechanisms of FXR regulation of liver functions.« less
Diverse Peptide Hormones Affecting Root Growth Identified in the Medicago truncatula Secreted Peptidome.

PubMed

Patel, Neha; Mohd-Radzman, Nadiatul A; Corcilius, Leo; Crossett, Ben; Connolly, Angela; Cordwell, Stuart J; Ivanovici, Ariel; Taylor, Katia; Williams, James; Binos, Steve; Mariani, Michael; Payne, Richard J; Djordjevic, Michael A

2018-01-01

Multigene families encoding diverse secreted peptide hormones play important roles in plant development. A need exists to efficiently elucidate the structures and post-translational-modifications of these difficult-to-isolate peptide hormones in planta so that their biological functions can be determined. A mass spectrometry and bioinformatics approach was developed to comprehensively analyze the secreted peptidome of Medicago hairy root cultures and xylem sap. We identified 759 spectra corresponding to the secreted products of twelve peptide hormones including four CEP ( C -TERMINALLY E NCODED P EPTIDE), two CLE ( CL V3/ E NDOSPERM SURROUNDING REGION RELATED) and six XAP ( X YLEM SAP A SSOCIATED P EPTIDE) peptides. The MtCEP1, MtCEP2, MtCEP5 and MtCEP8 peptides identified differed in post-translational-modifications. Most were hydroxylated at conserved proline residues but some MtCEP1 derivatives were tri-arabinosylated. In addition, many CEP peptides possessed unexpected N - and C -terminal extensions. The pattern of these extensions suggested roles for endo- and exoproteases in CEP peptide maturation. Longer than expected, hydroxylated and homogeneously modified mono- and tri-arabinosylated CEP peptides corresponding to their in vivo structures were chemically synthesized to probe the effect of these post-translational-modifications on function. The ability of CEP peptides to elevate root nodule number was increased by hydroxylation at key positions. MtCEP1 peptides with N -terminal extensions or with tri-arabinosylation modification, however, were unable to impart increased nodulation. The MtCLE5 and MtCLE17 peptides identified were of precise size, and inhibited main root growth and increased lateral root number. Six XAP peptides, each beginning with a conserved DY sulfation motif, were identified including MtXAP1a, MtXAP1b, MtXAP1c, MtXAP3, MtXAP5 and MtXAP7. MtXAP1a and MtXAP5 inhibited lateral root emergence. Transcriptional analyses demonstrated peptide hormone gene expression in the root vasculature and tip. Since hairy roots can be induced on many plants, their corresponding root cultures may represent ideal source materials to efficiently identify diverse peptide hormones in vivo in a broad range of species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Discovering Mercury Protein Modifications in Whole Proteomes Using Natural Isotope Distributions Observed in Liquid Chromatography-Tandem Mass Spectrometry

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

Polacco, Benjamin J.; Purvine, Samuel O.; Zink, Erika M.

2011-08-01

The identification of peptides that result from post-translational modifications is critical for understanding normal pathways of cellular regulation as well as identifying damage from, or exposures to xenobiotics, i.e. the exposome. However, because of their low abundance in proteomes, effective detection of modified peptides by mass spectrometry (MS) typically requires enrichment to eliminate false identifications. We present a new method for confidently identifying peptides with mercury (Hg)-containing adducts that is based on the influence of mercury’s seven stable isotopes on peptide isotope distributions detected by high-resolution MS. Using a pure protein and E. coli cultures exposed to phenyl mercuric acetate,more » we show the pattern of peak heights in isotope distributions from primary MS single scans efficiently identified Hg adducts in data from chromatographic separation coupled with tandem mass spectrometry with sensitivity and specificity greater than 90%. Isotope distributions are independent of peptide identifications based on peptide fragmentation (e.g. by SEQUEST), so both methods can be combined to eliminate false positives. Summing peptide isotope distributions across multiple scans improved specificity to 99.4% and sensitivity above 95%, affording identification of an unexpected Hg modification. We also illustrate the theoretical applicability of the method for detection of several less common elements including the essential element, selenium, as selenocysteine in peptides.« less
Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry

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

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

Histones play central roles in most chromosomal functions and both their basic biology and roles in disease have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here, we used state of the art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFindermore » as search engine, and LcMsSpectator as a data visualization tool. ProMex sums across retention time to maximize sensitivity and accuracy for low abundance species in MS1deconvolution. MSPathFinder searches the MS2 data against protein sequence databases with user-defined modifications. LcMsSpectator presents the results from ProMex and MSPathFinder in a format that allows quick manual evaluation of critical attributes for high-confidence identifications. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.« less
Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

PubMed

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2018-03-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.
Human Spermatozoa Contain Multiple Targets for Protein S-Nitrosylation: An Alternative Mechanism of the Modulation of Sperm Function by Nitric Oxide?

PubMed Central

Lefièvre, Linda; Chen, Yongjian; Conner, Sarah J; Scott, Joanna L; Publicover, Steve J; Ford, W Christopher L; Barratt, Christopher LR

2009-01-01

Nitric oxide (NO) enhances human sperm motility and capacitation associated with increased protein phosphorylation. NO activates soluble guanylyl cyclase, but can also modify protein function covalently via S-nitrosylation of cysteine. Remarkably, this mechanism remains unexplored in sperm although they depend on post-translational protein modification to achieve changes in function required for fertilisation. Our objective was to identify targets for S-nitrosylation in human sperm. Spermatozoa were incubated with NO donors and S-nitrosylated proteins were identified using the biotin switch assay and a proteomic approach using tandem mass spectrometry. 240 S-nitrosylated proteins were detected in sperm incubated with S-nitrosoglutathione. Minimal levels were observed in glutathione or untreated samples. Proteins identified consistently based on multiple peptides included established targets for S-nitrosylation in other cells e.g. tubulin,, glutathione-S-transferase and heat shock proteins but also novel targets including A-kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent anion-selective channel protein 3 and semenogelin 1 and 2. In situ localisation revealed S-nitrosylated targets on the post-acrosomal region of the head and throughout the flagellum. Potential targets for S-nitrosylation in human sperm include physiologically significant proteins not previously reported in other cells. Their identification will provide novel insight into the mechanism of action of NO in spermatozoa. PMID:17683036
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
Cross-cultural validation of instruments measuring health beliefs about colorectal cancer screening among Korean Americans.

PubMed

Lee, Shin-Young; Lee, Eunice E

2015-02-01

The purpose of this study was to report the instrument modification and validation processes to make existing health belief model scales culturally appropriate for Korean Americans (KAs) regarding colorectal cancer (CRC) screening utilization. Instrument translation, individual interviews using cognitive interviewing, and expert reviews were conducted during the instrument modification phase, and a pilot test and a cross-sectional survey were conducted during the instrument validation phase. Data analyses of the cross-sectional survey included internal consistency and construct validity using exploratory and confirmatory factor analysis. The main issues identified during the instrument modification phase were (a) cultural and linguistic translation issues and (b) newly developed items reflecting Korean cultural barriers. Cross-sectional survey analyses during the instrument validation phase revealed that all scales demonstrate good internal consistency reliability (Cronbach's alpha=.72~.88). Exploratory factor analysis showed that susceptibility and severity loaded on the same factor, which may indicate a threat variable. Items with low factor loadings in the confirmatory factor analysis may relate to (a) lack of knowledge about fecal occult blood testing and (b) multiple dimensions of the subscales. Methodological, sequential processes of instrument modification and validation, including translation, individual interviews, expert reviews, pilot testing and a cross-sectional survey, were provided in this study. The findings indicate that existing instruments need to be examined for CRC screening research involving KAs.
Translation of lifestyle modification programs focused on physical activity and dietary habits delivered in community settings.

PubMed

Stoutenberg, Mark; Stanzilis, Katie; Falcon, Ashley

2015-06-01

Lifestyle modification programs (LMPs) can provide individuals with behavioral skills to sustain long-term changes to their physical activity (PA) levels and dietary habits. Yet, there is much work to be done in the translation of these programs to community settings. This review identified LMPs that focused on changing both PA and dietary behaviors and examined common features and barriers faced in their translation to community settings. A search of multiple online databases was conducted to identify LMPs that included participants over the age of 18 who enrolled in LMPs, offered in community settings, and had the goal of improving both PA and dietary behaviors. Data were extracted on participant demographics, study design characteristics, and study outcome variables including changes in PA, dietary habits, body weight, and clinical outcomes. We identified 27 studies that met inclusion criteria. Despite high levels of retention and adherence to the interventions, varying levels of success were observed in increasing PA levels, improving dietary habits, reducing body weight, and improving clinic outcomes. LMPs addressing issues of PA and dietary habits can be successfully implemented in a community setting. However, inconsistent reporting of key components in the translation of these studies (participant recruitment, utilization of behavioral strategies) may limit their replication and advancement of future programs. Future efforts should better address issues such as identifying barriers to participation and program implementation, utilization of community resources, and evaluating changes across multiple health behaviors.
Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry.

PubMed

Bailey, Ulla-Maja; Punyadeera, Chamindie; Cooper-White, Justin J; Schulz, Benjamin L

2012-12-12

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. Copyright © 2012 Elsevier B.V. All rights reserved.
Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products: New Insights Into the Role of Leader and Core Peptides During Biosynthesis

PubMed Central

Yang, Xiao; van der Donk, Wilfred A.

2013-01-01

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

PubMed

Gitlin, Laura N; Chernett, Nancy L; Harris, Lynn Fields; Palmer, Delores; Hopkins, Paul; Dennis, Marie P

2008-10-01

We describe the translation of K. R. Lorig and colleagues' Chronic Disease Self-Management Program (CDSMP) for delivery in a senior center and evaluate pre-post benefits for African American participants. Modifications to the CDSMP included a name change; an additional introductory session; and course augmentations involving culturally relevant foods, stress reduction techniques, and communicating with racially/ethnically diverse physicians. We recruited participants from senior center members, area churches, and word of mouth. We conducted baseline and 4-month post-interviews. A total of 569 African American elders attended an introductory session, with 519 (91%) enrolling in the 6-session program. Of the 519, 444 (86%) completed >/=4 sessions and 414 (79%) completed pre-post interviews. We found small but statistically significant improvements for exercise (p =.001), use of cognitive management strategies (p =.001), energy/fatigue (p =.001), self-efficacy (p =.001), health distress (p =.001), and illness intrusiveness in different life domains (probabilities from.001-.021). We found no changes for health utilization. Outcomes did not differ by gender, number of sessions attended, number and type of chronic conditions, facilitator, leader, or recruitment site. The CDSMP can be translated for delivery by trained senior center personnel to African American elders. Participant benefits compare favorably to original trial outcomes. The translated program is replicable and may help to address health disparities.
Liganded and unliganded activation of estrogen receptor and hormone replacement therapies.

PubMed

Maggi, Adriana

2011-08-01

Over the past two decades, our understanding of estrogen receptor physiology in mammals widened considerably as we acquired a deeper appreciation of the roles of estrogen receptor alpha and beta (ERα and ERβ) in reproduction as well as in bone and metabolic homeostasis, depression, vascular disorders, neurodegenerative diseases and cancer. In addition, our insights on ER transcriptional functions in cells increased considerably with the demonstration that ER activity is not strictly dependent on ligand availability. Indeed, unliganded ERs may be transcriptionally active and post-translational modifications play a major role in this context. The finding that several intracellular transduction molecules may regulate ER transcriptional programs indicates that ERs may act as a hub where several molecular pathways converge: this allows to maintain ER transcriptional activity in tune with all cell functions. Likely, the biological relevant role of ER was favored by evolution as a mean of integration between reproductive and metabolic functions. We here review the post-translational modifications modulating ER transcriptional activity in the presence or in the absence of estrogens and underline their potential role for ER tissue-specific activities. In our opinion, a better comprehension of the variety of molecular events that control ER activity in reproductive and non-reproductive organs is the foundation for the design of safer and more efficacious hormone-based therapies, particularly for menopause. This article is part of a Special Issue entitled: Translating Nuclear receptors from health to disease. Copyright © 2011 Elsevier B.V. All rights reserved.
In the loop: how chromatin topology links genome structure to function in mechanisms underlying learning and memory.

PubMed

Watson, L Ashley; Tsai, Li-Huei

2017-04-01

Different aspects of learning, memory, and cognition are regulated by epigenetic mechanisms such as covalent DNA modifications and histone post-translational modifications. More recently, the modulation of chromatin architecture and nuclear organization is emerging as a key factor in dynamic transcriptional regulation of the post-mitotic neuron. For instance, neuronal activity induces relocalization of gene loci to 'transcription factories', and specific enhancer-promoter looping contacts allow for precise transcriptional regulation. Moreover, neuronal activity-dependent DNA double-strand break formation in the promoter of immediate early genes appears to overcome topological constraints on transcription. Together, these findings point to a critical role for genome topology in integrating dynamic environmental signals to define precise spatiotemporal gene expression programs supporting cognitive processes. Copyright © 2016 Elsevier Ltd. All rights reserved.
Web-ware bioinformatical analysis and structure modelling of N-terminus of human multisynthetase complex auxiliary component protein p43.

PubMed

Deineko, Viktor

2006-01-01

Human multisynthetase complex auxiliary component, protein p43 is an endothelial monocyte-activating polypeptide II precursor. In this study, comprehensive sequence analysis of N-terminus has been performed to identify structural domains, motifs, sites of post-translation modification and other functionally important parameters. The spatial structure model of full-chain protein p43 is obtained.
Conformational ensemble of human α-synuclein physiological form predicted by molecular simulations.

PubMed

Rossetti, G; Musiani, F; Abad, E; Dibenedetto, D; Mouhib, H; Fernandez, C O; Carloni, P

2016-02-17

We perform here enhanced sampling simulations of N-terminally acetylated human α-synuclein, an intrinsically disordered protein involved in Parkinson's disease. The calculations, consistent with experiments, suggest that the post-translational modification leads to the formation of a transient amphipathic α-helix. The latter, absent in the non-physiological form, alters protein dynamics at the N-terminal and intramolecular interactions.
Functional Characterization of CENP-A Post-Translational Modifications in Chromosome Segregation

DTIC Science & Technology

2015-07-01

for Experimental Biologist) meeting - Mitosis : Spindle Assembly and Function. Moreover I gave talk and did poster presentation in several meetings...Publications, Abstracts, and Presentations: 1. FASEB (Federation of American Societies for Experimental Biologist) meeting - Mitosis : Spindle Assembly and...that the amino terminal tail of CENP-A is sufficient for trimethylation. We also found increase in methylation of centromeric CENP-A towards mitosis
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

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

PubMed Central

Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

2016-01-01

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338
Chaperonin of Group I: Oligomeric Spectrum and Biochemical and Biological Implications.

PubMed

Vilasi, Silvia; Bulone, Donatella; Caruso Bavisotto, Celeste; Campanella, Claudia; Marino Gammazza, Antonella; San Biagio, Pier L; Cappello, Francesco; Conway de Macario, Everly; Macario, Alberto J L

2017-01-01

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans) has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on the oligomeric equilibrium and stability of the various forms of HSP60 in comparison with GroEL. We also discuss post-translational modifications associated with anti-cancer drugs to indicate the potential of Hsp60 in Medicine, as a biomarker and etiopathogenic factor.
Modifications and Trafficking of APP in the Pathogenesis of Alzheimer’s Disease

PubMed Central

Wang, Xin; Zhou, Xuan; Li, Gongying; Zhang, Yun; Wu, Yili; Song, Weihong

2017-01-01

Alzheimer’s disease (AD), the most common neurodegenerative disorder, is the leading cause of dementia. Neuritic plaque, one of the major characteristics of AD neuropathology, mainly consists of amyloid β (Aβ) protein. Aβ is derived from amyloid precursor protein (APP) by sequential cleavages of β- and γ-secretase. Although APP upregulation can promote AD pathogenesis by facilitating Aβ production, growing evidence indicates that aberrant post-translational modifications and trafficking of APP play a pivotal role in AD pathogenesis by dysregulating APP processing and Aβ generation. In this report, we reviewed the current knowledge of APP modifications and trafficking as well as their role in APP processing. More importantly, we discussed the effect of aberrant APP modifications and trafficking on Aβ generation and the underlying mechanisms, which may provide novel strategies for drug development in AD. PMID:28966576
Modifications and Trafficking of APP in the Pathogenesis of Alzheimer's Disease.

PubMed

Wang, Xin; Zhou, Xuan; Li, Gongying; Zhang, Yun; Wu, Yili; Song, Weihong

2017-01-01

Alzheimer's disease (AD), the most common neurodegenerative disorder, is the leading cause of dementia. Neuritic plaque, one of the major characteristics of AD neuropathology, mainly consists of amyloid β (Aβ) protein. Aβ is derived from amyloid precursor protein (APP) by sequential cleavages of β- and γ-secretase. Although APP upregulation can promote AD pathogenesis by facilitating Aβ production, growing evidence indicates that aberrant post-translational modifications and trafficking of APP play a pivotal role in AD pathogenesis by dysregulating APP processing and Aβ generation. In this report, we reviewed the current knowledge of APP modifications and trafficking as well as their role in APP processing. More importantly, we discussed the effect of aberrant APP modifications and trafficking on Aβ generation and the underlying mechanisms, which may provide novel strategies for drug development in AD.
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.
A Quadrupole Dalton-based multi-attribute method for product characterization, process development, and quality control of therapeutic proteins.

PubMed

Xu, Weichen; Jimenez, Rod Brian; Mowery, Rachel; Luo, Haibin; Cao, Mingyan; Agarwal, Nitin; Ramos, Irina; Wang, Xiangyang; Wang, Jihong

2017-10-01

During manufacturing and storage process, therapeutic proteins are subject to various post-translational modifications (PTMs), such as isomerization, deamidation, oxidation, disulfide bond modifications and glycosylation. Certain PTMs may affect bioactivity, stability or pharmacokinetics and pharmacodynamics profile and are therefore classified as potential critical quality attributes (pCQAs). Identifying, monitoring and controlling these PTMs are usually key elements of the Quality by Design (QbD) approach. Traditionally, multiple analytical methods are utilized for these purposes, which is time consuming and costly. In recent years, multi-attribute monitoring methods have been developed in the biopharmaceutical industry. However, these methods combine high-end mass spectrometry with complicated data analysis software, which could pose difficulty when implementing in a quality control (QC) environment. Here we report a multi-attribute method (MAM) using a Quadrupole Dalton (QDa) mass detector to selectively monitor and quantitate PTMs in a therapeutic monoclonal antibody. The result output from the QDa-based MAM is straightforward and automatic. Evaluation results indicate this method provides comparable results to the traditional assays. To ensure future application in the QC environment, this method was qualified according to the International Conference on Harmonization (ICH) guideline and applied in the characterization of drug substance and stability samples. The QDa-based MAM is shown to be an extremely useful tool for product and process characterization studies that facilitates facile understanding of process impact on multiple quality attributes, while being QC friendly and cost-effective.
Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.

PubMed

Wu, Vincent W; Dana, Craig M; Iavarone, Anthony T; Clark, Douglas S; Glass, N Louise

2017-01-17

The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. Pyroglutamate modification is the post-translational conversion of N-terminal glutamine or glutamate into a cyclized amino acid derivative. This modification is well studied in animal systems but poorly explored in fungal systems. In Neurospora crassa, we show that this modification takes place in the ER and is catalyzed by two well-conserved enzymes, ubiquitously conserved throughout the fungal kingdom. We demonstrate that the modification is important for the structural stability and aminopeptidase resistance of CBH-1 and GH5-1, two important cellulase enzymes utilized in industrial plant cell wall deconstruction. Many additional fungal proteins predicted in the genome of N. crassa and other filamentous fungi are predicted to carry an N-terminal pyroglutamate modification. Pyroglutamate addition may also be a useful way to stabilize secreted proteins and peptides, which can be easily produced in fungal production systems. Copyright © 2017 Wu et al.
Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals.

PubMed

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

2017-01-01

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed ( p < 0.05, fold change >1.25 or < -1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.
A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening.

PubMed

Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

2012-08-01

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.
Post-translational control of nitrate reductase activity responding to light and photosynthesis evolved already in the early vascular plants.

PubMed

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

2013-05-01

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

PubMed Central

Matthews, G; Colman, A

1991-01-01

We describe the use of a Xenopus laevis egg extract for the in vitro translation and post translational modification of membrane and secretory proteins. This extract is capable of the translation and segregation into membranes of microgram per millilitre levels of protein from added mRNAs. Signal sequences of segregated proteins are efficiently cleaved and appropriate N-linked glycosylation patterns are produced. The extract also supports the quantitative assembly of murine immunoglobulin heavy and light chains into tetramers, and two events which take place beyond the endoplasmic reticulum, mannose 6 phosphorylation of murine cathepsin D and O-linked glycosylation of coronavirus E1 protein, also occur, but at reduced efficiency. The stability of the membranes allows protease protection studies and quantitative centrifugal fractionation of segregated and unsegregated proteins to be performed. Conditions for the use of stored extract have also been determined. Images PMID:1754376
Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

PubMed Central

Leppek, Kathrin; Das, Rhiju; Barna, Maria

2017-01-01

RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424
An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

PubMed

Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

2016-11-18

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.
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
Extracellular and Intracellular Cyclophilin A, Native and Post-Translationally Modified, Show Diverse and Specific Pathological Roles in Diseases.

PubMed

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

2018-05-01

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

PubMed

Banerjee, Aditya; Roychoudhury, Aryadeep

2017-01-01

One of the major causes of significant crop loss throughout the world is the myriad of environmental stresses including drought, salinity, cold, heavy metal toxicity, and ultraviolet-B (UV-B) rays. Plants as sessile organisms have evolved various effective mechanism which enable them to withstand this plethora of stresses. Most of such regulatory mechanisms usually follow the abscisic-acid (ABA)-dependent pathway. In this review, we have primarily focussed on the basic leucine zipper (bZIP) transcription factors (TFs) activated by the ABA-mediated signalosome. Upon perception of ABA by specialized receptors, the signal is transduced via various groups of Ser/Thr kinases, which phosphorylate the bZIP TFs. Following such post-translational modification of TFs, they are activated so that they bind to specific cis-acting sequences called abscisic-acid-responsive elements (ABREs) or GC-rich coupling elements (CE), thereby influencing the expression of their target downstream genes. Several in silico techniques have been adopted so far to predict the structural features, recognize the regulatory modification sites, undergo phylogenetic analyses, and facilitate genome-wide survey of TF under multiple stresses. Current investigations on the epigenetic regulation that controls greater accessibility of the inducible regions of DNA of the target gene to the bZIP TFs exclusively under stress situations, along with the evolved stress memory responses via genomic imprinting mechanism, have been highlighted. The potentiality of overexpression of bZIP TFs, either in a homologous or in a heterologous background, in generating transgenic plants tolerant to various abiotic stressors have also been addressed by various groups. The present review will provide a coherent documentation on the functional characterization and regulation of bZIP TFs under multiple environmental stresses, with the major goal of generating multiple-stress-tolerant plant cultivars in near future.
SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle

PubMed Central

Figueroa-Romero, Claudia; Iñiguez-Lluhí, Jorge A.; Stadler, Julia; Chang, Chuang-Rung; Arnoult, Damien; Keller, Peter J.; Hong, Yu; Blackstone, Craig; Feldman, Eva L.

2009-01-01

Dynamin-related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP-binding, Middle, insert B, and C-terminal GTPase effector (GED) domains. Drp1 associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drp1 activity remain poorly understood but are likely to involve reversible post-translational modifications, such as conjugation of small ubiquitin-like modifier (SUMO) proteins. Through a detailed analysis, we find that Drp1 interacts with the SUMO-conjugating enzyme Ubc9 via multiple regions and demonstrate that Drp1 is a direct target of SUMO modification by all three SUMO isoforms. While Drp1 does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drp1 in response to staurosporine is unaffected by loss of SUMOylation, we find that Drp1 SUMOylation is enhanced in the context of the K38A mutation. This dominant-negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drp1 is linked to its activity cycle and is influenced by Drp1 localization.—Figueroa-Romero, C., Iñiguez-Lluhí, J. A., Stadler, J., Chang, C.-R., Arnoult, D., Keller, P. J., Hong, Y., Blackstone, C., Feldman, E. L. SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. PMID:19638400
Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications.

PubMed

Kang, Zhen; Huang, Hao; Zhang, Yunfeng; Du, Guocheng; Chen, Jian

2017-01-01

Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P. pastoris. Furthermore, the applications of P. pastoris towards synthetic biology were also discussed and prospected especially in the context of genome-scale metabolic pathway analysis.
Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.
Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

2013-01-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I

2013-12-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.
Agents that Stabilize Mutated von Hippel Lindau Protein Result in Differential Post-Translational Modification and Subcellular Localization

PubMed Central

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

2014-01-01

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

PubMed Central

2014-01-01

Background Salinity tolerance is a physiologically multi-faceted trait attributed to multiple mechanisms. Three barley (Hordeum vulgare) varieties contrasting in their salinity tolerance were used to assess the relative contribution of ionic, osmotic and oxidative stress components towards overall salinity stress tolerance in this species, both at the whole-plant and cellular levels. In addition, transcriptional changes in the gene expression profile were studied for key genes mediating plant ionic and oxidative homeostasis (NHX; RBOH; SOD; AHA and GORK), to compare a contribution of transcriptional and post-translational factors towards the specific components of salinity tolerance. Results Our major findings are two-fold. First, plant tissue tolerance was a dominating component that has determined the overall plant responses to salinity, with root K+ retention ability and reduced sensitivity to stress-induced hydroxyl radical production being the main contributing tolerance mechanisms. Second, it was not possible to infer which cultivars were salinity tolerant based solely on expression profiling of candidate genes at one specific time point. For the genes studied and the time point selected that transcriptional changes in the expression of these specific genes had a small role for barley’s adaptive responses to salinity. Conclusions For better tissue tolerance, sodium sequestration, K+ retention and resistance to oxidative stress all appeared to be crucial. Because these traits are highly interrelated, it is suggested that a major progress in crop breeding for salinity tolerance can be achieved only if these complementary traits are targeted at the same time. This study also highlights the essentiality of post translational modifications in plant adaptive responses to salinity. PMID:24774965
Targeting Palmitoyl Acyltransferases in Mutant NRAS-Driven Melanoma

DTIC Science & Technology

2015-10-01

activation in melanoma cells using chemical biology and functional genomic approaches. In the first year of the study, we have developed more potent...post-translational modification by adding a 16-carbon palmitate) is required for N-RAS proper membrane localization and its oncogenic activities ...RAS regulation could be a novel strategy to treat N-RAS mutant melanoma. We have developed chemical probes that covalently label the active sites of
Identification and Characterization of Post-Translational Modifications on EAF1 and EAF2 in Prostate Cancer

DTIC Science & Technology

2013-04-01

1nM R1881). All samples were normalized to renilla . B) Activity of PSA- luciferase in the presence of FOXA1 when EAF2 is over-expressed. All...samples performed in the presence of 1nM R1881. All samples were normalized to renilla . *=pɘ.05 All experiments were performed in C4-2 cells. FOXA1
Functional Characterization of CENP-A Post-Translational Modifications in Chromosome Segregation

DTIC Science & Technology

2016-09-01

our overall findings in discussion part, and finally we will explain major materials and methods we used. Results CENP-A α-amino methylation...centromere and kinetochore and accurate segregation of the genetic materials . Moreover, we established that centromere/kinetochore defects in the absence...developed. Materials and methods: Creation of CENP-A complete replacement RPE cells: RPE CENP-A knockout cell line generated by Don Cleaveland Lab7 used
Biosynthetic Tailoring of Microcin E492m: Post-Translational Modification Affords an Antibacterial Siderophore-Peptide Conjugate

PubMed Central

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

2008-01-01

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

PubMed Central

Kurotani, Atsushi; Sakurai, Tetsuya

2015-01-01

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups. PMID:26307970
In Silico Analysis of Correlations between Protein Disorder and Post-Translational Modifications in Algae.

PubMed

Kurotani, Atsushi; Sakurai, Tetsuya

2015-08-20

Recent proteome analyses have reported that intrinsically disordered regions (IDRs) of proteins play important roles in biological processes. In higher plants whose genomes have been sequenced, the correlation between IDRs and post-translational modifications (PTMs) has been reported. The genomes of various eukaryotic algae as common ancestors of plants have also been sequenced. However, no analysis of the relationship to protein properties such as structure and PTMs in algae has been reported. Here, we describe correlations between IDR content and the number of PTM sites for phosphorylation, glycosylation, and ubiquitination, and between IDR content and regions rich in proline, glutamic acid, serine, and threonine (PEST) and transmembrane helices in the sequences of 20 algae proteomes. Phosphorylation, O-glycosylation, ubiquitination, and PEST preferentially occurred in disordered regions. In contrast, transmembrane helices were favored in ordered regions. N-glycosylation tended to occur in ordered regions in most of the studied algae; however, it correlated positively with disordered protein content in diatoms. Additionally, we observed that disordered protein content and the number of PTM sites were significantly increased in the species-specific protein clusters compared to common protein clusters among the algae. Moreover, there were specific relationships between IDRs and PTMs among the algae from different groups.
CEP295 interacts with microtubules and is required for centriole elongation.

PubMed

Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C; Tang, Tang K

2016-07-01

Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. © 2016. Published by The Company of Biologists Ltd.
CEP295 interacts with microtubules and is required for centriole elongation

PubMed Central

Chang, Ching-Wen; Hsu, Wen-Bin; Tsai, Jhih-Jie; Tang, Chieh-Ju C.

2016-01-01

ABSTRACT Centriole duplication is a tightly ordered process during which procentrioles are assembled in G1-S and elongate during S and G2. Here, we show that human CEP295 (Drosophila Ana1) is not essential for initial cartwheel assembly, but is required to build distal half centrioles during S and G2. Using super-resolution and immunogold electron microscopy, we demonstrate that CEP295 is recruited to the proximal end of procentrioles in early S phase, when it is also localized at the centriolar microtubule wall that surrounds the human SAS6 cartwheel hub. Interestingly, depletion of CEP295 not only inhibits the recruitments of POC5 and POC1B to the distal half centrioles in G2, resulting in shorter centrioles, it also blocks the post-translational modification of centriolar microtubules (e.g. acetylation and glutamylation). Importantly, our results indicate that CEP295 directly interacts with microtubules, and that excess CEP295 could induce the assembly of overly long centrioles. Furthermore, exogenous expression of the N-terminal domain of CEP295 exerts a dominant-negative effect on centriole elongation. Collectively, these findings suggest that CEP295 is essential for building the distal half centrioles and for post-translational modification of centriolar microtubules. PMID:27185865
The Deep Thioredoxome in Chlamydomonas reinhardtii: New Insights into Redox Regulation.

PubMed

Pérez-Pérez, María Esther; Mauriès, Adeline; Maes, Alexandre; Tourasse, Nicolas J; Hamon, Marion; Lemaire, Stéphane D; Marchand, Christophe H

2017-08-07

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular processes. This study broadens not only the redox regulation to new enzymes involved in well-known thioredoxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce (aromatic amino acid biosynthesis, nuclear transport, etc). Moreover, we characterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.
Biosynthetic tailoring of microcin E492m: post-translational modification affords an antibacterial siderophore-peptide conjugate.

PubMed

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

2007-11-21

The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring Gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl)serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both a C10 model peptide and full-length MccE492. In the enzymatic product, the C-terminal serine residue is covalently attached to the C4' oxygen of the glucose moiety. Nonenzymatic and base-catalyzed migration of the peptide to the C6' position affords the C6' glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures.
novPTMenzy: a database for enzymes involved in novel post-translational modifications

PubMed Central

Khater, Shradha; Mohanty, Debasisa

2015-01-01

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

PubMed

Huang, Liang-Chin; Ross, Karen E; Baffi, Timothy R; Drabkin, Harold; Kochut, Krzysztof J; Ruan, Zheng; D'Eustachio, Peter; McSkimming, Daniel; Arighi, Cecilia; Chen, Chuming; Natale, Darren A; Smith, Cynthia; Gaudet, Pascale; Newton, Alexandra C; Wu, Cathy; Kannan, Natarajan

2018-04-25

Many bioinformatics resources with unique perspectives on the protein landscape are currently available. However, generating new knowledge from these resources requires interoperable workflows that support cross-resource queries. In this study, we employ federated queries linking information from the Protein Kinase Ontology, iPTMnet, Protein Ontology, neXtProt, and the Mouse Genome Informatics to identify key knowledge gaps in the functional coverage of the human kinome and prioritize understudied kinases, cancer variants and post-translational modifications (PTMs) for functional studies. We identify 32 functional domains enriched in cancer variants and PTMs and generate mechanistic hypotheses on overlapping variant and PTM sites by aggregating information at the residue, protein, pathway and species level from these resources. We experimentally test the hypothesis that S768 phosphorylation in the C-helix of EGFR is inhibitory by showing that oncogenic variants altering S768 phosphorylation increase basal EGFR activity. In contrast, oncogenic variants altering conserved phosphorylation sites in the 'hydrophobic motif' of PKCβII (S660F and S660C) are loss-of-function in that they reduce kinase activity and enhance membrane translocation. Our studies provide a framework for integrative, consistent, and reproducible annotation of the cancer kinomes.
Comprehensive Proteomics Analysis of Laticifer Latex Reveals New Insights into Ethylene Stimulation of Natural Rubber Production.

PubMed

Wang, Xuchu; Wang, Dan; Sun, Yong; Yang, Qian; Chang, Lili; Wang, Limin; Meng, Xueru; Huang, Qixing; Jin, Xiang; Tong, Zheng

2015-09-08

Ethylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.
NDE1 and NDEL1: twin neurodevelopmental proteins with similar ‘nature’ but different ‘nurture’

PubMed Central

Bradshaw, Nicholas J.; Hennah, William; Soares, Dinesh C.

2013-01-01

Nuclear distribution element 1 (NDE1, also known as NudE) and NDE-like 1 (NDEL1, also known as Nudel) are paralogous proteins essential for mitosis and neurodevelopment that have been implicated in psychiatric and neurodevelopmental disorders. The two proteins possess high sequence similarity and have been shown to physically interact with one another. Numerous lines of experimental evidence in vivo and in cell culture have demonstrated that these proteins share common functions, although instances of differing functions between the two have recently emerged. We review the key aspects of NDE1 and NDEL1 in terms of recent advances in structure elucidation and cellular function, with an emphasis on their differing mechanisms of post-translational modification. Based on a review of the literature and bioinformatics assessment, we advance the concept that the twin proteins NDE1 and NDEL1, while sharing a similar ‘nature’ in terms of their structure and basic functions, appear to be different in their ‘nurture’, the manner in which they are regulated both in terms of expression and of post-translational modification within the cell. These differences are likely to be of significant importance in understanding the specific roles of NDE1 and NDEL1 in neurodevelopment and disease. PMID:24093049
The language-related transcription factor FOXP2 is post-translationally modified with small ubiquitin-like modifiers.

PubMed

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

2016-02-12

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

PubMed Central

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

2016-01-01

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

PubMed

Hurtado-Guerrero, Ramon; Davies, Gideon J

2012-12-01

Enzymatic glycosylation of proteins, a post-transitional modification of great significance, is carried out by diverse glycosyltransferases (GTs) that harness activated sugar donors, typically nucleotide or lipid-phosphate linked species. Recent work has seen a major increase in the study of the 3D structure and reaction mechanism of these enzymes. Key advances include the dissection of the classical O-glycosylating and N-glycosylating apparatus, revealing unusual folds and hitherto unconsidered chemical mechanisms for acceptor activation. There has been considerable success in the application of kinetic isotope effects and quantum simulations to address the controversial issue of the reaction mechanism of retaining GTs. New roles for old modifications, exemplified by potential epigenetic roles for glycosylation, have been discovered and there has also been a plethora of studies into important mammalian glycosylations that play key roles in cellular biology, opening up new targets for chemical intervention approaches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Total chemical synthesis of modified histones

NASA Astrophysics Data System (ADS)

Qi, Yun-Kun; Ai, Hua-Song; Li, Yi-Ming; Yan, Baihui

2018-02-01

In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.
[Pain and emotional dysregulation: Cellular memory due to pain].

PubMed

Narita, Minoru; Watanabe, Moe; Hamada, Yusuke; Tamura, Hideki; Ikegami, Daigo; Kuzumaki, Naoko; Igarashi, Katsuhide

2015-08-01

Genetic factors are involved in determinants for the risk of psychiatric disorders, and neurological and neurodegenerative diseases. Chronic pain stimuli and intense pain have effects at a cellular and/or gene expression level, and will eventually induce "cellular memory due to pain", which means that tissue damage, even if only transient, can elicit epigenetically abnormal transcription/translation and post-translational modification in related cells depending on the degree or kind of injury or associated conditions. Such cell memory/transformation due to pain can cause an abnormality in a fundamental intracellular response, such as a change in the three-dimensional structure of DNA, transcription, or translation. On the other hand, pain is a multidimensional experience with sensory-discriminative and motivational-affective components. Recent human brain imaging studies have examined differences in activity in the nucleus accumbens between controls and patients with chronic pain, and have revealed that the nucleus accumbens plays a role in predicting the value of a noxious stimulus and its offset, and in the consequent changes in the motivational state. In this review, we provide a very brief overview of a comprehensive understanding of chronic pain associated with emotional dysregulation due to transcriptional regulation, epigenetic modification and miRNA regulation.
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
Differential expression of mucins 1-6 in papillary thyroid carcinoma: evidence for transformation-dependent post-translational modifications of MUC1 in situ.

PubMed

Magro, Gaetano; Schiappacassi, Monica; Perissinotto, Daniela; Corsaro, Antonella; Borghese, Cinzia; Belfiore, Antonino; Colombatti, Alfonso; Grasso, Sebastiano; Botti, Carlo; Bombardieri, Emilio; Perris, Roberto

2003-07-01

Mucins are primary glycoproteins of epithelia that are known to undergo major changes in their post-translational processing during neoplastic transformation. This study has examined the expression pattern of seven primary mucins, ie mucin (MUC) 1, 2, 3, 4, 5AC, 5B and 6, in normal, hyperplastic, benign neoplastic, and papillary-type carcinoma (PTC) tissues of the thyroid. MUC1 and MUC5B were the only mucins to be widely transcribed in both benign and malignant tissues. In contrast, MUC4 transcripts were undetectable in normal thyroids, and were present in only 40% of the hyperplastic and malignant thyroid tissues. In PTC, MUC1 was identified as a single mRNA transcript, rejecting the idea that this mucin may undergo transformation-dependent alternative splicing in thyroid tumours. The tissue distribution of MUC1 and MUC4 proteins was highly heterogeneous: this largely paralleled their mRNA expression profiles and supported the conclusion that whereas MUC1 was ubiquitously expressed in PTC, MUC4 was detectable in less than 20% of the cases analysed. In order to determine whether post-translational modifications of MUC1, putatively associated with malignancy, also occurred in the mucin produced by PTC, immunohistochemistry was performed with a panel of well-characterized anti-MUC1 antibodies in conjunction with digestion of the tissue sections with deglycosylating enzymes. These experiments, which were supported by immunochemical analyses of the MUC1 and MUC4 glycoforms extracted from tissues, collectively demonstrated markedly divergent MUC1 glycosylation profiles in normal and benign thyroid tissues when compared with PTC. Characteristically, these latter neoplastic cells produced mucin molecules carrying complex poly-N-lactosamine-type glycans capped with fucose and neuraminic acid residues. The present study also found evidence in PTC for the potential presence of proteolytically processed MUC1 isoforms which differ in their post-translational traits depending on whether they are retained on the cell surface or secreted into the extracellular space. It is proposed that the observed differences in the glycosylation properties of normal and neoplastic MUC1 may be exploitable as an ancillary tool in the diagnosis of PTC. Copyright 2003 John Wiley & Sons, Ltd.
Redox proteomics and the dynamic molecular landscape of the aging brain.

PubMed

Perluigi, Marzia; Swomley, Aaron M; Butterfield, D Allan

2014-01-01

It is well established that the risk to develop neurodegenerative disorders increases with chronological aging. Accumulating studies contributed to characterize the age-dependent changes either at gene and protein expression level which, taken together, show that aging of the human brain results from the combination of the normal decline of multiple biological functions with environmental factors that contribute to defining disease risk of late-life brain disorders. Finding the "way out" of the labyrinth of such complex molecular interactions may help to fill the gap between "normal" brain aging and development of age-dependent diseases. To this purpose, proteomics studies are a powerful tool to better understand where to set the boundary line of healthy aging and age-related disease by analyzing the variation of protein expression levels and the major post translational modifications that determine "protein" physio/pathological fate. Increasing attention has been focused on oxidative modifications due to the crucial role of oxidative stress in aging, in addition to the fact that this type of modification is irreversible and may alter protein function. Redox proteomics studies contributed to decipher the complexity of brain aging by identifying the proteins that were increasingly oxidized and eventually dysfunctional as a function of age. The purpose of this review is to summarize the most important findings obtained by applying proteomics approaches to murine models of aging with also a brief overview of some human studies, in particular those related to dementia. Copyright © 2014. Published by Elsevier B.V.
Characterizing the molecular architectures of chromatin-modifying complexes.

PubMed

Setiaputra, Dheva T; Yip, Calvin K

2017-11-01

Eukaryotic cells package their genome in the form of a DNA-protein complex known as chromatin. This organization not only condenses the genome to fit within the confines of the nucleus, but also provides a platform for a cell to regulate accessibility to different gene sequences. The basic packaging element of chromatin is the nucleosome, which consists of 146 base pairs of DNA wrapped around histone proteins. One major means that a cell regulates chromatin structure is by depositing post-translational modifications on nucleosomal histone proteins, and thereby altering internucleosomal interactions and/or binding to different chromatin associated factors. These chromatin modifications are often catalyzed by multi-subunit enzyme complexes, whose large size, sophisticated composition, and inherent conformational flexibility pose significant technical challenges to their biochemical and structural characterization. Multiple structural approaches including nuclear magnetic resonance spectroscopy, X-ray crystallography, single-particle electron microscopy, and crosslinking coupled to mass spectrometry are often used synergistically to probe the overall architecture, subunit organization, and catalytic mechanisms of these macromolecular assemblies. In this review, we highlight several recent chromatin-modifying complexes studies that embodies this multipronged structural approach, and explore common themes amongst them. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.
dbPAF: an integrative database of protein phosphorylation in animals and fungi.

PubMed

Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

2016-03-24

Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.
Cyclic AMP-dependent modification of gonad-selective TAF(II)105 in a human ovarian granulosa cell line.

PubMed

Wu, Yimin; Lu, Yunzhe; Hu, Yanfen; Li, Rong

2005-11-01

In response to gonadotropins, the elevated level of intracellular-cyclic AMP (cAMP) in ovarian granulosa cells triggers an ordered activation of multiple ovarian genes, which in turn promotes various ovarian functions including folliculogenesis and steroidogenesis. Identification and characterization of transcription factors that control ovarian gene expression are pivotal to the understanding of the molecular basis of the tissue-specific gene regulation programs. The recent discovery of the mouse TATA binding protein (TBP)-associated factor 105 (TAF(II)105) as a gonad-selective transcriptional co-activator strongly suggests that general transcription factors such as TFIID may play a key role in regulating tissue-specific gene expression. Here we show that the human TAF(II)105 protein is preferentially expressed in ovarian granulosa cells. We also identified a novel TAF(II)105 mRNA isoform that results from alternative exon inclusion and is predicted to encode a dominant negative mutant of TAF(II)105. Following stimulation by the adenylyl cyclase activator forskolin, TAF(II)105 in granulosa cells undergoes rapid and transient phosphorylation that is dependent upon protein kinase A (PKA). Thus, our work suggests that pre-mRNA processing and post-translational modification represent two important regulatory steps for the gonad-specific functions of human TAF(II)105. Copyright 2005 Wiley-Liss, Inc.
O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster.

PubMed

Park, Sujin; Lee, Yangsin; Pak, Jin Won; Kim, Hanbyeol; Choi, Hyeonjin; Kim, Jae-woo; Roth, Jürgen; Cho, Jin Won

2015-08-01

O-GlcNAcylation is a dynamic post-translational modification that takes place on ser/thr residues of nucleocytoplasmic proteins. O-GlcNAcylation regulates almost all cellular events as a nutrient sensor, a transcriptional and translational regulator, and a disease-related factor. Although the role of O-GlcNAcylation in insulin signaling and metabolism are well established, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we manipulated O-GlcNAcylation in Drosophila and found that it regulates autophagy through Akt/dFOXO signaling. We demonstrate that O-GlcNAcylation and the levels of O-GlcNAc transferase (OGT) are increased during starvation. Furthermore, Atg proteins and autolysosomes are increased in OGT-reduced flies without fasting. Atg proteins and autophagosomes are reduced in OGT-overexpressing flies. Our results suggest that not only autophagy gene expression but also autophagic structures are regulated by OGT through Akt and dFOXO. These data imply that O-GlcNAcylation is important in modulating autophagy as well as insulin signaling in Drosophila.
CRISPR-Mediated Epigenome Editing

PubMed Central

Enríquez, Paul

2016-01-01

Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current research gaps, forecasts future research opportunities, and argues that as epigenome editing technologies mature, overcoming critical challenges in delivery, specificity, and fidelity should clear the path to bring these technologies into the clinic. PMID:28018139
CRISPR-Mediated Epigenome Editing.

PubMed

Enríquez, Paul

2016-12-01

Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current research gaps, forecasts future research opportunities, and argues that as epigenome editing technologies mature, overcoming critical challenges in delivery, specificity, and fidelity should clear the path to bring these technologies into the clinic.
Protein S-glutathionylation: from current basics to targeted modifications.

PubMed

Popov, Doina

2014-10-01

The interaction between antioxidant glutathione and the free thiol in susceptible cysteine residues of proteins leads to reversible protein S-glutathionylation. This reaction ensures cellular homeostasis control (as a common redox-dependent post-translational modification associated with signal transduction) and intervenes in oxidative stress-related cardiovascular pathology (as initiated by redox imbalance). The purpose of this review is to evaluate the recent knowledge on protein S-glutathionylation in terms of chemistry, broad cellular intervention, specific quantification, and potential for therapeutic exploitation. The data bases searched were Medline and PubMed, from 2009 to 2014 (term: glutathionylation). Protein S-glutathionylation ensures protection of protein thiols against irreversible over-oxidation, operates as a biological redox switch in both cell survival (influencing kinases and protein phosphatases pathways) and cell death (by potentiation of apoptosis), and cross-talks with phosphorylation and with S-nitrosylation. Collectively, protein S-glutathionylation appears as a valuable biomarker for oxidative stress, with potential for translation into novel therapeutic strategies.
Non-degradative Ubiquitination of Protein Kinases

PubMed Central

Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

2016-01-01

Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329
Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.

PubMed

Uda, Munehiro; Kawasaki, Hiroaki; Iizumi, Kyoichi; Shigenaga, Ayako; Baba, Takeshi; Naito, Hisashi; Yoshioka, Toshitada; Yamakura, Fumiyuki

2015-11-01

Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.
Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

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

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less
POTAMOS mass spectrometry calculator: computer aided mass spectrometry to the post-translational modifications of proteins. A focus on histones.

PubMed

Vlachopanos, A; Soupsana, E; Politou, A S; Papamokos, G V

2014-12-01

Mass spectrometry is a widely used technique for protein identification and it has also become the method of choice in order to detect and characterize the post-translational modifications (PTMs) of proteins. Many software tools have been developed to deal with this complication. In this paper we introduce a new, free and user friendly online software tool, named POTAMOS Mass Spectrometry Calculator, which was developed in the open source application framework Ruby on Rails. It can provide calculated mass spectrometry data in a time saving manner, independently of instrumentation. In this web application we have focused on a well known protein family of histones whose PTMs are believed to play a crucial role in gene regulation, as suggested by the so called "histone code" hypothesis. The PTMs implemented in this software are: methylations of arginines and lysines, acetylations of lysines and phosphorylations of serines and threonines. The application is able to calculate the kind, the number and the combinations of the possible PTMs corresponding to a given peptide sequence and a given mass along with the full set of the unique primary structures produced by the possible distributions along the amino acid sequence. It can also calculate the masses and charges of a fragmented histone variant, which carries predefined modifications already implemented. Additional functionality is provided by the calculation of the masses of fragments produced upon protein cleavage by the proteolytic enzymes that are most widely used in proteomics studies. Copyright © 2014 Elsevier Ltd. All rights reserved.
Ancient Regulatory Role of Lysine Acetylation in Central Metabolism

DOE PAGES

Nakayasu, Ernesto S.; Burnet, Meagan C.; Walukiewicz, Hanna E.; ...

2017-11-28

ABSTRACT Lysine acetylation is a common protein post-translational modification in bacteria and eukaryotes. Unlike phosphorylation, whose functional role in signaling has been established, it is unclear what regulatory mechanism acetylation plays and whether it is conserved across evolution. By performing a proteomic analysis of 48 phylogenetically distant bacteria, we discovered conserved acetylation sites on catalytically essential lysine residues that are invariant throughout evolution. Lysine acetylation removes the residue’s charge and changes the shape of the pocket required for substrate or cofactor binding. Two-thirds of glycolytic and tricarboxylic acid (TCA) cycle enzymes are acetylated at these critical sites. Our data suggestmore » that acetylation may play a direct role in metabolic regulation by switching off enzyme activity. We propose that protein acetylation is an ancient and widespread mechanism of protein activity regulation. IMPORTANCE Post-translational modifications can regulate the activity and localization of proteins inside the cell. Similar to phosphorylation, lysine acetylation is present in both eukaryotes and prokaryotes and modifies hundreds to thousands of proteins in cells. However, how lysine acetylation regulates protein function and whether such a mechanism is evolutionarily conserved is still poorly understood. Here, we investigated evolutionary and functional aspects of lysine acetylation by searching for acetylated lysines in a comprehensive proteomic data set from 48 phylogenetically distant bacteria. We found that lysine acetylation occurs in evolutionarily conserved lysine residues in catalytic sites of enzymes involved in central carbon metabolism. Moreover, this modification inhibits enzymatic activity. Our observations suggest that lysine acetylation is an evolutionarily conserved mechanism of controlling central metabolic activity by directly blocking enzyme active sites.« less
DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodríguez-Romero, Adela, E-mail: adela@unam.mx; Hernández-Santoyo, Alejandra; Fuentes-Silva, Deyanira

This study describes the three-dimensional structure of the endogenous glycosylated allergen Hev b 2 (endo-β-1,3-glucanase), which exhibits three post-translational modifications that form a patch on the surface of the molecule that is proposed to be an allergenic IgE epitope. Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibitsmore » three post-translational modifications, including an N-terminal pyroglutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein.« less
Functional Interplay between Small Non-Coding RNAs and RNA Modification in the Brain.

PubMed

Leighton, Laura J; Bredy, Timothy W

2018-06-07

Small non-coding RNAs are essential for transcription, translation and gene regulation in all cell types, but are particularly important in neurons, with known roles in neurodevelopment, neuroplasticity and neurological disease. Many small non-coding RNAs are directly involved in the post-transcriptional modification of other RNA species, while others are themselves substrates for modification, or are functionally modulated by modification of their target RNAs. In this review, we explore the known and potential functions of several distinct classes of small non-coding RNAs in the mammalian brain, focusing on the newly recognised interplay between the epitranscriptome and the activity of small RNAs. We discuss the potential for this relationship to influence the spatial and temporal dynamics of gene activation in the brain, and predict that further research in the field of epitranscriptomics will identify interactions between small RNAs and RNA modifications which are essential for higher order brain functions such as learning and memory.

The Perseus computational platform for comprehensive analysis of (prote)omics data.

PubMed

Tyanova, Stefka; Temu, Tikira; Sinitcyn, Pavel; Carlson, Arthur; Hein, Marco Y; Geiger, Tamar; Mann, Matthias; Cox, Jürgen

2016-09-01

A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.
Mammalian O-Mannosylation Pathway: Glycan Structures, Enzymes, and Protein Substrates

PubMed Central

2015-01-01

The mammalian O-mannosylation pathway for protein post-translational modification is intricately involved in modulating cell–matrix interactions in the musculature and nervous system. Defects in enzymes of this biosynthetic pathway are causative for multiple forms of congenital muscular dystophy. The application of advanced genetic and biochemical technologies has resulted in remarkable progress in this field over the past few years, culminating with the publication of three landmark papers in 2013 alone. In this review, we will highlight recent progress focusing on the dramatic expansion of the set of genes known to be involved in O-mannosylation and disease processes, the concurrent acceleration of the rate of O-mannosylation pathway protein functional assignments, the tremendous increase in the number of proteins now known to be modified by O-mannosylation, and the recent progress in protein O-mannose glycan quantification and site assignment. Also, we attempt to highlight key outstanding questions raised by this abundance of new information. PMID:24786756
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
Huntington’s Disease: The Past, Present, and Future Search for Disease Modifiers

PubMed Central

Clabough, Erin B.D.

2013-01-01

Huntington’s disease (HD) is an autosomal dominant genetic disorder that specifically causes neurodegeneration of striatal neurons, resulting in a triad of symptoms that includes emotional, cognitive, and motor disturbances. The HD mutation causes a polyglutamine repeat expansion within the N-terminal of the huntingtin (Htt) protein. This expansion causes aggregate formation within the cytosol and nucleus due to the presence of misfolded mutant Htt, as well as altered interactions with Htt’s multiple binding partners, and changes in post-translational Htt modifications. The present review charts efforts toward a therapy that delays age of onset or slows symptom progression in patients affected by HD, as there is currently no effective treatment. Although silencing Htt expression appears promising as a disease modifying treatment, it should be attempted with caution in light of Htt’s essential roles in neural maintenance and development. Other therapeutic targets include those that boost aggregate dissolution, target excitotoxicity and metabolic issues, and supplement growth factors. PMID:23766742
Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

PubMed Central

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965
Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

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

Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar

2011-03-18

Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependentmore » functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.« less
Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

PubMed

Pang, Xiaodong; Zhou, Huan-Xiang

2014-05-01

Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.
Mass Spectrometric Immunoassay and Multiple Reaction Monitoring as Targeted MS-based Quantitative Approaches in Biomarker Development: Potential Applications to Cardiovascular Disease and Diabetes

PubMed Central

Yassine, Hussein; Borges, Chad R.; Schaab, Matthew R.; Billheimer, Dean; Stump, Craig; Reaven, Peter; Lau, Serrine S.; Nelson, Randall

2014-01-01

Type 2 diabetes (T2DM) is an important risk factor for cardiovascular disease (CVD)—the leading cause of death in the US. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed towards treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary mass spectrometry-based approaches—Mass Spectrometric Immunoassay (MSIA) and tandem mass spectrometry as multiple reaction monitoring (MRM)—for the analysis of plasma proteins and post translational modifications (PTMs) of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the HDL proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on high density lipoprotein (HDL) proteins from T2DM patients to provide examples of how these mass spectrometry approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice. PMID:23696124
Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

PubMed Central

Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

2014-01-01

To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields. PMID:25000943
Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

PubMed

Li, Jiang; Du, Xingrong; Shi, Hao; Deng, Kejing; Chi, Hongbo; Tao, Wufan

2015-12-25

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-translational Modification of Extremophilic Proteins: N-glycosylation in Archaea

DTIC Science & Technology

2014-12-02

Kaminski, Z. Guan, S. Yurist-Doutsch, J. Eichler. Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes...Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans, Applied and Environmental Microbiology, (11 2013...Archaea, N-‐linked oligosaccharides are assembled on dolichol phosphate prior to transfer of the glycan
[Animals' clever adaptation strategy for seasonal changes in environment].

PubMed

Ikegami, Keisuke; Yoshimura, Takashi

2015-08-01

Organisms living outside of tropical zones experience seasonal changes in environment. Organisms are using day length as a calendar to change their physiology and behavior such as seasonal breeding, hibernation, migration, and molting. A comparative biology approach revealed underlying mechanisms of vertebrate seasonal reproduction. Here we review the current understanding of vertebrate seasonal reproduction. We Aso describe the involvement of tissue-specific post-translational modification in functional diversification of a hormone.
A machine learning approach for predicting methionine oxidation sites.

PubMed

Aledo, Juan C; Cantón, Francisco R; Veredas, Francisco J

2017-09-29

The oxidation of protein-bound methionine to form methionine sulfoxide, has traditionally been regarded as an oxidative damage. However, recent evidences support the view of this reversible reaction as a regulatory post-translational modification. The perception that methionine sulfoxidation may provide a mechanism to the redox regulation of a wide range of cellular processes, has stimulated some proteomic studies. However, these experimental approaches are expensive and time-consuming. Therefore, computational methods designed to predict methionine oxidation sites are an attractive alternative. As a first approach to this matter, we have developed models based on random forests, support vector machines and neural networks, aimed at accurate prediction of sites of methionine oxidation. Starting from published proteomic data regarding oxidized methionines, we created a hand-curated dataset formed by 113 unique polypeptides of known structure, containing 975 methionyl residues, 122 of which were oxidation-prone (positive dataset) and 853 were oxidation-resistant (negative dataset). We use a machine learning approach to generate predictive models from these datasets. Among the multiple features used in the classification task, some of them contributed substantially to the performance of the predictive models. Thus, (i) the solvent accessible area of the methionine residue, (ii) the number of residues between the analyzed methionine and the next methionine found towards the N-terminus and (iii) the spatial distance between the atom of sulfur from the analyzed methionine and the closest aromatic residue, were among the most relevant features. Compared to the other classifiers we also evaluated, random forests provided the best performance, with accuracy, sensitivity and specificity of 0.7468±0.0567, 0.6817±0.0982 and 0.7557±0.0721, respectively (mean ± standard deviation). We present the first predictive models aimed to computationally detect methionine sites that may become oxidized in vivo in response to oxidative signals. These models provide insights into the structural context in which a methionine residue become either oxidation-resistant or oxidation-prone. Furthermore, these models should be useful in prioritizing methinonyl residues for further studies to determine their potential as regulatory post-translational modification sites.
Genomics, transcriptomics and proteomics to elucidate the pathogenesis of rheumatoid arthritis.

PubMed

Song, Xinqiang; Lin, Qingsong

2017-08-01

Rheumatoid arthritis is an autoimmune disease that affects several organs and tissues, predominantly the synovial joints. The pathogenesis of this disease is not completely understood, which maybe involved in the genomic variations, gene expression, protein translation and post-translational modifications. These system variations in genomics, transcriptomics and proteomics are dynamic in nature and their crosstalk is overwhelmingly complex, thus analyzing them separately may not be very informative. However, various '-omics' techniques developed in recent years have opened up new possibilities for clarifying disease pathways and thereby facilitating early diagnosis and specific therapies. This review examines how recent advances in the fields of genomics, transcriptomics and proteomics have contributed to our understanding of rheumatoid arthritis.
Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide

PubMed Central

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

2014-01-01

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H218O2, we verified incorporation of 18O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics. PMID:24939847
Absence of post-translational aspartyl beta-hydroxylation of epidermal growth factor domains in mice leads to developmental defects and an increased incidence of intestinal neoplasia.

PubMed

Dinchuk, Joseph E; Focht, Richard J; Kelley, Jennifer A; Henderson, Nancy L; Zolotarjova, Nina I; Wynn, Richard; Neff, Nicola T; Link, John; Huber, Reid M; Burn, Timothy C; Rupar, Mark J; Cunningham, Mark R; Selling, Bernard H; Ma, Jianhong; Stern, Andrew A; Hollis, Gregory F; Stein, Robert B; Friedman, Paul A

2002-04-12

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl beta-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, beta-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl beta-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.
Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

PubMed

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

2014-08-08

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Dynamic m6 A RNA Methylation in Photobiology.

PubMed

Robinson, Myles; Shah, Palak; Cui, Yan-Hong; He, Yu-Ying

2018-05-04

N 6 -methyladenosine (m 6 A) is the most abundant internal RNA modification among numerous post-transcriptional modifications identified in eukaryotic mRNA. m 6 A modification of RNA is catalyzed by the "writer" m 6 A methyltransferase enzyme complex, consisting of METTL3, METTL14, WTAP and KIAA1429. The m 6 A modification is reversible and can be removed by "eraser" m 6 A demethylase enzymes, namely, FTO and ALKBH5. The biological function of m 6 A modification on RNA is carried out by RNA-binding effector proteins called "readers." Varied functions of the reader proteins regulate mRNA metabolism by affecting stability, translation, splicing or nuclear export. The epitranscriptomic gene regulation by m 6 A RNA methylation regulates various pathways, which contribute to basic cellular processes essential for cell maintenance, development and cell fate, and affect response to external stimuli and stressors. In this review, we summarize the recent advances in the regulation and function of m 6 A RNA methylation, with a focus on UV-induced DNA damage response and the circadian clock machinery. Insights into the mechanisms of m 6 A RNA regulation and post-transcriptional regulatory function in these biological processes may facilitate the development of new preventive and therapeutic strategies for various diseases related to dysregulation of UV damage response and circadian rhythm. © 2018 The American Society of Photobiology.
An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

PubMed

Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

2016-06-28

Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing. Copyright © 2016 Elsevier B.V. All rights reserved.
Metabolic memory and chronic diabetes complications: potential role for epigenetic mechanisms.

PubMed

Intine, Robert V; Sarras, Michael P

2012-10-01

Recent estimates indicate that diabetes mellitus currently affects more than 10 % of the world's population. Evidence from both the laboratory and large scale clinical trials has revealed that prolonged hyperglycemia induces chronic complications which persist and progress unimpeded even when glycemic control is pharmaceutically achieved via the phenomenon of metabolic memory. The epigenome is comprised of all chromatin modifications including post translational histone modification, expression control via miRNAs and the methylation of cytosine within DNA. Modifications of these epigenetic marks not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters. As such, these processes have gained much attention as potential molecular mechanisms underlying metabolic memory and chronic diabetic complications. Here we present a review of the very recent literature published pertaining to this subject.

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.
Metrics for the Human Proteome Project 2016: Progress on Identifying and Characterizing the Human Proteome, Including Post-Translational Modifications.

PubMed

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

2016-11-04

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

PubMed

Kuhn, Claus-D

2016-05-01

tRNAs undergo multiple conformational changes during the translation cycle that are required for tRNA translocation and proper communication between the ribosome and translation factors. Recent structural data on how destabilized tRNAs utilize the CCA-adding enzyme to proofread themselves put a spotlight on tRNA flexibility beyond the translation cycle. In analogy to tRNA surveillance, this review finds that other processes also exploit versatile tRNA folding to achieve, amongst others, specific aminoacylation, translational regulation by riboswitches or a block of bacterial translation. tRNA flexibility is thereby not restricted to the hinges utilized during translation. In contrast, the flexibility of tRNA is distributed all over its L-shape and is actively exploited by the tRNA-interacting partners to discriminate one tRNA from another. Since the majority of tRNA modifications also modulate tRNA flexibility it seems that cells devote enormous resources to tightly sense and regulate tRNA structure. This is likely required for error-free protein synthesis. © 2016 WILEY Periodicals, Inc.
tRNA tKUUU, tQUUG, and tEUUC wobble position modifications fine-tune protein translation by promoting ribosome A-site binding.

PubMed

Rezgui, Vanessa Anissa Nathalie; Tyagi, Kshitiz; Ranjan, Namit; Konevega, Andrey L; Mittelstaet, Joerg; Rodnina, Marina V; Peter, Matthias; Pedrioli, Patrick G A

2013-07-23

tRNA modifications are crucial to ensure translation efficiency and fidelity. In eukaryotes, the URM1 and ELP pathways increase cellular resistance to various stress conditions, such as nutrient starvation and oxidative agents, by promoting thiolation and methoxycarbonylmethylation, respectively, of the wobble uridine of cytoplasmic (tK(UUU)), (tQ(UUG)), and (tE(UUC)). Although in vitro experiments have implicated these tRNA modifications in modulating wobbling capacity and translation efficiency, their exact in vivo biological roles remain largely unexplored. Using a combination of quantitative proteomics and codon-specific translation reporters, we find that translation of a specific gene subset enriched for AAA, CAA, and GAA codons is impaired in the absence of URM1- and ELP-dependent tRNA modifications. Moreover, in vitro experiments using native tRNAs demonstrate that both modifications enhance binding of tK(UUU) to the ribosomal A-site. Taken together, our data suggest that tRNA thiolation and methoxycarbonylmethylation regulate translation of genes with specific codon content.
Regulation of Mammary Stem Cell Quiescence via Post-Translational Modification of DeltaNp63alpha

DTIC Science & Technology

2014-02-01

mRNA expression in atopic dermatitis following narrow- band ultraviolet B phototherapy: results of a pilot study. J Dermatol Sci 44: 56– 58. 39. Lee...Change II site-directed mutagenesis kit ( Cat # 200523-5) with the following set of primers; sense primer 59-CGATGCTCTCGCTCCAGCACCCGC- CATCCCCTCC-39, and...Invitrogen, Cat #11668) according to manufacturers protocol. Kinome Screen The Silencer Select Human Kinase siRNA Library ( Cat - alog#4397918) from Ambion
Post-translational modifications are key players of the Legionella pneumophila infection strategy

PubMed Central

Michard, Céline; Doublet, Patricia

2015-01-01

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

PubMed Central

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

2009-01-01

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

PubMed Central

Barone, Rosario; Rappa, Francesca; Macaluso, Filippo; Caruso Bavisotto, Celeste; Sangiorgi, Claudia; Di Paola, Gaia; Tomasello, Giovanni; Di Felice, Valentina; Marcianò, Vito; Farina, Felicia; Zummo, Giovanni; Conway de Macario, Everly; J.L. Macario, Alberto; Cocchi, Massimo; Cappello, MD, Francesco; Marino Gammazza, Antonella

2016-01-01

Objectives: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. Methods: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. Results: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. Conclusions: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease. PMID:26795070
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.
Liganded and unliganded activation of estrogen receptor and hormone replacement therapies

PubMed Central

Maggi, Adriana

2011-01-01

Over the past two decades, our understanding of estrogen receptor physiology in mammals widened considerably as we acquired a deeper appreciation of the roles of estrogen receptor alpha and beta (ERα and ERβ) in reproduction as well as in bone and metabolic homeostasis, depression, vascular disorders, neurodegenerative diseases and cancer. In addition, our insights on ER transcriptional functions in cells increased considerably with the demonstration that ER activity is not strictly dependent on ligand availability. Indeed, unliganded ERs may be transcriptionally active and post-translational modifications play a major role in this context. The finding that several intracellular transduction molecules may regulate ER transcriptional programs indicates that ERs may act as a hub where several molecular pathways converge: this allows to maintain ER transcriptional activity in tune with all cell functions. Likely, the biological relevant role of ER was favored by evolution as a mean of integration between reproductive and metabolic functions. We here review the post-translational modifications modulating ER transcriptional activity in the presence or in the absence of estrogens and underline their potential role for ER tissue-specific activities. In our opinion, a better comprehension of the variety of molecular events that control ER activity in reproductive and non-reproductive organs is the foundation for the design of safer and more efficacious hormone-based therapies, particularly for menopause. PMID:21605666
Extensive and systematic rewiring of histone post-translational modifications in cancer model systems.

PubMed

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

2018-05-04

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

PubMed Central

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

2018-01-01

Abstract Histone post-translational modifications (PTMs) generate a complex combinatorial code that regulates gene expression and nuclear functions, and whose deregulation has been documented in different types of cancers. Therefore, the availability of relevant culture models that can be manipulated and that retain the epigenetic features of the tissue of origin is absolutely crucial for studying the epigenetic mechanisms underlying cancer and testing epigenetic drugs. In this study, we took advantage of quantitative mass spectrometry to comprehensively profile histone PTMs in patient tumor tissues, primary cultures and cell lines from three representative tumor models, breast cancer, glioblastoma and ovarian cancer, revealing an extensive and systematic rewiring of histone marks in cell culture conditions, which includes a decrease of H3K27me2/me3, H3K79me1/me2 and H3K9ac/K14ac, and an increase of H3K36me1/me2. While some changes occur in short-term primary cultures, most of them are instead time-dependent and appear only in long-term cultures. Remarkably, such changes mostly revert in cell line- and primary cell-derived in vivo xenograft models. Taken together, these results support the use of xenografts as the most representative models of in vivo epigenetic processes, suggesting caution when using cultured cells, in particular cell lines and long-term primary cultures, for epigenetic investigations. PMID:29618087
Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

PubMed

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

2018-01-05

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

PubMed

Xu, Yan-Ming; Huang, Dong-Yang; Chiu, Jen-Fu; Lau, Andy T Y

2012-05-04

Heat shock factors (HSFs) are vital for modulating stress and heat shock-related gene expression in cells. The activity of HSFs is controlled largely by post-translational modifications (PTMs). For example, basal phosphorylation of HSF1 on three serine sites suppresses the heat shock response, and hyperphosphorylation of HSF1 on several other serine and threonine sites by stress-activated kinases results in its activation, while acetylation on K80 inhibits its DNA-binding ability. Sumoylation of HSF2 on K82 regulates its DNA-binding ability, whereas sumoylation of HSF4B on K293 represses its transcriptional activity. With the advancement of proteomic technology, novel PTM sites on various HSFs have been identified with the use of tandem mass spectrometry (MS/MS), but the functions of many of these PTMs are still unclear. Yet, it should be noted that the discovery of these novel PTM sites provided the necessary evidence for the existence of these PTM marks in vivo. Followed by subsequent functional analysis, this would ultimately lead to a better understanding of these PTM marks. MS/MS-based proteomic approach is becoming a gold standard in PTM validation in the field of life science. Here, the recent literature of all known PTMs reported on human HSFs and the resulting functions will be discussed.
Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

PubMed

Lin, Yi-Han; Machner, Matthias P

2017-06-15

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.
SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

PubMed Central

Wu, Ching-Shyi; Ouyang, Jian; Mori, Eiichiro; Nguyen, Hai Dang; Maréchal, Alexandre; Hallet, Alexander; Chen, David J.; Zou, Lee

2014-01-01

The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR–Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11–RAD50–NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway. PMID:24990965
Chemical-agnostic hazard prediction: statistical inference of in ...

EPA Pesticide Factsheets

Toxicity pathways have been defined as normal cellular pathways that, when sufficiently perturbed as a consequence of chemical exposure, lead to an adverse outcome. If an exposure alters one or more normal biological pathways to an extent that leads to an adverse toxicity outcome, a significant correlation must exist between the exposure, the extent of pathway alteration, and the degree of adverse outcome. Biological pathways are regulated at multiple levels, including transcriptional, post-transcriptional, post-translational, and targeted degradation, each of which can affect the levels and extents of modification of proteins involved in the pathways. Significant alterations of toxicity pathways resulting from changes in regulation at any of these levels therefore are likely to be detectable as alterations in the proteome. We hypothesize that significant correlations between exposures, adverse outcomes, and changes in the proteome have the potential to identify putative toxicity pathways, facilitating selection of candidate targets for high throughput screening, even in the absence of a priori knowledge of either the specific pathways involved or the specific agents inducing the pathway alterations. We explored this hypothesis in vitro in BEAS-2B human airway epithelial cells exposed to different concentrations of Ni2+, Cd2+, and Cr6+, alone and in defined mixtures. Levels and phosphorylation status of a variety of signaling pathway proteins and cytokines were
Progress in Top-Down Proteomics and the Analysis of Proteoforms

PubMed Central

Toby, Timothy K.; Fornelli, Luca; Kelleher, Neil L.

2017-01-01

From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research. PMID:27306313
Epigenetic drugs that do not target enzyme activity.

PubMed

Owen, Dafydd R; Trzupek, John D

2014-06-01

While the installation and removal of epigenetic post-translational modifications or ‘marks’ on both DNA and histone proteins are the tangible outcome of enzymatically catalyzed processes, the role of the epigenetic reader proteins looks, at first, less obvious. As they do not catalyze a chemical transformation or process as such, their role is not enzymatic. However, this does not preclude them from being potential targets for drug discovery as their function is clearly correlated to transcriptional activity and as a class of proteins, they appear to have binding sites of sufficient definition and size to be inhibited by small molecules. This suggests that this third class of epigenetic proteins that are involved in the interpretation of post-translational marks (as opposed to the creation or deletion of marks) may represent attractive targets for drug discovery efforts. This review mainly summarizes selected publications, patent literature and company disclosures on these non-enzymatic epigenetic reader proteins from 2009 to the present. © 2014 Elsevier Ltd . All rights reserved.

Alterations of collagen matrix in weight-bearing bones during skeletal unloading

NASA Technical Reports Server (NTRS)

Shiiba, M.; Arnaud, S. B.; Tanzawa, H.; Uzawa, K.; Yamauchi, M.

2001-01-01

Skeletal unloading induces loss of bone mineral density in weight-bearing bones. The objectives of this study were to characterize the post-translational modifications of collagen of weight-bearing bones subjected to hindlimb unloading for 8 weeks. In unloaded bones, tibiae and femurs, while the overall amino acid composition was essentially identical in the unloaded and control tibiae and femurs, the collagen cross-link profile showed significant differences. Two major reducible cross-links (analyzed as dihydroxylysinonorleucine and hydroxylysinonorleucine) were increased in the unloaded bones. In addition, the ratios of the former to the latter as well as pyridinoline to deoxypyridinoline were significantly decreased in the unloaded bones indicating a difference in the extent of lysine hydroxylation at the cross-linking sites between these two groups. These results indicate that upon skeletal unloading the relative pool of newly synthesized collagen is increased and it is post-translationally altered. The alteration could be associated with impaired osteoblastic differentiation induced by skeletal unloading that results in a mineralization defect.
Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

PubMed Central

Malaker, Stacy A.; Penny, Sarah A.; Steadman, Lora G.; Myers, Paisley T.; Loke, Justin C; Raghavan, Manoj; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Cobbold, Mark

2017-01-01

Leukemias are highly immunogenic but have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these post-translationally modified neoantigens provide logical targets for cancer immunotherapy. PMID:28314751
Melanoma differentiation associated gene-7/interleukin-24 induces caspase-3 denitrosylation to facilitate the activation of cancer cell apoptosis.

PubMed

Tian, Hui; Zhang, De-Fang; Zhang, Bao-Fu; Li, Hui-Zhong; Zhang, Qing; Li, Lian-Tao; Pei, Dong-Sheng; Zheng, Jun-Nian

2015-03-01

Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) induces caspase-3 cleavage and subsequent activation via the intrinsic or extrinsic pathway to result in cancer cell-selective apoptosis, but whether mda-7/IL-24 may directly regulate caspase-3 through the post-translational modification remains unknown. Here, we reported that tumor-selective replicating adenovirus ZD55-IL-24 led to caspase-3 denitrosylation and subsequent activation, indicating that caspase-3 denitrosylation played a crucial role in ZD55-IL-24-induced cancer cell apoptosis. To confirm the relationship between caspase-3 denitrosylation and its activation in response to ZD55-IL-24, we treated carcinoma cells with the different nitric oxide (NO) regulators to modulate caspase-3 denitrosylation level, then observed the corresponding caspase-3 cleavage. We found that NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) promoted caspase-3 denitrosylation and caspase-3 cleavage, thereby exacerbating ZD55-IL-24-induced cancer cell apoptosis, whereas NO donor sodium nitroprusside (SNP) showed the opposite effect. Moreover, caspase-3 denitrosylation facilitated its downstream target poly ADP-ribose polymerase (PARP) degradation that further increased the apoptotic susceptibility. Although caspase-3 activation controlled by denitrosylation modification has emerged as an important regulator of programmed cell death, the detailed molecular mechanism by which caspase-3 exerts its denitrosylation modification in response to ZD55-IL-24 still needs to be elucidated. Thus, our results demonstrated that ZD55-IL-24 increased Fas expression to enhance thioredoxin reductase 2 (TrxR2), which was responsible for caspase-3 denitrosylation. Collectively, these findings elucidate that ZD55-IL-24 induces caspase-3 denitrosylation through Fas-mediated TrxR2 enhancement, thereby facilitating caspase-3 cleavage and the downstream caspase signaling pathway activation, which provides a novel insight into ZD55-IL-24-induced cancer-specific apoptosis by post-translational modification of the apoptotic executor caspase-3.
Bioinformatics analysis reveals biophysical and evolutionary insights into the 3-nitrotyrosine post-translational modification in the human proteome

PubMed Central

Ng, John Y.; Boelen, Lies; Wong, Jason W. H.

2013-01-01

Protein 3-nitrotyrosine is a post-translational modification that commonly arises from the nitration of tyrosine residues. This modification has been detected under a wide range of pathological conditions and has been shown to alter protein function. Whether 3-nitrotyrosine is important in normal cellular processes or is likely to affect specific biological pathways remains unclear. Using GPS-YNO2, a recently described 3-nitrotyrosine prediction algorithm, a set of predictions for nitrated residues in the human proteome was generated. In total, 9.27 per cent of the proteome was predicted to be nitratable (27 922/301 091). By matching the predictions against a set of curated and experimentally validated 3-nitrotyrosine sites in human proteins, it was found that GPS-YNO2 is able to predict 73.1 per cent (404/553) of these sites. Furthermore, of these sites, 42 have been shown to be nitrated endogenously, with 85.7 per cent (36/42) of these predicted to be nitrated. This demonstrates the feasibility of using the predicted dataset for a whole proteome analysis. A comprehensive bioinformatics analysis was subsequently performed on predicted and all experimentally validated nitrated tyrosine. This found mild but specific biophysical constraints that affect the susceptibility of tyrosine to nitration, and these may play a role in increasing the likelihood of 3-nitrotyrosine to affect processes, including phosphorylation and DNA binding. Furthermore, examining the evolutionary conservation of predicted 3-nitrotyrosine showed that, relative to non-nitrated tyrosine residues, 3-nitrotyrosine residues are generally less conserved. This suggests that, at least in the majority of cases, 3-nitrotyrosine is likely to have a deleterious effect on protein function and less likely to be important in normal cellular function. PMID:23389939
In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

PubMed

Schulze-Krebs, Anja; Canneva, Fabio; Schnepf, Rebecca; Dobner, Julia; Dieterich, Walburga; von Hörsten, Stephan

2016-01-15

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression. Copyright © 2015 Elsevier B.V. All rights reserved.
Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress*

PubMed Central

Tong, Jiefei; Taylor, Paul; Moran, Michael F.

2014-01-01

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S1104. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation. PMID:24797263
SUMOylation of HSP27 by small ubiquitin-like modifier 2/3 promotes proliferation and invasion of hepatocellular carcinoma cells.

PubMed

Ge, Haize; Du, Juan; Xu, Jingman; Meng, Xiangliang; Tian, Jinchuan; Yang, Jie; Liang, Huimin

2017-08-03

Primary hepatocellular carcinoma (PHC) is a major health problem worldwide and is one of the 10 most commonly diagnosed cancers in China. Heat shock protein 27 (HSP27) were found to be overexpressed in a wide range of malignancies including PHC, however, post-translational modification of HSP27 still needs exploration in PHC. Recently, SUMOylation, an important post-translational modification associating with the development of many kinds of cancers has been intensively studied. In the current study, mRNA and protein level of HSP27 in archived tumor samples representing various pathological characteristics of PHC were examined, and modification of HSP27 by SUMO2/3 was investigated. HSP27 were expressed abundantly in patients' tumor tissues, and found to be associated with pathological progression. Besides, HSP27 was also elevated significantly in liver cancer cell lines Huh7 and HepG2 compared with human hepatocyte cells L02. Furthermore, knockdown of HSP27 was found to be associated with the decreased proliferation and invasion ability in Huh7 and HepG2 cells. Immunofluorescence assay showed that HSP27 and SUMO2/3 were co-localized in the subcellular, and co-immunoprecipitation verified the interaction between HSP27 and SUMO2/3. Overexpression of SUMO2/3 upregulated the HSP27 protein level and promotes Huh7 and HepG2 cell proliferation and invasion, and vice versa when the SUMO2/3 was knockdown. Taken together, increased protein level of HSP27 through SUMO2/3-mediated SUMOylation plays crucial roles in the progression of PHC, and this finding may shed light on developing potential therapeutic targets for PHC.
Post-translational Maturation of Dystroglycan Is Necessary for Pikachurin Binding and Ribbon Synaptic Localization*

PubMed Central

Kanagawa, Motoi; Omori, Yoshihiro; Sato, Shigeru; Kobayashi, Kazuhiro; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Endo, Tamao; Furukawa, Takahisa; Toda, Tatsushi

2010-01-01

Pikachurin, the most recently identified ligand of dystroglycan, plays a crucial role in the formation of the photoreceptor ribbon synapse. It is known that glycosylation of dystroglycan is necessary for its ligand binding activity, and hypoglycosylation is associated with a group of muscular dystrophies that often involve eye abnormalities. Because little is known about the interaction between pikachurin and dystroglycan and its impact on molecular pathogenesis, here we characterize the interaction using deletion constructs and mouse models of muscular dystrophies with glycosylation defects (Largemyd and POMGnT1-deficient mice). Pikachurin-dystroglycan binding is calcium-dependent and relatively less sensitive to inhibition by heparin and high NaCl concentration, as compared with other dystroglycan ligand proteins. Using deletion constructs of the laminin globular domains in the pikachurin C terminus, we show that a certain steric structure formed by the second and the third laminin globular domains is necessary for the pikachurin-dystroglycan interaction. Binding assays using dystroglycan deletion constructs and tissue samples from Large-deficient (Largemyd) mice show that Large-dependent modification of dystroglycan is necessary for pikachurin binding. In addition, the ability of pikachurin to bind to dystroglycan prepared from POMGnT1-deficient mice is severely reduced, suggesting that modification of the GlcNAc-β1,2-branch on O-mannose is also necessary for the interaction. Immunofluorescence analysis reveals a disruption of pikachurin localization in the photoreceptor ribbon synapse of these model animals. Together, our data demonstrate that post-translational modification on O-mannose, which is mediated by Large and POMGnT1, is essential for pikachurin binding and proper localization, and suggest that their disruption underlies the molecular pathogenesis of eye abnormalities in a group of muscular dystrophies. PMID:20682766
Identifying the Machine Translation Error Types with the Greatest Impact on Post-editing Effort.

PubMed

Daems, Joke; Vandepitte, Sonia; Hartsuiker, Robert J; Macken, Lieve

2017-01-01

Translation Environment Tools make translators' work easier by providing them with term lists, translation memories and machine translation output. Ideally, such tools automatically predict whether it is more effortful to post-edit than to translate from scratch, and determine whether or not to provide translators with machine translation output. Current machine translation quality estimation systems heavily rely on automatic metrics, even though they do not accurately capture actual post-editing effort. In addition, these systems do not take translator experience into account, even though novices' translation processes are different from those of professional translators. In this paper, we report on the impact of machine translation errors on various types of post-editing effort indicators, for professional translators as well as student translators. We compare the impact of MT quality on a product effort indicator (HTER) with that on various process effort indicators. The translation and post-editing process of student translators and professional translators was logged with a combination of keystroke logging and eye-tracking, and the MT output was analyzed with a fine-grained translation quality assessment approach. We find that most post-editing effort indicators (product as well as process) are influenced by machine translation quality, but that different error types affect different post-editing effort indicators, confirming that a more fine-grained MT quality analysis is needed to correctly estimate actual post-editing effort. Coherence, meaning shifts, and structural issues are shown to be good indicators of post-editing effort. The additional impact of experience on these interactions between MT quality and post-editing effort is smaller than expected.
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
The Altered Hepatic Tubulin Code in Alcoholic Liver Disease

PubMed Central

Groebner, Jennifer L.; Tuma, Pamela L.

2015-01-01

The molecular mechanisms that lead to the progression of alcoholic liver disease have been actively examined for decades. Because the hepatic microtubule cytoskeleton supports innumerable cellular processes, it has been the focus of many such mechanistic studies. It has long been appreciated that α-tubulin is a major target for modification by highly reactive ethanol metabolites and reactive oxygen species. It is also now apparent that alcohol exposure induces post-translational modifications that are part of the natural repertoire, mainly acetylation. In this review, the modifications of the “tubulin code” are described as well as those adducts by ethanol metabolites. The potential cellular consequences of microtubule modification are described with a focus on alcohol-induced defects in protein trafficking and enhanced steatosis. Possible mechanisms that can explain hepatic dysfunction are described and how this relates to the onset of liver injury is discussed. Finally, we propose that agents that alter the cellular acetylation state may represent a novel therapeutic strategy for treating liver disease. PMID:26393662
The Altered Hepatic Tubulin Code in Alcoholic Liver Disease.

PubMed

Groebner, Jennifer L; Tuma, Pamela L

2015-09-18

The molecular mechanisms that lead to the progression of alcoholic liver disease have been actively examined for decades. Because the hepatic microtubule cytoskeleton supports innumerable cellular processes, it has been the focus of many such mechanistic studies. It has long been appreciated that α-tubulin is a major target for modification by highly reactive ethanol metabolites and reactive oxygen species. It is also now apparent that alcohol exposure induces post-translational modifications that are part of the natural repertoire, mainly acetylation. In this review, the modifications of the "tubulin code" are described as well as those adducts by ethanol metabolites. The potential cellular consequences of microtubule modification are described with a focus on alcohol-induced defects in protein trafficking and enhanced steatosis. Possible mechanisms that can explain hepatic dysfunction are described and how this relates to the onset of liver injury is discussed. Finally, we propose that agents that alter the cellular acetylation state may represent a novel therapeutic strategy for treating liver disease.
Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.
Building the Future: Post-transcriptional Regulation of Cell Fate Decisions Prior to the Xenopus Midblastula Transition.

PubMed

Sheets, Michael D

2015-01-01

In all animals, a critical period in early development is when embryonic cells switch from relying solely upon maternally deposited RNAs and proteins to relying upon molecules encoded by the zygotic genome. Xenopus embryos have served as a model for examining this switch, as well as the maternally controlled stages that prepare for it. In Xenopus, the robust activation of zygotic transcription occurs at the 12th cleavage division and is referred to as the midblastula transition (MBT). Prior to MBT, gene expression is regulated by post-transcriptional events including mRNA and protein localization, protein post-translational modification, and mRNA translation. After the MBT, appropriate transcriptional regulation of the zygotic genome becomes critical and predominates. However, it is important to realize that the first key cell fate decisions that have profound impacts on development occur prior to the MBT and these are governed by regulating the expression of maternally deposited regulatory mRNAs and proteins. In this chapter, I will discuss post-transcriptional mechanisms that function during the maternal stages of Xenopus development with an emphasis on mechanisms known to directly modulate cell fate decisions. Emerging approaches and technologies that will help better understand this phase of development will also be discussed. © 2015 Elsevier Inc. All rights reserved.
Plant peptide hormone signalling.

PubMed

Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi

2015-01-01

The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. © 2015 Authors; published by Portland Press Limited.
Structural insights into the role of diphthamide on elongation factor 2 in messenger RNA reading frame maintenance.

PubMed

Pellegrino, Simone; Demeshkina, Natalia; Mancera-Martinez, Eder; Melnikov, Sergey; Simonetti, Angelita; Myasnikov, Alexander; Yusupov, Marat; Yusupova, Gulnara; Hashem, Yaser

2018-06-07

One of the most critical steps of protein biosynthesis is the coupled movement of messenger RNA (mRNA), that encodes genetic information, with transfer RNAs (tRNAs) on the ribosome. In eukaryotes this process is catalyzed by a conserved G-protein, the elongation factor 2 (eEF2), which carries a unique post-translational modification, called diphthamide, found in all eukaryotic species. Here we present near-atomic resolution cryo-EM structures of yeast 80S ribosome complexes containing mRNA, tRNA and eEF2 trapped in different GTP-hydrolysis states which provide further structural insights on the role of diphthamide in the mechanism of translation fidelity in eukaryotes. Copyright © 2018. Published by Elsevier Ltd.
Staufen1 senses overall transcript secondary structure to regulate translation

PubMed Central

Ricci, Emiliano P; Kucukural, Alper; Cenik, Can; Mercier, Blandine C; Singh, Guramrit; Heyer, Erin E; Ashar-Patel, Ami; Peng, Lingtao; Moore, Melissa J

2015-01-01

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3′ untranslated regions (UTRs) or in ‘strongly distal’ 3′ UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3′ UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins. PMID:24336223
Modification of orthogonal tRNAs: unexpected consequences for sense codon reassignment.

PubMed

Biddle, Wil; Schmitt, Margaret A; Fisk, John D

2016-12-01

Breaking the degeneracy of the genetic code via sense codon reassignment has emerged as a way to incorporate multiple copies of multiple non-canonical amino acids into a protein of interest. Here, we report the modification of a normally orthogonal tRNA by a host enzyme and show that this adventitious modification has a direct impact on the activity of the orthogonal tRNA in translation. We observed nearly equal decoding of both histidine codons, CAU and CAC, by an engineered orthogonal M. jannaschii tRNA with an AUG anticodon: tRNA Opt We suspected a modification of the tRNA Opt AUG anticodon was responsible for the anomalous lack of codon discrimination and demonstrate that adenosine 34 of tRNA Opt AUG is converted to inosine. We identified tRNA Opt AUG anticodon loop variants that increase reassignment of the histidine CAU codon, decrease incorporation in response to the histidine CAC codon, and improve cell health and growth profiles. Recognizing tRNA modification as both a potential pitfall and avenue of directed alteration will be important as the field of genetic code engineering continues to infiltrate the genetic codes of diverse organisms. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Global Survey of Protein Expression during Gonadal Sex Determination in Mice*

PubMed Central

Ewen, Katherine; Baker, Mark; Wilhelm, Dagmar; Aitken, R. John; Koopman, Peter

2009-01-01

The development of an embryo as male or female depends on differentiation of the gonads as either testes or ovaries. A number of genes are known to be important for gonadal differentiation, but our understanding of the regulatory networks underpinning sex determination remains fragmentary. To advance our understanding of sexual development beyond the transcriptome level, we performed the first global survey of the mouse gonad proteome at the time of sex determination by using two-dimensional nanoflow LC-MS/MS. The resulting data set contains a total of 1037 gene products (154 non-redundant and 883 redundant proteins) identified from 620 peptides. Functional classification and biological network construction suggested that the identified proteins primarily serve in RNA post-transcriptional modification and trafficking, protein synthesis and folding, and post-translational modification. The data set contains potential novel regulators of gonad development and sex determination not revealed previously by transcriptomics and proteomics studies and more than 60 proteins with potential links to human disorders of sexual development. PMID:19617587

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