Constant pH molecular dynamics of proteins in explicit solvent with proton tautomerism.

PubMed

Goh, Garrett B; Hulbert, Benjamin S; Zhou, Huiqing; Brooks, Charles L

2014-07-01

pH is a ubiquitous regulator of biological activity, including protein-folding, protein-protein interactions, and enzymatic activity. Existing constant pH molecular dynamics (CPHMD) models that were developed to address questions related to the pH-dependent properties of proteins are largely based on implicit solvent models. However, implicit solvent models are known to underestimate the desolvation energy of buried charged residues, increasing the error associated with predictions that involve internal ionizable residue that are important in processes like hydrogen transport and electron transfer. Furthermore, discrete water and ions cannot be modeled in implicit solvent, which are important in systems like membrane proteins and ion channels. We report on an explicit solvent constant pH molecular dynamics framework based on multi-site λ-dynamics (CPHMD(MSλD)). In the CPHMD(MSλD) framework, we performed seamless alchemical transitions between protonation and tautomeric states using multi-site λ-dynamics, and designed novel biasing potentials to ensure that the physical end-states are predominantly sampled. We show that explicit solvent CPHMD(MSλD) simulations model realistic pH-dependent properties of proteins such as the Hen-Egg White Lysozyme (HEWL), binding domain of 2-oxoglutarate dehydrogenase (BBL) and N-terminal domain of ribosomal protein L9 (NTL9), and the pKa predictions are in excellent agreement with experimental values, with a RMSE ranging from 0.72 to 0.84 pKa units. With the recent development of the explicit solvent CPHMD(MSλD) framework for nucleic acids, accurate modeling of pH-dependent properties of both major class of biomolecules-proteins and nucleic acids is now possible. © 2013 Wiley Periodicals, Inc.

Process evaluation of the Hunter Illawarra Kids Challenge Using Parent Support study: a multisite randomized controlled trial for the management of child obesity.

PubMed

Jones, Rachel A; Warren, Janet M; Okely, Anthony D; Collins, Clare E; Morgan, Philip J; Cliff, Dylan P; Burrows, Tracy; Cleary, Jane; Baur, Louise A

2010-11-01

The purposes of this article are to (a) outline findings from secondary or process outcome data of the Hunter Illawarra Kids Challenge Using Parent Support (HIKCUPS) study and (b) inform the design and development of future research interventions and practice in the management of child obesity. Data were collected by means of facilitator evaluations, independent session observation, attendance records, and parent questionnaires. Internal validity and reliability of the program delivery were high. All parents reported positive changes in their children as a result of the physical activity program, the dietary modification program, or both. Most participants completed the home activities, but more than half reported that finding time to do them was problematic. Facilitator review indicated that future programs should specifically cater to children of similar age or same sex, allow adequate time for explanation of complex nutritional concepts, and use intrinsic motivators for participants. Recommendations for future interventions, specifically the implementation of subsequent HIKCUPS or other multisite effectiveness studies, are detailed.

The IRS-1 signaling system.

PubMed

Myers, M G; Sun, X J; White, M F

1994-07-01

Insulin-receptor substrate 1 (IRS-1) is a principal substrate of the receptor tyrosine kinase for insulin and insulin-like growth factor 1, and a substrate for a tyrosine kinase activated by interleukin 4. IRS-1 undergoes multisite tyrosine phosphorylation and mediates downstream signals by 'docking' various proteins that contain Src homology 2 domains. IRS-1 appears to be a unique molecule; however, 4PS, a protein found mainly in hemopoietic cells, may represent another member of this family.

In silico concurrent multisite pH titration in proteins.

PubMed

Hu, Hao; Shen, Lin

2014-07-30

The concurrent proton binding at multiple sites in macromolecules such as proteins and nucleic acids is an important yet challenging problem in biochemistry. We develop an efficient generalized Hamiltonian approach to attack this issue. Based on the previously developed generalized-ensemble methods, an effective potential energy is constructed which combines the contributions of all (relevant) protonation states of the molecule. The effective potential preserves important phase regions of all states and, thus, allows efficient sampling of these regions in one simulation. The need for intermediate states in alchemical free energy simulations is greatly reduced. Free energy differences between different protonation states can be determined accurately and enable one to construct the grand canonical partition function. Therefore, the complicated concurrent multisite proton titration process of protein molecules can be satisfactorily simulated. Application of this method to the simulation of the pKa of Glu49, Asp50, and C-terminus of bovine pancreatic trypsin inhibitor shows reasonably good agreement with published experimental work. This method provides an unprecedented vivid picture of how different protonation states change their relative population upon pH titration. We believe that the method will be very useful in deciphering the molecular mechanism of pH-dependent biomolecular processes in terms of a detailed atomistic description. Copyright © 2014 Wiley Periodicals, Inc.

How Can Multi-Site Evaluations Be Participatory?

ERIC Educational Resources Information Center

Lawrenz, Frances; Huffman, Douglas

2003-01-01

Multi-site evaluations are becoming increasingly common in federal funding portfolios. Although much thought has been given to multi-site evaluation, there has been little emphasis on how it might interact with participatory evaluation. Therefore, this paper reviews several National Science Foundation educational, multi-site evaluations for the…

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

PubMed

Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2006-02-07

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Long-term effect of modification of dietary protein intake on the progression of diabetic nephropathy: a randomised controlled trial.

PubMed

Koya, D; Haneda, M; Inomata, S; Suzuki, Y; Suzuki, D; Makino, H; Shikata, K; Murakami, Y; Tomino, Y; Yamada, K; Araki, S I; Kashiwagi, A; Kikkawa, R

2009-10-01

There is currently insufficient evidence to recommend a low-protein diet for type 2 diabetic patients with diabetic nephropathy. We assessed whether a low-protein diet could prevent the progression of diabetic nephropathy. This was a multi-site parallel randomised controlled trial for prevention of diabetic nephropathy progression among 112 Japanese type 2 diabetic patients with overt nephropathy. It was conducted in Japan from 1 December 1997 to 30 April 2006. The participants were randomly assigned using a central computer-generated schedule to either low-protein diet (0.8 g kg(-1) day(-1)) and normal-protein diet (1.2 g kg(-1) day(-1)), and were followed for 5 years. The participants and investigators were not blinded to the assignment. The primary outcomes were the annual change in estimated GFR and creatinine clearance, the incidence of doubling of serum creatinine and the time to doubling of baseline serum creatinine. The study was completed by 47 (84%) of 56 participants in the low-protein diet group and 41 (73%) of 56 participants in the normal-diet group. During the study period, the difference in mean annual change in estimated GFR between the low-protein diet and the normal-protein diet groups was -0.3 ml min(-1) 1.73 m(-2) (95% CI -3.9, 4.4; p = 0.93). The difference in mean annual change in creatinine clearance between the low-protein diet and the normal-protein diet groups was -0.006 ml s(-1) 1.73 m(-2) (95% CI -0.089, 0.112; p = 0.80). A doubling of serum creatinine was reached in 16 patients of the low-protein group (34.0%), compared with 15 in the normal-protein group (36.6%), the difference between groups being -2.6% (95% CI -22.6, 17.5; p = 0.80). The time to doubling of serum creatinine was similar in both groups (p = 0.66). It is extremely difficult to get patients to follow a long-term low-protein diet. Although in the low-protein group overall protein intake was slightly (but not significantly) lower, it did not confer renoprotection. ClinicalTrials.gov NCT00448526. Research grant from the Ministry of Health, Labour and Welfare of Japan.

Conducting a Multisite Education Research Project: Strategies to Overcome the Barriers to Achieve the Benefits.

PubMed

Beischel, Kelly P; Hart, Julie; Turkelson, Sandra L

2016-01-01

Multisite education research projects have many benefits as well as perceived barriers. In this article, we share our experiences with a multisite education research project and the barriers we overcame to reap the benefits. The outcome of our research resulted in increased rigor, role-modeling professional collaboration, and promotion of future multisite education studies. The strategies presented in this article will help alleviate perceived barriers and ameliorate the process of conducting multisite education research studies.

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

Protein mass analysis of histones.

PubMed

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

2003-09-01

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

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

DOEpatents

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

2015-03-03

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

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

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

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

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

Controlled levels of protein modification through a chromatography-mediated bioconjugation

DOE PAGES

Kwant, Richard L.; Jaffe, Jake; Palmere, Peter J.; ...

2015-02-27

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety usingmore » an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.« less

Multi-site pain and working conditions as predictors of work ability in a 4-year follow-up among food industry employees.

PubMed

Neupane, S; Virtanen, P; Leino-Arjas, P; Miranda, H; Siukola, A; Nygård, C-H

2013-03-01

We investigated the separate and joint effects of multi-site musculoskeletal pain and physical and psychosocial exposures at work on future work ability. A survey was conducted among employees of a Finnish food industry company in 2005 (n = 1201) and a follow-up survey in 2009 (n = 734). Information on self-assessed work ability (current work ability on a scale from 0 to 10; 7 = poor work ability), multi-site musculoskeletal pain (pain in at least two anatomical areas of four), leisure-time physical activity, body mass index and physical and psychosocial exposures was obtained by questionnaire. The separate and joint effects of multi-site pain and work exposures on work ability at follow-up, among subjects with good work ability at baseline, were assessed by logistic regression, and p-values for the interaction derived. Compared with subjects with neither multi-site pain nor adverse work exposure, multi-site pain at baseline increased the risk of poor work ability at follow-up, allowing for age, gender, occupational class, body mass index and leisure-time physical activity. The separate effects of the work exposures on work ability were somewhat smaller than those of multi-site pain. Multi-site pain had an interactive effect with work environment and awkward postures, such that no association of multi-site pain with poor work ability was seen when work environment was poor or awkward postures present. The decline in work ability connected with multi-site pain was not increased by exposure to adverse physical or psychosocial factors at work. © 2012 European Federation of International Association for the Study of Pain Chapters.

PS2-06: Best Practices for Advancing Multi-site Chart Abstraction Research

PubMed Central

Blick, Noelle; Cole, Deanna; King, Colleen; Riordan, Rick; Von Worley, Ann; Yarbro, Patty

2012-01-01

Background/Aims Multi-site chart abstraction studies are becoming increasingly common within the HMORN. Differences in systems among HMORN sites can pose significant obstacles to the success of these studies. It is therefore crucial to standardize abstraction activities by following best practices for multi-site chart abstraction, as consistency of processes across sites will increase efficiencies and enhance data quality. Methods Over the past few months the authors have been meeting to identify obstacles to multi-site chart abstraction and to address ways in which multi-site chart abstraction processes can be systemized and standardized. The aim of this workgroup is to create a best practice guide for multi-site chart abstraction studies. Focus areas include: abstractor training, format for chart abstraction (database, paper, etc), data quality, redaction, mechanism for transferring data, site specific access to medical records, IRB/HIPAA concerns, and budgetary issues. Results The results of the workgroup’s efforts (the best practice guide) will be presented by a panel of experts at the 2012 HMORN conference. The presentation format will also focus on discussion among attendees to elicit further input and to identify areas that need to be further addressed. Subsequently, the best practice guide will be posted on the HMORN website. Discussion The best practice guide for multi-site chart abstraction studies will establish sound guidelines and serve as an aid to researchers embarking on multi-site chart abstraction studies. Efficiencies and data quality will be further enhanced with standardized multi-site chart abstraction practices.

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

NASA Astrophysics Data System (ADS)

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

1984-11-01

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

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

PubMed Central

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

2016-01-01

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

Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation

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

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

2012-02-01

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

Progress and Promise of Attention-Deficit Hyperactivity Disorder Pharmacogenetics

PubMed Central

Froehlich, Tanya E.; McGough, James J.; Stein, Mark A.

2010-01-01

One strategy for understanding variability in attention-deficit hyperactivity disorder (ADHD) medication response, and therefore redressing the current trial-and-error approach to ADHD medication management, is to identify genetic moderators of treatment. This article summarizes ADHD pharmacogenetic investigative efforts to date, which have primarily focused on short-term response to methylphenidate and largely been limited by modest sample sizes. The most well studied genes include the dopamine transporter and dopamine D4 receptor, with additional genes that have been significantly associated with stimulant medication response including the adrenergic α2A-receptor, catechol-O-methyltransferase, D5 receptor, noradrenaline (norepinephrine) transporter protein 1 and synaptosomal-associated protein 25 kDa. Unfortunately, results of current ADHD pharmacogenetic studies have not been entirely consistent, possibly due to differences in study design, medication dosing regimens and outcome measures. Future directions for ADHD pharmacogenetics investigations may include examination of drug-metabolizing enzymes and a wider range of stimulant and non-stimulant medications. In addition, researchers are increasingly interested in going beyond the individual candidate gene approach to investigate gene-gene interactions or pathways, effect modification by additional environmental exposures and whole genome approaches. Advancements in ADHD pharmacogenetics will be facilitated by multi-site collaborations to obtain larger sample sizes using standardized protocols. Although ADHD pharmacogenetic efforts are still in a relatively early stage, their potential clinical applications may include the development of treatment efficacy and adverse effect prediction algorithms that incorporate the interplay of genetic and environmental factors, as well as the development of novel ADHD treatments. PMID:20088618

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

PubMed Central

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

2015-01-01

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

On multi-site damage identification using single-site training data

NASA Astrophysics Data System (ADS)

Barthorpe, R. J.; Manson, G.; Worden, K.

2017-11-01

This paper proposes a methodology for developing multi-site damage location systems for engineering structures that can be trained using single-site damaged state data only. The methodology involves training a sequence of binary classifiers based upon single-site damage data and combining the developed classifiers into a robust multi-class damage locator. In this way, the multi-site damage identification problem may be decomposed into a sequence of binary decisions. In this paper Support Vector Classifiers are adopted as the means of making these binary decisions. The proposed methodology represents an advancement on the state of the art in the field of multi-site damage identification which require either: (1) full damaged state data from single- and multi-site damage cases or (2) the development of a physics-based model to make multi-site model predictions. The potential benefit of the proposed methodology is that a significantly reduced number of recorded damage states may be required in order to train a multi-site damage locator without recourse to physics-based model predictions. In this paper it is first demonstrated that Support Vector Classification represents an appropriate approach to the multi-site damage location problem, with methods for combining binary classifiers discussed. Next, the proposed methodology is demonstrated and evaluated through application to a real engineering structure - a Piper Tomahawk trainer aircraft wing - with its performance compared to classifiers trained using the full damaged-state dataset.

Automated Solid-Phase Protein Modification with Integrated Enzymatic Digest for Reaction Validation: Application of a Compartmented Microfluidic Reactor for Rapid Optimization and Analysis of Protein Biotinylation

PubMed Central

Fraas, Regina; Diehm, Juliane; Franzreb, Matthias

2017-01-01

Protein modification by covalent coupling of small ligands or markers is an important prerequisite for the use of proteins in many applications. Well-known examples are the use of proteins with fluorescent markers in many in vivo experiments or the binding of biotinylated antibodies via biotin–streptavidin coupling in the frame of numerous bioassays. Multiple protocols were established for the coupling of the respective molecules, e.g., via the C and N-terminus, or via cysteines and lysines exposed at the protein surface. Still, in most cases the conditions of these standard protocols are only an initial guess. Optimization of the coupling parameters like reagent concentrations, pH, or temperature may strongly increase coupling yield and the biological activity of the modified protein. In order to facilitate the process of optimizing coupling conditions, a method was developed which uses a compartmented microfluidic reactor for the rapid screening of different coupling conditions. In addition, the system allows for the integration of an enzymatic digest of the modified protein directly after modification. In combination with a subsequent MALDI-TOF analysis of the resulting fragments, this gives a fast and detailed picture not only of the number and extent of the generated modifications but also of their position within the protein sequence. The described process was demonstrated for biotinylation of green fluorescent protein. Different biotin-excesses and different pH-values were tested in order to elucidate the influence on the modification extent and pattern. In addition, the results of solid-phase based modifications within the microfluidic reactor were compared to modification patterns resulting from coupling trials with unbound protein. As expected, modification patterns of immobilized proteins showed clear differences to the ones of dissolved proteins. PMID:29181376

A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins.

PubMed

Orr, Asuka A; Gonzalez-Rivera, Juan C; Wilson, Mark; Bhikha, P Reena; Wang, Daiqi; Contreras, Lydia M; Tamamis, Phanourios

2018-02-01

There are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a computational protocol for the characterization of interactions between proteins and RNA containing post-transcriptional modifications. Given an RNA-protein complex structure, potential RNA modified ribonucleoside positions, and molecular mechanics parameters for capturing energetics of RNA modifications, our protocol operates in two stages. In the first stage, a decision-making tool, comprising short simulations and interaction energy calculations, performs a fast and efficient search in a high-throughput fashion, through a list of different types of RNA modifications categorized into trees according to their structural and physicochemical properties, and selects a subset of RNA modifications prone to interact with the target protein. In the second stage, RNA modifications that are selected as recognized by the protein are examined in-detail using all-atom simulations and free energy calculations. We implement and experimentally validate this protocol in a test case involving the study of RNA modifications in complex with Escherichia coli (E. coli) protein Polynucleotide Phosphorylase (PNPase), depicting the favorable interaction between 8-oxo-7,8-dihydroguanosine (8-oxoG) RNA modification and PNPase. Further advancement of the protocol can broaden our understanding of protein interactions with all known RNA modifications in several systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

PubMed

Ghiraldi-Lopes, Luciana D; Campanerut-Sá, Paula Az; Meneguello, Jean E; Seixas, Flávio Av; Lopes-Ortiz, Mariana A; Scodro, Regiane Bl; Pires, Claudia Ta; da Silva, Rosi Z; Siqueira, Vera Ld; Nakamura, Celso V; Cardoso, Rosilene F

2017-08-01

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

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

PubMed

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

2015-04-29

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

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

The Role of Involvement and Use in Multisite Evaluations

ERIC Educational Resources Information Center

Lawrenz, Frances; King, Jean A.; Ooms, Ann

2011-01-01

A cross-case analysis of four National Science Foundation (NSF) case studies identified both unique details and common themes related to promoting the use and influence of multisite evaluations. The analysis provided evidence of diverse evaluation use by stakeholders and suggested that people taking part in the multisite evaluations perceived…

Multi-Sited Global Ethnography and Travel: Gendered Journeys in Three Registers

ERIC Educational Resources Information Center

Epstein, Debbie; Fahey, Johannah; Kenway, Jane

2013-01-01

This paper joins a barely begun conversation about multi-sited and global ethnography in educational research; a conversation that is likely to intensify along with growing interest in the links between education, globalisation, internationalisation and transnationalism. Drawing on an ongoing multi-sited global ethnography of elite schools and…

Work-related determinants of multi-site musculoskeletal pain among employees in the health care sector.

PubMed

Neupane, Subas; Nygård, Clas-Håkan; Oakman, Jodi

2016-06-16

Work-related musculoskeletal pain is a major occupational problem. Those with pain in multiple sites usually report worse health outcomes than those with pain in one site. This study explored prevalence and associated predictors of multi-site pain in health care sector employees. Survey responses from 1348 health care sector employees across three organisations (37% response rate) collected data on job satisfaction, work life balance, psychosocial and physical hazards, general health and work ability. Musculoskeletal discomfort was measured across 5 body regions with pain in ≥ 2 sites defined as multi-site pain. Generalized linear models were used to identify relationships between work-related factors and multi-site pain. Over 52% of the employees reported pain in multiple body sites and 19% reported pain in one site. Poor work life balance (PRR = 2.33, 95% CI = 1.06-5.14). physical (PRR = 7.58, 95% CI = 4.89-11.77) and psychosocial (PRR = 1.59, 95% CI = 1.00-2.57) hazard variables were related to multi-site pain (after controlling for age, gender, health and work ability. Older employees and females were more likely to report multi-site pain. Effective risk management of work related multi-site pain must include identification and control of psychosocial and physical hazards.

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

PubMed Central

Verma, Vikash; Mallik, Leena; Hariadi, Rizal F.; Sivaramakrishnan, Sivaraj; Skiniotis, Georgios; Joglekar, Ajit P.

2015-01-01

DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis. PMID:26348722

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

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.

A Mixed Methods Sampling Methodology for a Multisite Case Study

ERIC Educational Resources Information Center

Sharp, Julia L.; Mobley, Catherine; Hammond, Cathy; Withington, Cairen; Drew, Sam; Stringfield, Sam; Stipanovic, Natalie

2012-01-01

The flexibility of mixed methods research strategies makes such approaches especially suitable for multisite case studies. Yet the utilization of mixed methods to select sites for these studies is rarely reported. The authors describe their pragmatic mixed methods approach to select a sample for their multisite mixed methods case study of a…

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

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

Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

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

Zhao, Huaying, E-mail: zhaoh3@mail.nih.gov; Schuck, Peter, E-mail: zhaoh3@mail.nih.gov

2015-01-01

Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysicalmore » techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.« less

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

USDA-ARS?s Scientific Manuscript database

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

Surface modification of protein enhances encapsulation in chitosan nanoparticles

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

How Institutional and University Counselor Policies Effectively Respond to Victims of Cyber Violent Acts: A Multisite Case Study

ERIC Educational Resources Information Center

Richards, Gretchen M.

2012-01-01

This multisite case study examined how institutional and university counselor policies effectively respond to cyber violent acts. Stake's (2006) multisite case study methodology was used to identify seven themes from current literature. Two sites with four participants were selected. The participants included two counseling directors and the…

Biological stress systems, adverse life events and the onset of chronic multisite musculoskeletal pain: a 6-year cohort study.

PubMed

Generaal, Ellen; Vogelzangs, Nicole; Macfarlane, Gary J; Geenen, Rinie; Smit, Johannes H; de Geus, Eco J C N; Penninx, Brenda W J H; Dekker, Joost

2016-05-01

Dysregulated biological stress systems and adverse life events, independently and in interaction, have been hypothesised to initiate chronic pain. We examine whether (1) function of biological stress systems, (2) adverse life events, and (3) their combination predict the onset of chronic multisite musculoskeletal pain. Subjects (n=2039) of the Netherlands Study of Depression and Anxiety, free from chronic multisite musculoskeletal pain at baseline, were identified using the Chronic Pain Grade Questionnaire and followed up for the onset of chronic multisite musculoskeletal pain over 6 years. Baseline assessment of biological stress systems comprised function of the hypothalamic-pituitary-adrenal axis (1-h cortisol awakening response, evening levels, postdexamethasone levels), the immune system (basal and lipopolysaccharide-stimulated inflammation) and the autonomic nervous system (heart rate, pre-ejection period, SD of the normal-to-normal interval, respiratory sinus arrhythmia). The number of recent adverse life events was assessed at baseline using the List of Threatening Events Questionnaire. Hypothalamic-pituitary-adrenal axis, immune system and autonomic nervous system functioning was not associated with onset of chronic multisite musculoskeletal pain, either by itself or in interaction with adverse life events. Adverse life events did predict onset of chronic multisite musculoskeletal pain (HR per event=1.14, 95% CI 1.04 to 1.24, p=0.005). This longitudinal study could not confirm that dysregulated biological stress systems increase the risk of developing chronic multisite musculoskeletal pain. Adverse life events were a risk factor for the onset of chronic multisite musculoskeletal pain, suggesting that psychosocial factors play a role in triggering the development of this condition. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Supramolecular PEGylation of biopharmaceuticals

PubMed Central

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

2016-01-01

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

Exploiting Multisite Gateway and pENFRUIT plasmid collection for fruit genetic engineering.

PubMed

Estornell, Leandro H; Granell, Antonio; Orzaez, Diego

2012-01-01

MultiSite Gateway cloning techniques based on homologous recombination facilitate the combinatorial assembly of basic genetic pieces (i.e., promoters, CDS, and terminators) into gene expression or gene silencing cassettes. pENFRUIT is a collection of MultiSite Triple Gateway Entry vectors dedicated to genetic engineering in fruits. It comprises a number of fruit-operating promoters as well as C-terminal tags adapted to the Gateway standard. In this way, flanking regulatory/labeling sequences can be easily Gateway-assembled with a given gene of interest for its ectopic expression or silencing in fruits. The resulting gene constructs can be analyzed in stable transgenic plants or in transient expression assays, the latter allowing fast testing of the increasing number of combinations arising from MultiSite methodology. A detailed description of the use of MultiSite cloning methodology for the assembly of pENFRUIT elements is presented.

Simulation of large-scale rule-based models

PubMed Central

Colvin, Joshua; Monine, Michael I.; Faeder, James R.; Hlavacek, William S.; Von Hoff, Daniel D.; Posner, Richard G.

2009-01-01

Motivation: Interactions of molecules, such as signaling proteins, with multiple binding sites and/or multiple sites of post-translational covalent modification can be modeled using reaction rules. Rules comprehensively, but implicitly, define the individual chemical species and reactions that molecular interactions can potentially generate. Although rules can be automatically processed to define a biochemical reaction network, the network implied by a set of rules is often too large to generate completely or to simulate using conventional procedures. To address this problem, we present DYNSTOC, a general-purpose tool for simulating rule-based models. Results: DYNSTOC implements a null-event algorithm for simulating chemical reactions in a homogenous reaction compartment. The simulation method does not require that a reaction network be specified explicitly in advance, but rather takes advantage of the availability of the reaction rules in a rule-based specification of a network to determine if a randomly selected set of molecular components participates in a reaction during a time step. DYNSTOC reads reaction rules written in the BioNetGen language which is useful for modeling protein–protein interactions involved in signal transduction. The method of DYNSTOC is closely related to that of StochSim. DYNSTOC differs from StochSim by allowing for model specification in terms of BNGL, which extends the range of protein complexes that can be considered in a model. DYNSTOC enables the simulation of rule-based models that cannot be simulated by conventional methods. We demonstrate the ability of DYNSTOC to simulate models accounting for multisite phosphorylation and multivalent binding processes that are characterized by large numbers of reactions. Availability: DYNSTOC is free for non-commercial use. The C source code, supporting documentation and example input files are available at http://public.tgen.org/dynstoc/. Contact: dynstoc@tgen.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19213740

Shared worlds: multi-sited ethnography and nursing research.

PubMed

Molloy, Luke; Walker, Kim; Lakeman, Richard

2017-03-22

Background Ethnography, originally developed for the study of supposedly small-scale societies, is now faced with an increasingly mobile, changing and globalised world. Cultural identities can exist without reference to a specific location and extend beyond regional and national boundaries. It is therefore no longer imperative that the sole object of the ethnographer's practice should be a geographically bounded site. Aim To present a critical methodological review of multi-sited ethnography. Discussion Understanding that it can no longer be taken with any certainty that location alone determines culture, multi-sited ethnography provides a method of contextualising multi-sited social phenomena. The method enables researchers to examine social phenomena that are simultaneously produced in different locations. It has been used to undertake cultural analysis of diverse areas such as organ trafficking, global organisations, technologies and anorexia. Conclusion The authors contend that multi-sited ethnography is particularly suited to nursing research as it provides researchers with an ethnographic method that is more relevant to the interconnected world of health and healthcare services. Implications for practice Multi-sited ethnography provides nurse researchers with an approach to cultural analysis in areas such as the social determinants of health, healthcare services and the effects of health policies across multiple locations.

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.

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

PubMed

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

2013-07-25

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

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

PubMed Central

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

1994-01-01

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

Tools for phospho- and glycoproteomics of plasma membranes.

PubMed

Wiśniewski, Jacek R

2011-07-01

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

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

PubMed

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

2007-08-27

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

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

PubMed Central

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

2007-01-01

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

Yeast One-Hybrid Gγ Recruitment System for Identification of Protein Lipidation Motifs

PubMed Central

Fukuda, Nobuo; Doi, Motomichi; Honda, Shinya

2013-01-01

Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs. PMID:23922919

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

PubMed

Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

2017-04-01

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Psychosocial stress and multi-site musculoskeletal pain: a cross-sectional survey of patient care workers.

PubMed

Sembajwe, Grace; Tveito, Torill Helene; Hopcia, Karen; Kenwood, Christopher; O'Day, Elizabeth Tucker; Stoddard, Anne M; Dennerlein, Jack T; Hashimoto, Dean; Sorensen, Glorian

2013-03-01

The aim of this study was to assess the relationship between psychosocial factors at work and multi-site musculoskeletal pain among patient care workers. In a survey of 1,572 workers from two hospitals, occupational psychosocial factors and health outcomes of workers with single and multi-site pain were evaluated using items from the Job Content Questionnaire that was designed to measure psychological demands, decision latitude, and social support. An adapted Nordic Questionnaire provided data on the musculoskeletal pain outcome. Covariates included body mass index, age, gender, and occupation. The analyses revealed statistically significant associations between psychosocial demands and multi-site musculoskeletal pain among patient care associates, nurses, and administrative personnel, both men and women. Supervisor support played a significant role for nurses and women. These results remained statistically significant after adjusting for covariates. These results highlight the associations between workplace psychosocial strain and multi-site musculoskeletal pain, setting the stage for future longitudinal explorations. Copyright 2013, SLACK Incorporated.

A comparison of single- and multi-site calibration and validation: a case study of SWAT in the Miyun Reservoir watershed, China

NASA Astrophysics Data System (ADS)

Bai, Jianwen; Shen, Zhenyao; Yan, Tiezhu

2017-09-01

An essential task in evaluating global water resource and pollution problems is to obtain the optimum set of parameters in hydrological models through calibration and validation. For a large-scale watershed, single-site calibration and validation may ignore spatial heterogeneity and may not meet the needs of the entire watershed. The goal of this study is to apply a multi-site calibration and validation of the Soil andWater Assessment Tool (SWAT), using the observed flow data at three monitoring sites within the Baihe watershed of the Miyun Reservoir watershed, China. Our results indicate that the multi-site calibration parameter values are more reasonable than those obtained from single-site calibrations. These results are mainly due to significant differences in the topographic factors over the large-scale area, human activities and climate variability. The multi-site method involves the division of the large watershed into smaller watersheds, and applying the calibrated parameters of the multi-site calibration to the entire watershed. It was anticipated that this case study could provide experience of multi-site calibration in a large-scale basin, and provide a good foundation for the simulation of other pollutants in followup work in the Miyun Reservoir watershed and other similar large areas.

Increasing the ranks of academic researchers in mental health: a multisite approach to postdoctoral fellowship training.

PubMed

O'Hara, Ruth; Cassidy-Eagle, Erin L; Beaudreau, Sherry A; Eyler, Lisa T; Gray, Heather L; Giese-Davis, Janine; Hubbard, Jeffrey; Yesavage, Jerome A

2010-01-01

This report highlights the use of multisite training for psychiatry and psychology postdoctoral fellows developing careers in academic clinical research in the field of mental health. The objective is to describe a model of training for young investigators to establish independent academic clinical research careers, including (1) program structure and eligibility, (2) program goals and development of a multisite curriculum, (3) use of technology for implementing the program across multiple sites, and (4) advantages and challenges of this multisite approach. In 2000, in collaboration with the Veterans Affairs (VA) Mental Illness Research, Education and Clinical Centers (MIRECCs), the VA Office of Academic Affiliations launched the Special Fellowship Program in Advanced Psychiatry and Psychology. Each of the 10 currently participating VA sites across the United States is affiliated with a MIRECC and an academic medical institution. In the first five years of this fellowship program, 83 fellows (34 psychiatrists and 49 psychologists) have participated. The success of this multisite approach is evidenced by the 58 fellows who have already graduated from the program: 70% have entered academic clinical research positions, and over 25 have obtained independent extramural grant support from the VA or the National Institutes of Health. Multisite training results in a greater transfer of knowledge and capitalizes on the nationwide availability of experts, creating unique networking and learning opportunities for trainees. The VA's multisite fellowship program plays a valuable role in preparing substantial numbers of psychiatry and psychology trainees for a range of academic clinical research and leadership positions in the field of mental health.

Reduced hypothalamic-pituitary-adrenal axis activity in chronic multi-site musculoskeletal pain: partly masked by depressive and anxiety disorders.

PubMed

Generaal, Ellen; Vogelzangs, Nicole; Macfarlane, Gary J; Geenen, Rinie; Smit, Johannes H; Penninx, Brenda W J H; Dekker, Joost

2014-07-09

Studies on hypothalamic-pituitary-adrenal axis (HPA-axis) function amongst patients with chronic pain show equivocal results and well-controlled cohort studies are rare in this field. The goal of our study was to examine whether HPA-axis dysfunction is associated with the presence and the severity of chronic multi-site musculoskeletal pain. Data are from the Netherlands Study of Depression and Anxiety including 1125 subjects with and without lifetime depressive and anxiety disorders. The Chronic Pain Grade questionnaire was used to determine the presence and severity of chronic multi-site musculoskeletal pain. Subjects were categorized into a chronic multi-site musculoskeletal pain group (n = 471) and a control group (n = 654). Salivary cortisol samples were collected to assess HPA-axis function (awakening level, 1-h awakening response, evening level, diurnal slope and post-dexamethasone level). In comparison with the control group, subjects with chronic multi-site musculoskeletal pain showed significantly lower cortisol level at awakening, lower evening level and a blunted diurnal slope. Lower cortisol level at awakening and a blunted diurnal slope appeared to be restricted to those without depressive and/or anxiety disorders, who also showed a lower 1-h awakening response. Our results suggest hypocortisolemia in chronic multi-site musculoskeletal pain. However, if chronic pain is accompanied by a depressive or anxiety disorder, typically related to hypercortisolemia, the association between cortisol levels and chronic multi-site musculoskeletal pain appears to be partly masked. Future studies should take psychopathology into account when examining HPA-axis function in chronic pain.

Reduced hypothalamic-pituitary-adrenal axis activity in chronic multi-site musculoskeletal pain: partly masked by depressive and anxiety disorders

PubMed Central

2014-01-01

Background Studies on hypothalamic-pituitary-adrenal axis (HPA-axis) function amongst patients with chronic pain show equivocal results and well-controlled cohort studies are rare in this field. The goal of our study was to examine whether HPA-axis dysfunction is associated with the presence and the severity of chronic multi-site musculoskeletal pain. Methods Data are from the Netherlands Study of Depression and Anxiety including 1125 subjects with and without lifetime depressive and anxiety disorders. The Chronic Pain Grade questionnaire was used to determine the presence and severity of chronic multi-site musculoskeletal pain. Subjects were categorized into a chronic multi-site musculoskeletal pain group (n = 471) and a control group (n = 654). Salivary cortisol samples were collected to assess HPA-axis function (awakening level, 1-h awakening response, evening level, diurnal slope and post-dexamethasone level). Results In comparison with the control group, subjects with chronic multi-site musculoskeletal pain showed significantly lower cortisol level at awakening, lower evening level and a blunted diurnal slope. Lower cortisol level at awakening and a blunted diurnal slope appeared to be restricted to those without depressive and/or anxiety disorders, who also showed a lower 1-h awakening response. Conclusions Our results suggest hypocortisolemia in chronic multi-site musculoskeletal pain. However, if chronic pain is accompanied by a depressive or anxiety disorder, typically related to hypercortisolemia, the association between cortisol levels and chronic multi-site musculoskeletal pain appears to be partly masked. Future studies should take psychopathology into account when examining HPA-axis function in chronic pain. PMID:25007969

Coordination and Management of Multisite Complementary and Alternative Medicine (CAM) Therapies: Experience from a Multisite Reflexology Intervention Trial

PubMed Central

Rahbar, Mohammad H.; Wyatt, Gwen; Sikorskii, Alla; Victorson, David; Ardjomand-Hessabi, Manouchehr

2011-01-01

Background Multisite randomized clinical trials allow for increased research collaboration among investigators and expedite data collection efforts. As a result, government funding agencies typically look favorably upon this approach. As the field of complementary and alternative medicine (CAM) continues to evolve, so do increased calls for the use of more rigorous study design and trial methodologies, which can present challenges for investigators. Purpose To describe the processes involved in the coordination and management of a multisite randomized clinical trial of a CAM intervention. Methods Key aspects related to the coordination and management of a multisite CAM randomized clinical trial are presented, including organizational and site selection considerations, recruitment concerns and issues related to data collection and randomization to treatment groups. Management and monitoring of data, as well as quality assurance procedures are described. Finally, a real world perspective is shared from a recently conducted multisite randomized clinical trial of reflexology for women diagnosed with advanced breast cancer. Results The use of multiple sites in the conduct of CAM-based randomized clinical trials can provide an efficient, collaborative and robust approach to study coordination and data collection that maximizes efficiency and ensures the quality of results. Conclusions Multisite randomized clinical trial designs can offer the field of CAM research a more standardized and efficient approach to examine the effectiveness of novel therapies and treatments. Special attention must be given to intervention fidelity, consistent data collection and ensuring data quality. Assessment and reporting of quantitative indicators of data quality should be required. PMID:21664296

Multi-site precipitation downscaling using a stochastic weather generator

NASA Astrophysics Data System (ADS)

Chen, Jie; Chen, Hua; Guo, Shenglian

2018-03-01

Statistical downscaling is an efficient way to solve the spatiotemporal mismatch between climate model outputs and the data requirements of hydrological models. However, the most commonly-used downscaling method only produces climate change scenarios for a specific site or watershed average, which is unable to drive distributed hydrological models to study the spatial variability of climate change impacts. By coupling a single-site downscaling method and a multi-site weather generator, this study proposes a multi-site downscaling approach for hydrological climate change impact studies. Multi-site downscaling is done in two stages. The first stage involves spatially downscaling climate model-simulated monthly precipitation from grid scale to a specific site using a quantile mapping method, and the second stage involves the temporal disaggregating of monthly precipitation to daily values by adjusting the parameters of a multi-site weather generator. The inter-station correlation is specifically considered using a distribution-free approach along with an iterative algorithm. The performance of the downscaling approach is illustrated using a 10-station watershed as an example. The precipitation time series derived from the National Centers for Environment Prediction (NCEP) reanalysis dataset is used as the climate model simulation. The precipitation time series of each station is divided into 30 odd years for calibration and 29 even years for validation. Several metrics, including the frequencies of wet and dry spells and statistics of the daily, monthly and annual precipitation are used as criteria to evaluate the multi-site downscaling approach. The results show that the frequencies of wet and dry spells are well reproduced for all stations. In addition, the multi-site downscaling approach performs well with respect to reproducing precipitation statistics, especially at monthly and annual timescales. The remaining biases mainly result from the non-stationarity of NCEP precipitation. Overall, the proposed approach is efficient for generating multi-site climate change scenarios that can be used to investigate the spatial variability of climate change impacts on hydrology.

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

PubMed

Kurien, Biji T; Scofield, R Hal

2007-03-21

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

High prevalence of daily and multi-site pain – a cross-sectional population-based study among 3000 Danish adolescents

PubMed Central

2013-01-01

Background Daily pain and multi-site pain are both associated with reduction in work ability and health-related quality of life (HRQoL) among adults. However, no population-based studies have yet investigated the prevalence of daily and multi-site pain among adolescents and how these are associated with respondent characteristics. The purpose of this study was to investigate the prevalence of self-reported daily and multi-site pain among adolescents aged 12–19 years and associations of almost daily pain and multi-site pain with respondent characteristics (sex, age, body mass index, HRQoL and sports participation). Methods A population-based cross-sectional study was conducted among 4,007 adolescents aged 12–19 years in Denmark. Adolescents answered an online questionnaire during physical education lessons. The questionnaire contained a mannequin divided into 12 regions on which the respondents indicated their current pain sites and pain frequency (rarely, monthly, weekly, more than once per week, almost daily pain), characteristics, sports participation and HRQoL measured by the EuroQoL 5D. Multivariate regression was used to calculate the odds ratio for the association between almost daily pain, multi-site pain and respondent characteristics. Results The response rate was 73.7%. A total of 2,953 adolescents (62% females) answered the questionnaire. 33.3% reported multi-site pain (pain in >1 region) while 19.8% reported almost daily pain. 61% reported current pain in at least one region with knee and back pain being the most common sites. Female sex (OR: 1.35-1.44) and a high level of sports participation (OR: 1.51-2.09) were associated with increased odds of having almost daily pain and multi-site pain. Better EQ-5D score was associated with decreased odds of having almost daily pain or multi-site pain (OR: 0.92-0.94). Conclusion In this population-based cohort of school-attending Danish adolescents, nearly two out of three reported current pain and, on average, one out of three reported pain in more than one body region. Female sex, and high level of sports participation were associated with increased odds of having almost daily pain and multi-site pain. The study highlights an important health issue that calls for investigations to improve our understanding of adolescent pain and our capacity to prevent and treat this condition. PMID:24252440

Ethics Review for a Multi-Site Project Involving Tribal Nations in the Northern Plains.

PubMed

Angal, Jyoti; Petersen, Julie M; Tobacco, Deborah; Elliott, Amy J

2016-04-01

Increasingly, Tribal Nations are forming ethics review panels, which function separately from institutional review boards (IRBs). The emergence of strong community representation coincides with a widespread effort supported by the U.S. Department of Health & Human Services and other federal agencies to establish a single IRB for all multi-site research. This article underscores the value of a tribal ethics review board and describes the tribal oversight for the Safe Passage Study-a multi-site, community-based project in the Northern Plains. Our experience demonstrates the benefits of tribal ethics review and makes a strong argument for including tribal oversight in future regulatory guidance for multi-site, community-based research. © The Author(s) 2016.

Structure and Modification of Electrode Materials for Protein Electrochemistry.

PubMed

Jeuken, Lars J C

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

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

PubMed

Cowan, Don A; Fernandez-Lafuente, Roberto

2011-09-10

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

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

PubMed

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

2018-05-07

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

Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids.

PubMed

Martin, Rey W; Des Soye, Benjamin J; Kwon, Yong-Chan; Kay, Jennifer; Davis, Roderick G; Thomas, Paul M; Majewska, Natalia I; Chen, Cindy X; Marcum, Ryan D; Weiss, Mary Grace; Stoddart, Ashleigh E; Amiram, Miriam; Ranji Charna, Arnaz K; Patel, Jaymin R; Isaacs, Farren J; Kelleher, Neil L; Hong, Seok Hoon; Jewett, Michael C

2018-03-23

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Chemical methods for encoding and decoding of posttranslational modifications

PubMed Central

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

2016-01-01

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

The ability of multi-site, multi-depth sacral lateral branch blocks to anesthetize the sacroiliac joint complex.

PubMed

Dreyfuss, Paul; Henning, Troy; Malladi, Niriksha; Goldstein, Barry; Bogduk, Nikolai

2009-01-01

To determine the physiologic effectiveness of multi-site, multi-depth sacral lateral branch injections. Double-blind, randomized, placebo-controlled study. Outpatient pain management center. Twenty asymptomatic volunteers. The dorsal innervation to the sacroiliac joint (SIJ) is from the L5 dorsal ramus and the S1-3 lateral branches. Multi-site, multi-depth lateral branch blocks were developed to compensate for the complex regional anatomy that limited the effectiveness of single-site, single-depth lateral branch injections. Bilateral multi-site, multi-depth lateral branch green dye injections and subsequent dissection on two cadavers revealed a 91% accuracy with this technique. Session 1: 20 asymptomatic subjects had a 25-g spinal needle probe their interosseous (IO) and dorsal sacroiliac (DSI) ligaments. The inferior dorsal SIJ was entered and capsular distension with contrast medium was performed. Discomfort had to occur with each provocation maneuver and a contained arthrogram was necessary to continue in the study. Session 2: 1 week later; computer randomized, double-blind multi-site, multi-depth lateral branch blocks injections were performed. Ten subjects received active (bupivicaine 0.75%) and 10 subjects received sham (normal saline) multi-site, multi-depth lateral branch injections. Thirty minutes later, provocation testing was repeated with identical methodology used in session 1. Presence or absence of pain for ligamentous probing and SIJ capsular distension. Seventy percent of the active group had an insensate IO and DSI ligaments, and inferior dorsal SIJ vs 0-10% of the sham group. Twenty percent of the active vs 10% of the sham group did not feel repeat capsular distension. Six of seven subjects (86%) retained the ability to feel repeat capsular distension despite an insensate dorsal SIJ complex. Multi-site, multi-depth lateral branch blocks are physiologically effective at a rate of 70%. Multi-site, multi-depth lateral branch blocks do not effectively block the intra-articular portion of the SIJ. There is physiological evidence that the intra-articular portion of the SIJ is innervated from both ventral and dorsal sources. Comparative multi-site, multi-depth lateral branch blocks should be considered a potentially valuable tool to diagnose extra-articular SIJ pain and determine if lateral branch radiofrequency neurotomy may assist one with SIJ pain.

Partnership-driven Resources to Improve and Enhance Research (PRIMER): a survey of community-engaged researchers and creation of an online toolkit.

PubMed

Dolor, Rowena J; Greene, Sarah M; Thompson, Ella; Baldwin, Laura-Mae; Neale, Anne Victoria

2011-08-01

This project aimed to develop an open-access website providing adaptable resources to facilitate best practices for multisite research from initiation to closeout. methods: A web-based assessment was sent to the leadership of the Clinical and Translational Science Award (CTSA) Community Engagement Key Functions Committee (n= 38) and the CTSA-affiliated Primary Care Practice-based Research Networks (PBRN, n= 55). Respondents rated the benefits and barriers of multisite research, the utility of available resources, and indicated their level of interest in unavailable resources. Then, existing research resources were evaluated for relevance to multisite research, adaptability to other projects, and source credibility. Fifty-five (59%) of invited participants completed the survey. Top perceived benefits of multisite research were the ability to conduct community-relevant research through academic-community partnerships (34%) and accelerating translation of research into practice (31%). Top perceived barriers were lack of research infrastructure to support PBRNs and community partners (31%) and inadequate funding to support multisite collaborations (26%). Over 200 resources were evaluated, of which 120 unique resources were included in the website. The PRIMER Research Toolkit (http://www.researchtoolkit.org) provides an array of peer-reviewed resources to facilitate translational research for the conduct of multisite studies within PBRNs and community-based organizations. © 2011 Wiley Periodicals, Inc.

Partnership‐Driven Resources to Improve and Enhance Research (PRIMER): A Survey of Community‐Engaged Researchers and Creation of an Online Toolkit

PubMed Central

Dolor, Rowena J.; Greene, Sarah M.; Thompson, Ella; Baldwin, Laura‐Mae; Neale, Anne Victoria

2011-01-01

Abstract Objective: This project aimed to develop an open‐access website providing adaptable resources to facilitate best practices for multisite research from initiation to closeout. Methods: A web‐based assessment was sent to the leadership of the Clinical and Translational Science Award (CTSA) Community Engagement Key Functions Committee (n= 38) and the CTSA‐affiliated Primary Care Practice‐based Research Networks (PBRN, n= 55). Respondents rated the benefits and barriers of multisite research, the utility of available resources, and indicated their level of interest in unavailable resources. Then, existing research resources were evaluated for relevance to multisite research, adaptability to other projects, and source credibility. Results: Fifty‐five (59%) of invited participants completed the survey. Top perceived benefits of multisite research were the ability to conduct community‐relevant research through academic–community partnerships (34%) and accelerating translation of research into practice (31%). Top perceived barriers were lack of research infrastructure to support PBRNs and community partners (31%) and inadequate funding to support multisite collaborations (26%). Over 200 resources were evaluated, of which 120 unique resources were included in the website. Conclusion: The PRIMER Research Toolkit (http://www.researchtoolkit.org) provides an array of peer‐reviewed resources to facilitate translational research for the conduct of multisite studies within PBRNs and community‐based organizations. Clin Trans Sci 2011; Volume 4: 259–265 PMID:21884512

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

PubMed

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

2017-01-01

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

Sequence tagging reveals unexpected modifications in toxicoproteomics

PubMed Central

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

2010-01-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-03-01

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

Chemical Posttranslational Modification with Designed Rhodium(II) Catalysts.

PubMed

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

2016-01-01

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

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

Challenges and lessons learned in conducting comparative-effectiveness trials.

PubMed

Herrick, Linda M; Locke, G Richard; Zinsmeister, Alan R; Talley, Nicholas J

2012-05-01

The current health-care environment is demanding evidence-based medicine that relies on clinical trials as the basis for decisions. Clinician investigators are more often finding that they are personally responsible for coordinating large, multisite trials. We present strategies for successful implementation and management of multisite clinical trials and knowledge gained through an international, multisite randomized clinical trial. Topics include team composition, regulatory requirements, study organization and governance, communication strategies, recruitment and retention efforts, budget, technology transfer, and publication.

Challenges and Lessons Learned in Conducting Comparative-Effectiveness Trials

PubMed Central

Herrick, Linda M.; Locke, G. Richard; Zinsmeister, Alan R.; Talley, Nicholas J.

2014-01-01

The current health-care environment is demanding evidence-based medicine that relies on clinical trials as the basis for decisions. Clinician investigators are more often finding that they are personally responsible for coordinating large, multisite trials. We present strategies for successful implementation and management of multisite clinical trials and knowledge gained through an international, multisite randomized clinical trial. Topics include team composition, regulatory requirements, study organization and governance, communication strategies, recruitment and retention efforts, budget, technology transfer, and publication. PMID:22552235

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

PubMed Central

Mahajan, Rohit; Gerace, Larry; Melchior, Frauke

1998-01-01

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

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

PubMed

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

2017-02-21

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

A view inside the nature of protein crystals

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

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

PubMed

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

2018-06-21

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

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

PubMed

Deming, Timothy J

2017-03-15

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

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-09-01

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

Modifications of Glycans: Biological Significance and Therapeutic Opportunities

PubMed Central

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

2012-01-01

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

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

PubMed Central

Addona, Terri A; Abbatiello, Susan E; Schilling, Birgit; Skates, Steven J; Mani, D R; Bunk, David M; Spiegelman, Clifford H; Zimmerman, Lisa J; Ham, Amy-Joan L; Keshishian, Hasmik; Hall, Steven C; Allen, Simon; Blackman, Ronald K; Borchers, Christoph H; Buck, Charles; Cardasis, Helene L; Cusack, Michael P; Dodder, Nathan G; Gibson, Bradford W; Held, Jason M; Hiltke, Tara; Jackson, Angela; Johansen, Eric B; Kinsinger, Christopher R; Li, Jing; Mesri, Mehdi; Neubert, Thomas A; Niles, Richard K; Pulsipher, Trenton C; Ransohoff, David; Rodriguez, Henry; Rudnick, Paul A; Smith, Derek; Tabb, David L; Tegeler, Tony J; Variyath, Asokan M; Vega-Montoto, Lorenzo J; Wahlander, Åsa; Waldemarson, Sofia; Wang, Mu; Whiteaker, Jeffrey R; Zhao, Lei; Anderson, N Leigh; Fisher, Susan J; Liebler, Daniel C; Paulovich, Amanda G; Regnier, Fred E; Tempst, Paul; Carr, Steven A

2010-01-01

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma. PMID:19561596

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

PubMed Central

Chang, Chuang-Rung; Blackstone, Craig

2017-01-01

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

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

PubMed Central

Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

2016-01-01

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

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

PubMed

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

2012-05-01

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

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

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

PubMed

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

2017-04-20

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

Tools and Methods for Risk Management in Multi-Site Engineering Projects

NASA Astrophysics Data System (ADS)

Zhou, Mingwei; Nemes, Laszlo; Reidsema, Carl; Ahmed, Ammar; Kayis, Berman

In today's highly global business environment, engineering and manufacturing projects often involve two or more geographically dispersed units or departments, research centers or companies. This paper attempts to identify the requirements for risk management in a multi-site engineering project environment, and presents a review of the state-of-the-art tools and methods that can be used to manage risks in multi-site engineering projects. This leads to the development of a risk management roadmap, which will underpin the design and implementation of an intelligent risk mapping system.

Biological Stress Systems, Adverse Life Events, and the Improvement of Chronic Multisite Musculoskeletal Pain Across a 6-Year Follow-Up.

PubMed

Generaal, Ellen; Vogelzangs, Nicole; Macfarlane, Gary J; Geenen, Rinie; Smit, Johannes H; de Geus, Eco J C N; Dekker, Joost; Penninx, Brenda W J H

2017-02-01

Dysfunction of biological stress systems and adverse life events, independently and in interaction, have been hypothesized to predict chronic pain persistence. Conversely, these factors may hamper the improvement of chronic pain. Longitudinal evidence is currently lacking. We examined whether: 1) function of biological stress systems, 2) adverse life events, and 3) their combination predict the improvement of chronic multisite musculoskeletal pain. Subjects of the Netherlands Study of Depression and Anxiety (NESDA) with chronic multisite musculoskeletal pain at baseline (N = 665) were followed-up 2, 4, and 6 years later. The Chronic Pain Grade Questionnaire was used to determine improvement (not meeting the criteria) of chronic multisite musculoskeletal pain at follow-up. Baseline assessment of biological stress systems included function of hypothalamic-pituitary-adrenal axis (1-hour cortisol awakening response, evening level, and post dexamethasone level), the immune system (basal and lipopolysaccharide-stimulated inflammatory markers), the autonomic nervous system (heart rate, pre-ejection period, SD of the normal-to-normal interval, and respiratory sinus arrhythmia). The number of adverse life events were assessed at baseline and 2-year follow-up using the List of Threatening Events Questionnaire. We showed that hypothalamic-pituitary-adrenal axis, immune system, and autonomic nervous system functioning and adverse life events were not associated with the improvement of chronic multisite musculoskeletal pain, either as a main effect or in interaction. This longitudinal study could not confirm that biological stress system dysfunction and adverse life events affect the course of chronic multisite musculoskeletal pain. Biological stress systems and adverse life events are not associated with the improvement of chronic multisite musculoskeletal pain over 6 years of follow-up. Other determinants should thus be considered in future research to identify in which persons pain symptoms will improve. Copyright © 2016 American Pain Society. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2018-06-01

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

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

PubMed Central

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

2011-01-01

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

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

PubMed Central

Konrad, Zvia; Eichler, Jerry

2002-01-01

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

Multisite occupation of divalent dopants in barium and strontium titanates

NASA Astrophysics Data System (ADS)

Zulueta, Yohandys A.; Nguyen, Minh Tho

2018-10-01

Based on recent experimental and theoretical proofs of calcium multisite occupation in barium titanate, we investigated a mixed incorporation mechanism for divalent dopants in barium and strontium titanates (BaTiO3 and SrTiO3). Our present theoretical results demonstrated the multisite occupation of divalent dopants in both perovskite structures. We determined the dependences of the solution, binding energies, and final solution energies with respect to the ionic radii of the dopants. Calculated results obtained based on classical simulations showed that the divalent dopants can occupy both A- and Ti- cation sites in ATiO3 perovskite structures. Such a multisite occupation has direct implications for other experimental findings regarding BaTiO3, such as non-stabilization of the tetragonal phase, shifts in the Curie temperature, intensification of the diffuse phase transition, and shifts in the absorption of ultraviolet light to the visible range in photocatalytic applications related to solar cells for producing energy.

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

PubMed

Wani, Revati; Murray, Brion W

2017-01-01

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

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

PubMed Central

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

2017-01-01

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

New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

PubMed

Broad, W; Ling, Q; Jarvis, P

2016-01-01

Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. Copyright © 2016 Elsevier Inc. All rights reserved.

RNA-modifying proteins as anticancer drug targets.

PubMed

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

2018-06-01

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

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

PubMed

Running, William E; Reilly, James P

2010-10-01

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

Beyond histones - the expanding roles of protein lysine methylation.

PubMed

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

2017-09-01

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

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

PubMed

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

2011-10-01

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

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

PubMed Central

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

2011-01-01

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

Proteomic analyses of the environmental toxicity of carcinogenic chemicals

EPA Science Inventory

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

Biopharmaceuticals from plants: a multitude of options for posttranslational modifications.

PubMed

Warzecha, Heribert

2008-01-01

In 1982 the first recombinant therapeutic, human insulin, was introduced into the market and started a new branch of pharmaceutical development, manufacture, and therapy options. To date, more than 130 recombinant protein therapeutics have been approved by the US Food and Drug Administration (FDA) and many more are being developed world wide. With the increasing number of protein therapeutics the number of potential production organisms is also expanding, and posttranslational modification of proteins has become a topic of special focus. One major difference between small-molecule drugs and protein therapeutics is that the latter are reliant on a host organism for their production and this can have a large influence on the final structure and can ultimately affect the pharmacokinetics, immunogenicity, and the function of the protein depending on the production process. Plants can be efficiently used as production systems for recombinant proteins thereby offering a variety of options for transgene targeting and modification. This review is intended to give an overview about the potential of plants to serve as a production system for therapeutic and prophylactic biopharmaceuticals with respect to posttranslational modifications.

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

PubMed Central

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

2015-01-01

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

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

PubMed Central

Sullenger, Bruce A.

2013-01-01

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

Reduced-complexity multi-site rainfall generation: one million years over night using the model TripleM

NASA Astrophysics Data System (ADS)

Breinl, Korbinian; Di Baldassarre, Giuliano; Girons Lopez, Marc

2017-04-01

We assess uncertainties of multi-site rainfall generation across spatial scales and different climatic conditions. Many research subjects in earth sciences such as floods, droughts or water balance simulations require the generation of long rainfall time series. In large study areas the simulation at multiple sites becomes indispensable to account for the spatial rainfall variability, but becomes more complex compared to a single site due to the intermittent nature of rainfall. Weather generators can be used for extrapolating rainfall time series, and various models have been presented in the literature. Even though the large majority of multi-site rainfall generators is based on similar methods, such as resampling techniques or Markovian processes, they often become too complex. We think that this complexity has been a limit for the application of such tools. Furthermore, the majority of multi-site rainfall generators found in the literature are either not publicly available or intended for being applied at small geographical scales, often only in temperate climates. Here we present a revised, and now publicly available, version of a multi-site rainfall generation code first applied in 2014 in Austria and France, which we call TripleM (Multisite Markov Model). We test this fast and robust code with daily rainfall observations from the United States, in a subtropical, tropical and temperate climate, using rain gauge networks with a maximum site distance above 1,000km, thereby generating one million years of synthetic time series. The modelling of these one million years takes one night on a recent desktop computer. In this research, we first start the simulations with a small station network of three sites and progressively increase the number of sites and the spatial extent, and analyze the changing uncertainties for multiple statistical metrics such as dry and wet spells, rainfall autocorrelation, lagged cross correlations and the inter-annual rainfall variability. Our study contributes to the scientific community of earth sciences and the ongoing debate on extreme precipitation in a changing climate by making a stable, and very easily applicable, multi-site rainfall generation code available to the research community and providing a better understanding of the performance of multi-site rainfall generation depending on spatial scales and climatic conditions.

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

PubMed

Wu, Wei; Hua, Yufei; Lin, Qinlu

2014-03-01

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

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

PubMed Central

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

2016-01-01

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

Role of Carbonyl Modifications on Aging-Associated Protein Aggregation

PubMed Central

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

2016-01-01

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

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95.

PubMed

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

2017-04-01

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

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.

Sulfenic acid chemistry, detection and cellular lifetime☆

PubMed Central

Gupta, Vinayak; Carroll, Kate S.

2014-01-01

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

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

PubMed

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

2017-04-01

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

Analysis Concerning the Inspection Threshold for Multi-Site Damage.

DOT National Transportation Integrated Search

1993-12-01

Periodic inspections, at a prescribed interval, for Multi-Site Damage (MS) in longitudinal fuselage lap-joints start when the aircraft has accumulated a certain number of flights, the inspection threshold. The work reported here was an attempt to obt...

Multisite tumor sampling enhances the detection of intratumor heterogeneity at all different temporal stages of tumor evolution.

PubMed

Erramuzpe, Asier; Cortés, Jesús M; López, José I

2018-02-01

Intratumor heterogeneity (ITH) is an inherent process of tumor development that has received much attention in previous years, as it has become a major obstacle for the success of targeted therapies. ITH is also temporally unpredictable across tumor evolution, which makes its precise characterization even more problematic since detection success depends on the precise temporal snapshot at which ITH is analyzed. New and more efficient strategies for tumor sampling are needed to overcome these difficulties which currently rely entirely on the pathologist's interpretation. Recently, we showed that a new strategy, the multisite tumor sampling, works better than the routine sampling protocol for the ITH detection when the tumor time evolution was not taken into consideration. Here, we extend this work and compare the ITH detections of multisite tumor sampling and routine sampling protocols across tumor time evolution, and in particular, we provide in silico analyses of both strategies at early and late temporal stages for four different models of tumor evolution (linear, branched, neutral, and punctuated). Our results indicate that multisite tumor sampling outperforms routine protocols in detecting ITH at all different temporal stages of tumor evolution. We conclude that multisite tumor sampling is more advantageous than routine protocols in detecting intratumor heterogeneity.

Analysis of sDMA modifications of PIWI proteins

PubMed Central

Honda, Shozo; Kirino, Yoriko; Kirino, Yohei

2015-01-01

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

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

PubMed

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

2015-08-04

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

Identifying DNA methylation in a nanochannel

NASA Astrophysics Data System (ADS)

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

2016-01-01

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

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

PubMed

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

2013-06-01

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

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

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

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

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/

Culture and Influence in Multisite Evaluation

ERIC Educational Resources Information Center

Kirkhart, Karen E.

2011-01-01

Understanding the influence of multisite evaluation requires careful consideration of cultural context. The author illustrates dimensions of influence and culture with excerpts from four National Science Foundation evaluation case studies and summarizes what influence teaches everyone about culture and what culture teaches everyone about…

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

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2008-07-01

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

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.

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.

Sex-specific impact of early-life adversity on chronic pain: a large population-based study in Japan.

PubMed

Yamada, Keiko; Matsudaira, Ko; Tanaka, Eizaburo; Oka, Hiroyuki; Katsuhira, Junji; Iso, Hiroyasu

2017-01-01

Responses to early-life adversity may differ by sex. We investigated the sex-specific impact of early-life adversity on chronic pain, chronic multisite pain, and somatizing tendency with chronic pain. We examined 4229 respondents aged 20-79 years who participated in the Pain Associated Cross-Sectional Epidemiological Survey in Japan. Outcomes were: 1) chronic pain prevalence, 2) multisite pain (≥3 sites) prevalence, and 3) multiple somatic symptoms (≥3 symptoms) among respondents with chronic pain related to the presence or absence of early-life adversity. Multivariable-adjusted odds ratios (ORs) were calculated with 95% confidence intervals using a logistic regression model including age, smoking status, exercise routine, sleep time, body mass index, household expenditure, and the full distribution of scores on the Mental Health Inventory-5. We further adjusted for pain intensity when we analyzed the data for respondents with chronic pain. The prevalence of chronic pain was higher among respondents reporting the presence of early-life adversity compared with those reporting its absence, with multivariable ORs of 1.62 (1.22-2.15, p <0.01) in men and 1.47 (1.13-1.90, p <0.01) in women. Among women with chronic pain, early-life adversity was associated with multisite pain and multiple somatic symptoms; multivariable ORs were 1.78 (1.22-2.60, p <0.01) for multisite pain and 1.89 (1.27-2.83, p <0.01) for ≥3 somatic symptoms. No associations were observed between early-life adversity and chronic multisite pain or multiple somatic symptoms among men with chronic pain. Early-life adversity may be linked to a higher prevalence of chronic pain among both sexes and to multisite pain and somatizing tendency among women with chronic pain.

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

PubMed

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

2012-01-01

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

Proteomic methods for analysis of S-nitrosation⋄

PubMed Central

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

2007-01-01

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

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

Expansion of the gateway multisite recombination cloning toolkit.

PubMed

Shearin, Harold K; Dvarishkis, Alisa R; Kozeluh, Craig D; Stowers, R Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

Expansion of the Gateway MultiSite Recombination Cloning Toolkit

PubMed Central

Shearin, Harold K.; Dvarishkis, Alisa R.; Kozeluh, Craig D.; Stowers, R. Steven

2013-01-01

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters. PMID:24204935

Multi-level assessment protocol (MAP) for adoption in multi-site clinical trials

PubMed Central

Guydish, J.; Manser, S.T.; Jessup, M.; Tajima, B.; Sears, C.; Montini, T.

2010-01-01

The National Institute on Drug Abuse (NIDA) Clinical Trials Network (CTN) is intended to test promising drug abuse treatment models in multi-site clinical trials, and to support adoption of new interventions into clinical practice. Using qualitative research methods we asked: How might the technology of multi-site clinical trials be modified to better support adoption of tested interventions? A total of 42 participants, representing 8 organizational levels ranging from clinic staff to clinical trial leaders, were interviewed about their role in the clinical trial, its interactions with clinics, and intervention adoption. Among eight clinics participating in the clinical trial, we found adoption of the tested intervention in one clinic only. In analysis of interview data we identified four conceptual themes which are likely to affect adoption and may be informative in future multi-site clinical trials. We offer the conclusion that planning for adoption in the early stages of protocol development will better serve the aim of integrating new interventions into practice. PMID:20890376

A new MCM modification cycle regulates DNA replication initiation

PubMed Central

Wei, Lei; Zhao, Xiaolan

2016-01-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1 and activated in S phase to initiate replication. During this transition, the only known chemical change on MCM is the gain of multi-site phosphorylation that promotes cofactor recruitment. As replication initiation is intimately linked to multiple biological cues, additional changes on MCM can provide further regulatory points. Here, we describe a yeast MCM sumoylation cycle that negatively regulates replication. MCM subunits undergo sumoylation upon loading at origins in G1 prior to MCM phosphorylation. MCM sumoylation levels then decline as MCM phosphorylation levels rise, suggesting an inhibitory role in replication. Indeed, increasing MCM sumoylation impairs replication initiation through promoting the recruitment of a phosphatase that reduces MCM phosphorylation and activation. MCM sumoylation thus counterbalances kinase-based regulation to ensure accurate control of replication initiation. PMID:26854664

A new MCM modification cycle regulates DNA replication initiation.

PubMed

Wei, Lei; Zhao, Xiaolan

2016-03-01

The MCM DNA helicase is a central regulatory target during genome replication. MCM is kept inactive during G1, and it initiates replication after being activated in S phase. During this transition, the only known chemical change to MCM is the gain of multisite phosphorylation that promotes cofactor recruitment. Because replication initiation is intimately linked to multiple biological cues, additional changes to MCM can provide further regulatory points. Here, we describe a yeast MCM SUMOylation cycle that regulates replication. MCM subunits undergo SUMOylation upon loading at origins in G1 before MCM phosphorylation. MCM SUMOylation levels then decline as MCM phosphorylation levels rise, thus suggesting an inhibitory role of MCM SUMOylation during replication. Indeed, increasing MCM SUMOylation impairs replication initiation, partly through promoting the recruitment of a phosphatase that decreases MCM phosphorylation and activation. We propose that MCM SUMOylation counterbalances kinase-based regulation, thus ensuring accurate control of replication initiation.

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

PubMed

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

2011-12-01

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

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

PubMed

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

2018-06-16

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

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.

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.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

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

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

PubMed

Sharma, Rajal; Zhou, Ming-Ming

2015-09-01

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

Diagonal chromatography to study plant protein modifications.

PubMed

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

2016-08-01

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

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

PubMed

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

2017-01-01

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

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

PubMed Central

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

2017-01-01

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

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

PubMed

Feltes, Bruno César; Bonatto, Diego

2015-01-01

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

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

PubMed Central

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

2015-01-01

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

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

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

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

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

2010-01-18

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

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

PubMed

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

2015-10-21

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

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

Adaptive Landscape Flattening Accelerates Sampling of Alchemical Space in Multisite λ Dynamics.

PubMed

Hayes, Ryan L; Armacost, Kira A; Vilseck, Jonah Z; Brooks, Charles L

2017-04-20

Multisite λ dynamics (MSλD) is a powerful emerging method in free energy calculation that allows prediction of relative free energies for a large set of compounds from very few simulations. Calculating free energy differences between substituents that constitute large volume or flexibility jumps in chemical space is difficult for free energy methods in general, and for MSλD in particular, due to large free energy barriers in alchemical space. This study demonstrates that a simple biasing potential can flatten these barriers and introduces an algorithm that determines system specific biasing potential coefficients. Two sources of error, deep traps at the end points and solvent disruption by hard-core potentials, are identified. Both scale with the size of the perturbed substituent and are removed by sharp biasing potentials and a new soft-core implementation, respectively. MSλD with landscape flattening is demonstrated on two sets of molecules: derivatives of the heat shock protein 90 inhibitor geldanamycin and derivatives of benzoquinone. In the benzoquinone system, landscape flattening leads to 2 orders of magnitude improvement in transition rates between substituents and robust solvation free energies. Landscape flattening opens up new applications for MSλD by enabling larger chemical perturbations to be sampled with improved precision and accuracy.

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2016-04-29

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

Multi-Site Quality Assurance Project Plan for Wisconsin Public Service Corporation, Peoples Gas Light and Coke Company, and North Shore Gas

EPA Pesticide Factsheets

This Multi-Site QAPP presents the organization, data quality objectives (DQOs), a set of anticipated activities, sample analysis, data handling and specific Quality Assurance/Quality Control (QA/QC) procedures associated with Studies done in EPA Region 5

A Multisite Evaluation of Reading Is Fundamental: Summary Report.

ERIC Educational Resources Information Center

Parker, Ronald K.; And Others

Reading Is Fundamental (RIF) is a national program that seeks to motivate children to read. Implemented by local sponsors and aimed at disadvantaged groups, it offers participating children a wide selection of attractive paperback books about interesting people, places, and events. The multisite evaluation summarized in this report studied…

A Multisite Evaluation of Reading Is Fundamental: Technical Report.

ERIC Educational Resources Information Center

Parker, Ronald K.; And Others

Reading Is Fundamental (RIF) is a national program that seeks to motivate children to read. Implemented by local sponsors and aimed at disadvantaged groups, it offers participating children a wide selection of attractive paperbound books about interesting people, places, and events. The multisite evaluation discussed in this technical report…

Brief Treatments for Cannabis Dependence: Findings From a Randomized Multisite Trial

ERIC Educational Resources Information Center

Babor, Thomas F.

2004-01-01

This study evaluated the efficacy of 2 brief interventions for cannabis-dependent adults. A multisite randomized controlled trial compared cannabis use outcomes across 3 study conditions: (a) 2 sessions of motivational enhancement therapy (MET); (b) 9 sessions of multicomponent therapy that included MET, cognitive-behavioral therapy, and case…

Network cyberinfrastructure as a shared platform to support multi-site research

USDA-ARS?s Scientific Manuscript database

Multi-site research across the Long-term Agroecosystem Research (LTAR) network requires access to data and information. We present some existing examples where you can get data from across the network and summarize the rich inventory of measurements taken across LTAR sites. But data management suppo...

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.

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.

Systematic prediction of control proteins and their DNA binding sites

PubMed Central

Sorokin, Valeriy; Severinov, Konstantin; Gelfand, Mikhail S.

2009-01-01

We present here the results of a systematic bioinformatics analysis of control (C) proteins, a class of DNA-binding regulators that control time-delayed transcription of their own genes as well as restriction endonuclease genes in many type II restriction-modification systems. More than 290 C protein homologs were identified and DNA-binding sites for ∼70% of new and previously known C proteins were predicted by a combination of phylogenetic footprinting and motif searches in DNA upstream of C protein genes. Additional analysis revealed that a large proportion of C protein genes are translated from leaderless RNA, which may contribute to time-delayed nature of genetic switches operated by these proteins. Analysis of genetic contexts of newly identified C protein genes revealed that they are not exclusively associated with restriction-modification genes; numerous instances of associations with genes originating from mobile genetic elements were observed. These instances might be vestiges of ancient horizontal transfers and indicate that during evolution ancestral restriction-modification system genes were the sites of mobile elements insertions. PMID:19056824

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

PubMed Central

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

2017-01-01

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

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

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

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

PLMD: An updated data resource of protein lysine modifications.

PubMed

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

2017-05-20

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

Role of O-GlcNAcylation in nutritional sensing, insulin resistance and in mediating the benefits of exercise.

PubMed

Myslicki, Jason P; Belke, Darrell D; Shearer, Jane

2014-11-01

The purpose of this review is to highlight the role of O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification in metabolic disease states and to summarize current knowledge of how exercise affects this important post-translational signalling pathway. O-GlcNAc modification is an intracellular tool capable of integrating energy supply with demand. The accumulation of excess energy associated with obesity and insulin resistance is mediated, in part, by the hexosamine biosynthetic pathway (HBP), which results in the O-GlcNAcylation of a myriad of proteins, thereby affecting their respective function, stability, and localization. Insulin resistance is related to the excessive O-GlcNAcylation of key metabolic proteins causing a chronic blunting of insulin signalling pathways and precipitating the accompanying pathologies, such as heart and kidney disease. Lifestyle modifications such as diet and exercise also modify the pathway. Exercise is a front-line and cost-effective therapeutic approach for insulin resistance, and recent work shows that the intervention can alter O-GlcNAc gene expression, signalling, and protein modification. However, there is currently no consensus on the effect of frequency, intensity, type, and duration of exercise on O-GlcNAc modification, the HBP, and its related enzymes. On one end of the spectrum, mild, prolonged swim training reduces O-GlcNAcylation, while on the other end, higher intensity treadmill running increases cardiac protein O-GlcNAc modification. Clearly, a balance between acute and chronic stress of exercise is needed to reap the benefits of the intervention on O-GlcNAc signalling.

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

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMOmore » substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.« less

Resilience of biochemical activity in protein domains in the face of structural divergence.

PubMed

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

2014-06-01

Recent studies point to the prevalence of the evolutionary phenomenon of drastic structural transformation of protein domains while continuing to preserve their basic biochemical function. These transformations span a wide spectrum, including simple domains incorporated into larger structural scaffolds, changes in the structural core, major active site shifts, topological rewiring and extensive structural transmogrifications. Proteins from biological conflict systems, such as toxin-antitoxin, restriction-modification, CRISPR/Cas, polymorphic toxin and secondary metabolism systems commonly display such transformations. These include endoDNases, metal-independent RNases, deaminases, ADP ribosyltransferases, immunity proteins, kinases and E1-like enzymes. In eukaryotes such transformations are seen in domains involved in chromatin-related peptide recognition and protein/DNA-modification. Intense selective pressures from 'arms-race'-like situations in conflict and macromolecular modification systems could favor drastic structural divergence while preserving function. Published by Elsevier Ltd.

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

Treatment of chronically depressed patients: A multisite randomized controlled trial testing the effectiveness of 'Cognitive Behavioral Analysis System of Psychotherapy' (CBASP) for chronic depressions versus usual secondary care

PubMed Central

Wiersma, Jenneke E; van Schaik, Digna JF; van Oppen, Patricia; McCullough, James P; Schoevers, Robert A; Dekker, Jack J; Blom, Marc BJ; Maas, Kristel; Smit, Johannes H; Penninx, Brenda WJH; Beekman, Aartjan TF

2008-01-01

Background 'Cognitive Behavioral Analysis System of Psychotherapy' (CBASP) is a form of psychotherapy specifically developed for patients with chronic depression. In a study in the U.S., remarkable favorable effects of CBASP have been demonstrated. However, no other studies have as yet replicated these findings and CBASP has not been tested outside the United States. This protocol describes a randomized controlled trial on the effectiveness of CBASP in the Netherlands. Methods/Design The purpose of the present paper is to report the study protocol of a multisite randomized controlled trial testing the effectiveness of 'Cognitive Behavioral Analysis System of Psychotherapy' (CBASP) for chronic depression in the Netherlands. In this study, CBASP in combination with medication, will be tested versus usual secondary care in combination with medication. The aim is to recruit 160 patients from three mental health care organizations. Depressive symptoms will be assessed at baseline, after 8 weeks, 16 weeks, 32 weeks and 52 weeks, using the 28-item Inventory for Depressive Symptomatology (IDS). Effect modification by co morbid anxiety, alcohol consumption, general and social functioning and working alliance will be tested. GEE analyses of covariance, controlling for baseline value and center will be used to estimate the overall treatment effectiveness (difference in IDS score) at post-treatment and follow up. The primary analysis will be by 'intention to treat' using double sided tests. An economic analysis will compare the two groups in terms of mean costs and cost-effectiveness from a societal perspective. Discussion The study will provide an answer to the question whether the favorable effects of CBASP can be replicated outside the US. Trial Registration The Dutch Cochrane Center, NTR1090. PMID:18366729

Label-free proteomics assisted by affinity enrichment for elucidating the chemical reactivity of the liver mitochondrial proteome toward adduction by the lipid electrophile 4-hydroxy-2-nonenal (HNE)

NASA Astrophysics Data System (ADS)

Maier, Claudia

2016-03-01

The analysis of oxidative stress-induced post-translational modifications remains challenging due to the chemical diversity of these modifications, the possibility of the presence of positional isomers and the low stoichiometry of the modified proteins present in a cell or tissue proteome. Alcoholic liver disease (ALD) is a multifactorial disease in which mitochondrial dysfunction and oxidative stress have been identified as being critically involved in the progression of the disease from steatosis to cirrhosis. Ethanol metabolism leads to increased levels of reactive oxygen species (ROS), glutathione depletion and lipid peroxidation. Posttranslational modification of proteins by electrophilic products of lipid peroxidation has been associated with governing redox-associated signaling mechanisms, but also as contributing to protein dysfunction leading to organelle and liver injury. In particular the prototypical α,β-unsaturated aldehyde, 4-hydroxy-2-nonenal (HNE), has been extensively studied as marker of increased oxidative stress in hepatocytes. In this study, we combined a LC-MS label-free quantification method and affinity enrichment to assess the dose-dependent insult by HNE on the proteome of rat liver mitochondria. We used a carbonyl-selective probe, the ARP probe, to label HNE-protein adducts and to perform affinity capture at the protein level. Using LC-MS to obtain protein abundance estimates, a list of protein targets was obtained with increasing concentration of HNE used in the exposure studies. In parallel, we performed affinity capture at the peptide level to acquire site-specific information. Examining the concentration-dependence of the protein modifications, we observed distinct reactivity profiles for HNE-protein adduction. Pathway analysis indicated that proteins associated with metabolic processes, including amino acid, fatty acid and glyoxylate and dicarboxylate metabolism, bile acid synthesis and TCA cycle, showed enhanced reactivity to HNE adduction. Whereas, proteins associated with oxidative phosphorylation displayed retardation toward HNE adduction. We provide a list of 31 protein targets with a total of 61 modification sites that may guide future targeted LC-MS assays to monitor disease progression and/or intervention in preclinical models of ALD and possibly other liver diseases with oxidative stress component.

Protein arginine methylation: a prominent modification and its demethylation.

PubMed

Wesche, Juste; Kühn, Sarah; Kessler, Benedikt M; Salton, Maayan; Wolf, Alexander

2017-09-01

Arginine methylation of histones is one mechanism of epigenetic regulation in eukaryotic cells. Methylarginines can also be found in non-histone proteins involved in various different processes in a cell. An enzyme family of nine protein arginine methyltransferases catalyses the addition of methyl groups on arginines of histone and non-histone proteins, resulting in either mono- or dimethylated-arginine residues. The reversibility of histone modifications is an essential feature of epigenetic regulation to respond to changes in environmental factors, signalling events, or metabolic alterations. Prominent histone modifications like lysine acetylation and lysine methylation are reversible. Enzyme family pairs have been identified, with each pair of lysine acetyltransferases/deacetylases and lysine methyltransferases/demethylases operating complementarily to generate or erase lysine modifications. Several analyses also indicate a reversible nature of arginine methylation, but the enzymes facilitating direct removal of methyl moieties from arginine residues in proteins have been discussed controversially. Differing reports have been seen for initially characterized putative candidates, like peptidyl arginine deiminase 4 or Jumonji-domain containing protein 6. Here, we review the most recent cellular, biochemical, and mass spectrometry work on arginine methylation and its reversible nature with a special focus on putative arginine demethylases, including the enzyme superfamily of Fe(II) and 2-oxoglutarate-dependent oxygenases.

The tomato UV-damaged DNA binding protein 1 (DDB1) plays a role in organ size control via an epigenetic manner

USDA-ARS?s Scientific Manuscript database

Epigenetic regulation, including various covalent modifications of histone proteins and methylation of cytosine bases in DNA, participates broadly in many fundamentally physiological and developmental processes. The repressed or active states of transcription resulted from epigenetic modifications a...

Simultaneous quantification of oil and protein in cottonseed by low-field time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cottonseed quality traits is likely to be achieved through a combination of genetic modification, manipulation of nutrient allocation and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relation...

Non-destructive quantification of oil and protein in cottonseed by time-domain nuclear magnetic resonance

USDA-ARS?s Scientific Manuscript database

Modification of cotton seed quality traits is likely to be achieved through a combination of genetic modification, nutrient allocation, and selective breeding. Oil and protein stores comprise the majority of mass of cottonseed embryos. A more comprehensive understanding of the relationship between f...

Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

DOE PAGES

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

2017-01-25

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

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications.

PubMed

Porter, Joseph J; Mehl, Ryan A

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described.

Testing the Feasibility of Fidelity Evaluation in a Multisite, Multiprogram Initiative

ERIC Educational Resources Information Center

Cornish, Disa Lubker; Losch, Mary E.; Avery, Mitchell

2016-01-01

Monitoring fidelity of implementation is a critical task when initiating evidence-based programs. This pilot study sought to identify best practices in a fidelity monitoring process and determine the feasibility of continuing a fidelity monitoring process with a multisite, multiprogram initiative. A fidelity log was created for each of 11…

Intraclass Correlations for Three-Level Multi-Site Cluster-Randomized Trials of Science Achievement

ERIC Educational Resources Information Center

Westine, Carl D.

2015-01-01

A cluster-randomized trial (CRT) relies on random assignment of intact clusters to treatment conditions, such as classrooms or schools (Raudenbush & Bryk, 2002). One specific type of CRT, a multi-site CRT (MSCRT), is commonly employed in educational research and evaluation studies (Spybrook & Raudenbush, 2009; Spybrook, 2014; Bloom,…

A Multi-Site Study on Medical School Selection, Performance, Motivation and Engagement

ERIC Educational Resources Information Center

Wouters, A.; Croiset, G.; Schripsema, N. R.; Cohen-Schotanus, J.; Spaai, G. W.; Hulsman, R. L.; Kusurkar, R. A.

2017-01-01

Medical schools seek ways to improve their admissions strategies, since the available methods prove to be suboptimal for selecting the best and most motivated students. In this multi-site cross-sectional questionnaire study, we examined the value of (different) selection procedures compared to a weighted lottery procedure, which includes direct…

Development and Implementation of a Multisite Evaluation for the Women, Co-Occurring Disorders and Violence Study

ERIC Educational Resources Information Center

Giard, Julienne; Hennigan, Karen; Huntington, Nicholas; Vogel, Wendy; Rinehart, Debbie; Mazelis, Ruta; Nadlicki, Terri; Veysey, Bonita M.

2005-01-01

In this article we describe the development and implementation of the Substance Abuse and Mental Health Services Administration's (SAMHSA's) multisite Women, Co-Occurring Disorders and Violence Study (WCDVS), highlighting some of the challenges encountered, decisions made, and lessons learned. Four themes are discussed. First, the unique…

Learning "about" and "from" Variation in Program Impacts Using Multisite Trials. MDRC Working Papers on Research Methodology

ERIC Educational Resources Information Center

Raudenbush, Stephen W.; Bloom, Howard S.

2015-01-01

The present paper, which is intended for a diverse audience of evaluation researchers, applied social scientists, and research funders, provides a broad overview of the conceptual and statistical issues involved in using multisite randomized trials to learn "about" and "from" variation in program effects across…

Power Calculations for Moderators in Multi-Site Cluster Randomized Trials

ERIC Educational Resources Information Center

Spybrook, Jessaca; Kelcey, Ben; Dong, Nianbo

2016-01-01

Cluster randomized trials (CRTs), or studies in which intact groups of individuals are randomly assigned to a condition, are becoming more common in evaluation studies of educational programs. A specific type of CRT in which clusters are randomly assigned to treatment within blocks or sites, known as multisite cluster randomized trials (MSCRTs),…

Learning about and from a Distribution of Program Impacts Using Multisite Trials

ERIC Educational Resources Information Center

Raudenbush, Stephen W.; Bloom, Howard S.

2015-01-01

The present article provides a synthesis of the conceptual and statistical issues involved in using multisite randomized trials to learn about and from a distribution of heterogeneous program impacts across individuals and/or program sites. Learning "about" such a distribution involves estimating its mean value, detecting and quantifying…

Statistical Analysis for Multisite Trials Using Instrumental Variables with Random Coefficients

ERIC Educational Resources Information Center

Raudenbush, Stephen W.; Reardon, Sean F.; Nomi, Takako

2012-01-01

Multisite trials can clarify the average impact of a new program and the heterogeneity of impacts across sites. Unfortunately, in many applications, compliance with treatment assignment is imperfect. For these applications, we propose an instrumental variable (IV) model with person-specific and site-specific random coefficients. Site-specific IV…

How Does a Multi-Site Institution Plan Effectively?

ERIC Educational Resources Information Center

Jefferson, Curtis F.

A multi-site college plans effectively by having in place planning and evaluation systems that enhance its capability to respond effectively to changes in the environment in order to continue to provide high quality educational programs and services for the people in its service area. The effectiveness of these systems is dependent on clearly…

Toward Better Research on--and Thinking about--Evaluation Influence, Especially in Multisite Evaluations

ERIC Educational Resources Information Center

Mark, Melvin M.

2011-01-01

Evaluation is typically carried out with the intention of making a difference in the understandings and actions of stakeholders and decision makers. The author provides a general review of the concepts of evaluation "use," evaluation "influence," and "influence pathways," with connections to multisite evaluations. The study of evaluation influence…

Site Matters: Multisite Randomized Trial of Motivational Enhancement Therapy in Community Drug Abuse Clinics

ERIC Educational Resources Information Center

Ball, Samuel A.; Martino, Steve; Nich, Charla; Frankforter, Tami L.; Van Horn, Deborah; Crits-Christoph, Paul; Woody, George E.; Obert, Jeanne L.; Farentinos, Christiane; Carroll, Kathleen M.

2007-01-01

The effectiveness of motivational enhancement therapy (MET) in comparison with counseling as usual (CAU) for increasing retention and reducing substance use was evaluated in a multisite randomized clinical trial. Participants were 461 outpatients treated by 31 therapists within 1 of 5 outpatient substance abuse programs. There were no retention…

A Multisite Psychotherapy and Medication Trial for Depressed Adolescents: Background and Benefits

ERIC Educational Resources Information Center

Kratochvil, Christopher J.; Simons, Anne; Vitiello, Benedetto; Walkup, John; Emslie, Graham; Rosenberg, David; March, John S.

2005-01-01

The Treatment for Adolescents With Depression Study (TADS) is an NIMH-supported multisite clinical trial that compares the effectiveness of a depression-specific cognitive behavioral therapy (CBT), medication management with fluoxetine (FLX), the combination of CBT and FLX (COMB), and medical management with pill placebo (PBO). TADS was…

Nonlinear instabilities of multi-site breathers in Klein-Gordon lattices

DOE PAGES

Cuevas-Maraver, Jesus; Kevrekidis, Panayotis G.; Pelinovsky, Dmitry E.

2016-08-01

Here, we explore the possibility of multi-site breather states in a nonlinear Klein–Gordon lattice to become nonlinearly unstable, even if they are found to be spectrally stable. The mechanism for this nonlinear instability is through the resonance with the wave continuum of a multiple of an internal mode eigenfrequency in the linearization of excited breather states. For the nonlinear instability, the internal mode must have its Krein signature opposite to that of the wave continuum. This mechanism is not only theoretically proposed, but also numerically corroborated through two concrete examples of the Klein–Gordon lattice with a soft (Morse) and amore » hard (Φ 4) potential. Compared to the case of the nonlinear Schrödinger lattice, the Krein signature of the internal mode relative to that of the wave continuum may change depending on the period of the multi-site breather state. For the periods for which the Krein signatures of the internal mode and the wave continuum coincide, multi-site breather states are observed to be nonlinearly stable.« less

Distribution of Bell-inequality violation versus multiparty-quantum-correlation measures

NASA Astrophysics Data System (ADS)

Sharma, Kunal; Das, Tamoghna; Sen (de), Aditi; Sen, Ujjwal

Violation of a Bell inequality guarantees the existence of quantum correlations in a shared quantum state. A pure bipartite quantum state, having nonvanishing quantum correlation, always violates a Bell inequality. Such correspondence is absent for multipartite pure quantum states in the case of multipartite correlation function Bell inequalities with two settings at each site. We establish a connection between the monogamy of Bell-inequality violation and multiparty quantum correlations for shared multisite quantum states. We believe that the relation is generic, as it is true for a number of different multisite measures that are defined from radically different perspectives. Precisely, we quantify the multisite-quantum-correlation content in the states by generalized geometric measure, a genuine multisite entanglement measure, as well as three monogamybased multiparty-quantum-correlation measures, viz., 3-tangle, quantum-discord score, and quantum-work-deficit score. We find that generalized Greenberger-Horne-Zeilinger states and another single-parameter family of states, which we refer to as the special Greenberger-Horne-Zeilinger states, have the status of extremal states in such relations.

Non-Gaussian spatiotemporal simulation of multisite daily precipitation: downscaling framework

NASA Astrophysics Data System (ADS)

Ben Alaya, M. A.; Ouarda, T. B. M. J.; Chebana, F.

2018-01-01

Probabilistic regression approaches for downscaling daily precipitation are very useful. They provide the whole conditional distribution at each forecast step to better represent the temporal variability. The question addressed in this paper is: how to simulate spatiotemporal characteristics of multisite daily precipitation from probabilistic regression models? Recent publications point out the complexity of multisite properties of daily precipitation and highlight the need for using a non-Gaussian flexible tool. This work proposes a reasonable compromise between simplicity and flexibility avoiding model misspecification. A suitable nonparametric bootstrapping (NB) technique is adopted. A downscaling model which merges a vector generalized linear model (VGLM as a probabilistic regression tool) and the proposed bootstrapping technique is introduced to simulate realistic multisite precipitation series. The model is applied to data sets from the southern part of the province of Quebec, Canada. It is shown that the model is capable of reproducing both at-site properties and the spatial structure of daily precipitations. Results indicate the superiority of the proposed NB technique, over a multivariate autoregressive Gaussian framework (i.e. Gaussian copula).

Distribution of Bell-inequality violation versus multiparty-quantum-correlation measures

NASA Astrophysics Data System (ADS)

Sharma, Kunal; Das, Tamoghna; SenDe, Aditi; Sen, Ujjwal

2016-06-01

Violation of a Bell inequality guarantees the existence of quantum correlations in a shared quantum state. A pure bipartite quantum state, having nonvanishing quantum correlation, always violates a Bell inequality. Such correspondence is absent for multipartite pure quantum states in the case of multipartite correlation function Bell inequalities with two settings at each site. We establish a connection between the monogamy of Bell-inequality violation and multiparty quantum correlations for shared multisite quantum states. We believe that the relation is generic, as it is true for a number of different multisite measures that are defined from radically different perspectives. Precisely, we quantify the multisite-quantum-correlation content in the states by generalized geometric measure, a genuine multisite entanglement measure, as well as three monogamy-based multiparty-quantum-correlation measures, viz., 3-tangle, quantum-discord score, and quantum-work-deficit score. We find that generalized Greenberger-Horne-Zeilinger states and another single-parameter family of states, which we refer to as the special Greenberger-Horne-Zeilinger states, have the status of extremal states in such relations.

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.

Managing personal health information in distributed research network environments.

PubMed

Bredfeldt, Christine E; Butani, Amy L; Pardee, Roy; Hitz, Paul; Padmanabhan, Sandy; Saylor, Gwyn

2013-10-08

Studying rare outcomes, new interventions and diverse populations often requires collaborations across multiple health research partners. However, transferring healthcare research data from one institution to another can increase the risk of data privacy and security breaches. A working group of multi-site research programmers evaluated the need for tools to support data security and data privacy. The group determined that data privacy support tools should: 1) allow for a range of allowable Protected Health Information (PHI); 2) clearly identify what type of data should be protected under the Health Insurance Portability and Accountability Act (HIPAA); and 3) help analysts identify which protected health information data elements are allowable in a given project and how they should be protected during data transfer. Based on these requirements we developed two performance support tools to support data programmers and site analysts in exchanging research data. The first tool, a workplan template, guides the lead programmer through effectively communicating the details of multi-site programming, including how to run the program, what output the program will create, and whether the output is expected to contain protected health information. The second performance support tool is a checklist that site analysts can use to ensure that multi-site program output conforms to expectations and does not contain protected health information beyond what is allowed under the multi-site research agreements. Together the two tools create a formal multi-site programming workflow designed to reduce the chance of accidental PHI disclosure.

A P&T Committee’s Transition to a Complete Electronic Meeting System—A Multisite Institution Experience

PubMed Central

Al-Jedai, Ahmed H.; Algain, Roaa A.; Alghamidi, Said A.; Al-Jazairi, Abdulrazaq S.; Amin, Rashid; Bin Hussain, Ibrahim Z.

2017-01-01

Purpose In the last few decades, changes to formulary management processes have taken place in institutions with closed formulary systems. However, many P&T committees continued to operate using traditional paper-based systems. Paper-based systems have many limitations, including confidentiality, efficiency, open voting, and paper wastage. This becomes more challenging when dealing with a multisite P&T committee that handles formulary matters across the whole health care system. In this paper, we discuss the implementation of the first paperless, completely electronic, Web-based formulary management system across a large health care system in the Middle East. Summary We describe the transitioning of a multisite P&T committee in a large tertiary care institution from a paper-based to an all-electronic system. The challenges and limitations of running a multisite P&T committee utilizing a paper system are discussed. The design and development of a Web-based committee floor management application that can be used from notebooks, tablets, and hand-held devices is described. Implementation of a flexible, interactive, easy-to-use, and efficient electronic formulary management system is explained in detail. Conclusion The development of an electronic P&T committee meeting system that encompasses electronic document sharing, voting, and communication could help multisite health care systems unify their formularies across multiple sites. Our experience might not be generalizable to all institutions because this depends heavily on system features, existing processes and workflow, and implementation across different sites. PMID:29018301

A P&T Committee's Transition to a Complete Electronic Meeting System-A Multisite Institution Experience.

PubMed

Al-Jedai, Ahmed H; Algain, Roaa A; Alghamidi, Said A; Al-Jazairi, Abdulrazaq S; Amin, Rashid; Bin Hussain, Ibrahim Z

2017-10-01

In the last few decades, changes to formulary management processes have taken place in institutions with closed formulary systems. However, many P&T committees continued to operate using traditional paper-based systems. Paper-based systems have many limitations, including confidentiality, efficiency, open voting, and paper wastage. This becomes more challenging when dealing with a multisite P&T committee that handles formulary matters across the whole health care system. In this paper, we discuss the implementation of the first paperless, completely electronic, Web-based formulary management system across a large health care system in the Middle East. We describe the transitioning of a multisite P&T committee in a large tertiary care institution from a paper-based to an all-electronic system. The challenges and limitations of running a multisite P&T committee utilizing a paper system are discussed. The design and development of a Web-based committee floor management application that can be used from notebooks, tablets, and hand-held devices is described. Implementation of a flexible, interactive, easy-to-use, and efficient electronic formulary management system is explained in detail. The development of an electronic P&T committee meeting system that encompasses electronic document sharing, voting, and communication could help multisite health care systems unify their formularies across multiple sites. Our experience might not be generalizable to all institutions because this depends heavily on system features, existing processes and workflow, and implementation across different sites.

Voxel-based morphometric multisite collaborative study on schizophrenia.

PubMed

Segall, Judith M; Turner, Jessica A; van Erp, Theo G M; White, Tonya; Bockholt, H Jeremy; Gollub, Randy L; Ho, Beng C; Magnotta, Vince; Jung, Rex E; McCarley, Robert W; Schulz, S Charles; Lauriello, John; Clark, Vince P; Voyvodic, James T; Diaz, Michele T; Calhoun, Vince D

2009-01-01

Regional gray matter (GM) abnormalities are well known to exist in patients with chronic schizophrenia. Voxel-based morphometry (VBM) has been previously used on structural magnetic resonance images (MRI) data to characterize these abnormalities. Two multisite schizophrenia studies, the Functional Biomedical Informatics Research Network and the Mind Clinical Imaging Consortium, which include 9 data collection sites, are evaluating the efficacy of pooling structural imaging data across imaging centers. Such a pooling of data could yield the increased statistical power needed to elucidate effects that may not be seen with smaller samples. VBM analyses were performed to evaluate the consistency of patient versus control gray matter concentration (GMC) differences across the study sites, as well as the effects of combining multisite data. Integration of data from both studies yielded a large sample of 503 subjects, including 266 controls and 237 patients diagnosed with schizophrenia, schizoaffective or schizophreniform disorder. The data were analyzed using the combined sample, as well as analyzing each of the 2 multisite studies separately. A consistent pattern of reduced relative GMC in schizophrenia patients compared with controls was found across all study sites. Imaging center-specific effects were evaluated using a region of interest analysis. Overall, the findings support the use of VBM in combined multisite studies. This analysis of schizophrenics and controls from around the United States provides continued supporting evidence for GM deficits in the temporal lobes, anterior cingulate, and frontal regions in patients with schizophrenia spectrum disorders.

Mass Spectrometry of Intact Proteins Reveals +98 u Chemical Artifacts Following Precipitation in Acetone.

PubMed

Güray, Melda Z; Zheng, Shi; Doucette, Alan A

2017-02-03

Protein precipitation in acetone is frequently employed ahead of mass spectrometry for sample preconcentration and purification. Unfortunately, acetone is not chemically inert; mass artifacts have previously been observed on glycine-containing peptides when exposed to acetone under acidic conditions. We herein report a distinct chemical modification occurring at the level of intact proteins when incubated in acetone. This artifact manifests as one or more satellite peaks in the MS spectrum of intact protein, spaced 98 u above the mass of the unmodified protein. Other artifacts (+84, +112 u) also appear upon incubation of proteins or peptides in acetone. The reaction is pH-sensitive, being suppressed when proteins are exposed to acetone under acidic conditions. The +98 u artifact is speculated to originate through an intermediate product of aldol condensation of acetone to form diacetone alcohol and mesityl oxide. A +98 u product could originate from nucleophilic attack on mesityl oxide or through condensation with diacetone alcohol. Given the extent of modification possible upon exposure of proteins to acetone, particularly following overnight solvent exposure or incubation at room temperature, an awareness of the variables influencing this novel modification is valued by proteomics researchers who employ acetone precipitation for protein purification.

Immunohistochemical evidence for an increased oxidative stress and carbonyl modification of proteins in diabetic glomerular lesions.

PubMed

Suzuki, D; Miyata, T; Saotome, N; Horie, K; Inagi, R; Yasuda, Y; Uchida, K; Izuhara, Y; Yagame, M; Sakai, H; Kurokawa, K

1999-04-01

Advanced glycation end products (AGE) include a variety of protein adducts whose accumulation has been implicated in tissue damage associated with diabetic nephropathy (DN). It was recently demonstrated that among AGE, glycoxidation products, whose formation is closely linked to oxidation, such as carboxymethyllysine (CML) and pentosidine, accumulate in expanded mesangial matrix and nodular lesions in DN, in colocalization with malondialdehyde-lysine (MDA-lysine), a lipoxidation product, whereas pyrraline, another AGE structure whose deposition is rather independent from oxidative stress, was not found within diabetic glomeruli. Because CML, pentosidine, and MDA-lysine are all formed under oxidative stress by carbonyl amine chemistry between protein amino group and carbonyl compounds, their colocalization suggests a local oxidative stress and increased protein carbonyl modification in diabetic glomerular lesions. To address this hypothesis, human renal tissues from patients with DN or IgA nephropathy were examined with specific antibodies to characterize most, if not all, carbonyl modifications of proteins by autoxidation products of carbohydrates, lipids, and amino acids: CML (derived from carbohydrates, lipids, and amino acid), pentosidine (derived from carbohydrates), MDA-lysine (derived from lipids), 4-hydroxynonenal-protein adduct (derived from lipids), and acrolein-protein adduct (derived from lipids and amino acid). All of the protein adducts were identified in expanded mesangial matrix and nodular lesions in DN. In IgA nephropathy, another primary glomerular disease leading to end-stage renal failure, despite positive staining for MDA-lysine and 4-hydroxynonenal-protein adduct in the expanded mesangial area, CML, pentosidine, and acrolein-protein adduct immunoreactivities were only faint in glomeruli. These data suggest a broad derangement in nonenzymatic biochemistry in diabetic glomerular lesions, and implicate an increased local oxidative stress and carbonyl modification of proteins in diabetic glomerular tissue damage ("carbonyl stress").

Covalent modification of proteins by cocaine

NASA Astrophysics Data System (ADS)

Deng, Shi-Xian; Bharat, Narine; Fischman, Marian C.; Landry, Donald W.

2002-03-01

Cocaine covalently modifies proteins through a reaction in which the methyl ester of cocaine acylates the -amino group of lysine residues. This reaction is highly specific in vitro, because no other amino acid reacts with cocaine, and only cocaine's methyl ester reacts with the lysine side chain. Covalently modified proteins were present in the plasma of rats and human subjects chronically exposed to cocaine. Modified endogenous proteins are immunogenic, and specific antibodies were elicited in mouse and detected in the plasma of human subjects. Covalent modification of proteins could explain cocaine's autoimmune effects and provide a new biochemical approach to cocaine's long-term actions.

Kinetic memory based on the enzyme-limited competition.

PubMed

Hatakeyama, Tetsuhiro S; Kaneko, Kunihiko

2014-08-01

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

Perceived sleep deficit is a strong predictor of RLS in multisite pain - A population based study in middle aged females.

PubMed

Stehlik, Romana; Ulfberg, Jan; Zou, Ding; Hedner, Jan; Grote, Ludger

2017-10-01

Chronic pain conditions as well as Restless Legs Syndrome (RLS) are known to be associated with subjectively and objectively disturbed sleep. RLS has been recently described as highly prevalent in multisite pain and the role of sleep as a modifying factor in this RLS phenotype is unknown. This study aimed to investigate if perceived sleep deficit and other sleep related parameters predict RLS in subjects with multisite pain. We have recently demonstrated a strong association between Restless Legs Syndrome (RLS) and number of pain locations. In the current analysis we hypothesized that impaired sleep predicts RLS in subjects with multisite pain. Questionnaire-based data from 2727 randomly selected women aged 18-64 years were used to analyze RLS symptoms, self-reported sleep quality, and the degree of daytime sleepiness (Epworth Sleepiness Scale (ESS)) in relation to type, degree and localization of body pain. Potential confounders including anthropometrics, pain localization, co-morbidities, and medication were adjusted for in the Generalized Linear Models (GLM). Perceived sleep deficit ≥90min (OR 2.4 (1.5-3.8), p<0.001) and frequent nocturnal awakenings (OR 2.3 (1.4-3.6), p<0.001) were the strongest sleep related predictors for RLS in subjects with multisite pain. Additional factors include prolonged sleep latency (≥30min, OR 1.8 (1.1-2.8), p=0.01) and daytime symptoms like elevated daytime sleepiness (ESS score ≥9, OR 1.8 (1.2-2.7), p=0.005). Accordingly, RLS diagnosis was associated with impaired sleep quality (TST (Total Sleep Time) -8.2min, sleep latency +8.0min, and number of awakenings from sleep +0.4, p<0.01). ESS score increased with RLS diagnosis (+0.74, p<0.01) and number of pain locations (0.5, 1.7, and 1.8 for 1, 3, and 5 pain areas, p<0.001). In addition, confounders like pain severity, the history of psychiatric disease, and current smoking were associated with impaired sleep quality in this group of females. Perceived sleep deficit and sleep fragmentation are the strongest sleep related predictors of RLS in multisite pain. Potential implication of our results are that clinical management programmes of RLS in subjects with multisite pain need to consider both sleep quality and sleep quantity for individually tailored treatment regimes. RLS, pain, and sleep disorders are highly interrelated. Our study strongly suggests that clinical management of RLS in patients with multisite pain needs to consider sleep quality as an independent risk factor. Copyright © 2017 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

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

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging*

PubMed Central

Ramallo Guevara, Carina; Philipp, Oliver; Hamann, Andrea; Werner, Alexandra; Osiewacz, Heinz D.; Rexroth, Sascha; Rögner, Matthias; Poetsch, Ansgar

2016-01-01

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. PMID:26884511

Palmitoylation as a Functional Regulator of Neurotransmitter Receptors

PubMed Central

Naumenko, Vladimir S.

2018-01-01

The majority of neuronal proteins involved in cellular signaling undergo different posttranslational modifications significantly affecting their functions. One of these modifications is a covalent attachment of a 16-C palmitic acid to one or more cysteine residues (S-palmitoylation) within the target protein. Palmitoylation is a reversible modification, and repeated cycles of palmitoylation/depalmitoylation might be critically involved in the regulation of multiple signaling processes. Palmitoylation also represents a common posttranslational modification of the neurotransmitter receptors, including G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LICs). From the functional point of view, palmitoylation affects a wide span of neurotransmitter receptors activities including their trafficking, sorting, stability, residence lifetime at the cell surface, endocytosis, recycling, and synaptic clustering. This review summarizes the current knowledge on the palmitoylation of neurotransmitter receptors and its role in the regulation of receptors functions as well as in the control of different kinds of physiological and pathological behavior. PMID:29849559

Protein Tyrosine Nitration: Role in Aging.

PubMed

Chakravarti, Bulbul; Chakravarti, Deb N

2017-01-01

Aging is the inevitable fate of all living organisms, but the molecular basis of physiological aging is poorly understood. Oxidative stress is believed to play a key role in the aging process. In addition to Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS) are generated during aerobic metabolism in living organisms. Although protein damage and functional modification by ROS have been demonstrated in details, fewer studies have been reported on protein damage by RNS and its implication in the aging process. Proteins undergoing tyrosine nitration are associated with pathophysiology of several diseases, as well as physiological aging. The purpose of the current review article is to provide a brief summary of the biochemical mechanisms of tyrosine nitration, methodologies used for the detection of these modified proteins, effect of RNS induced post translational modification on biological functions and the putative role of tyrosine nitrated proteins in the aging process. Published studies on the role of RNS in age related functional alteration of various organs/ tissues were critically reviewed and evaluated. Covalent modification of various proteins by tyrosine nitration is associated with modification of biological functions of various organs/tissues such as skeletal muscle, heart, brain and liver due to aging. This information will be helpful to further investigate the interplay of different biochemical pathways and networks involved in the tyrosine nitration of various proteins due to aging with the ultimate goal to prevent the detrimental effects of RNS on the functional activities of these proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

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

2013-01-01

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

The Expanding Landscape of the Thiol Redox Proteome*

PubMed Central

Yang, Jing; Carroll, Kate S.; Liebler, Daniel C.

2016-01-01

Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO2H), S-sulfonylation (-SO3H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes. Here, we review chemical proteomic strategies used to expand the landscape of thiol redox modifications. PMID:26518762

Alpha-A crystallin: quantitation of C-terminal modification during lens aging

NASA Technical Reports Server (NTRS)

Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

1994-01-01

Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

Surface Modification of Biomaterials: A Quest for Blood Compatibility

PubMed Central

de Mel, Achala; Cousins, Brian G.; Seifalian, Alexander M.

2012-01-01

Cardiovascular implants must resist thrombosis and intimal hyperplasia to maintain patency. These implants when in contact with blood face a challenge to oppose the natural coagulation process that becomes activated. Surface protein adsorption and their relevant 3D confirmation greatly determine the degree of blood compatibility. A great deal of research efforts are attributed towards realising such a surface, which comprise of a range of methods on surface modification. Surface modification methods can be broadly categorized as physicochemical modifications and biological modifications. These modifications aim to modulate platelet responses directly through modulation of thrombogenic proteins or by inducing antithrombogenic biomolecules that can be biofunctionalised onto surfaces or through inducing an active endothelium. Nanotechnology is recognising a great role in such surface modification of cardiovascular implants through biofunctionalisation of polymers and peptides in nanocomposites and through nanofabrication of polymers which will pave the way for finding a closer blood match through haemostasis when developing cardiovascular implants with a greater degree of patency. PMID:22693509

Identification of O-linked β-d-N-acetylglucosamine-Modified Proteins from Arabidopsis

PubMed Central

Xu, Shou-Ling; Chalkley, Robert J.; Wang, Zhi-Yong; Burlingame, Alma L.

2013-01-01

The posttranslational modification of proteins with O-linked β-d-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

PubMed Central

McClure, Michelle; DeLucas, Lawrence J.; Wilson, Landon; Ray, Marjorie; Rowe, Steven M.; Wu, Xiaoyun; Dai, Qun; Hong, Jeong S.; Sorscher, Eric J.; Kappes, John C.; Barnes, Stephen

2012-01-01

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease. PMID:22119790

Protein kinase D displays intrinsic Tyr autophosphorylation activity: insights into mechanism and regulation.

PubMed

Cobbaut, Mathias; Derua, Rita; Parker, Peter J; Waelkens, Etienne; Janssens, Veerle; Van Lint, Johan

2018-06-22

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P+1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation. This article is protected by copyright. All rights reserved. This article is protected by copyright. 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

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

Chemical and Biological Tools for the Preparation of Modified Histone Proteins

PubMed Central

Howard, Cecil J.; Yu, Ruixuan R.; Gardner, Miranda L.; Shimko, John C.; Ottesen, Jennifer J.

2016-01-01

Eukaryotic chromatin is a complex and dynamic system in which the DNA double helix is organized and protected by interactions with histone proteins. This system is regulated through, a large network of dynamic post-translational modifications (PTMs) exists to ensure proper gene transcription, DNA repair, and other processes involving DNA. Homogenous protein samples with precisely characterized modification sites are necessary to better understand the functions of modified histone proteins. Here, we discuss sets of chemical and biological tools that have been developed for the preparation of modified histones, with a focus on the appropriate choice of tool for a given target. We start with genetic approaches for the creation of modified histones, including the incorporation of genetic mimics of histone modifications, chemical installation of modification analogs, and the use of the expanded genetic code to incorporate modified amino acids. Additionally, we will cover the chemical ligation techniques that have been invaluable in the generation of complex modified histones that are indistinguishable from the natural counterparts. Finally, we will end with a prospectus on future directions of synthetic chromatin in living systems. PMID:25863817

Finding Efficiency in the Design of Large Multisite Evaluations: Estimating Variances for Science Achievement Studies

ERIC Educational Resources Information Center

Westine, Carl D.

2016-01-01

Little is known empirically about intraclass correlations (ICCs) for multisite cluster randomized trial (MSCRT) designs, particularly in science education. In this study, ICCs suitable for science achievement studies using a three-level (students in schools in districts) MSCRT design that block on district are estimated and examined. Estimates of…

Childhood Traumatic Grief: A Multi-Site Empirical Examination of the Construct and Its Correlates

ERIC Educational Resources Information Center

Brown, Elissa J.; Amaya-Jackson, Lisa; Cohen, Judith; Handel, Stephanie; De Bocanegra, Heike Thiel; Zatta, Eileen; Goodman, Robin F.; Mannarino, Anthony

2008-01-01

This study evaluated the construct of childhood traumatic grief (CTG) and its correlates through a multi-site assessment of 132 bereaved children and adolescents. Youth completed a new measure of the characteristics, attributions, and reactions to exposure to death (CARED), as well as measures of CTG, posttraumatic stress disorder (PTSD),…

Positive Family Intervention for Severe Challenging Behavior I: A Multisite Randomized Clinical Trial

ERIC Educational Resources Information Center

Durand, V. Mark; Hieneman, Meme; Clarke, Shelley; Wang, Mo; Rinaldi, Melissa L.

2013-01-01

The present study was a multisite randomized clinical trial assessing the effects of adding a cognitive-behavioral intervention to positive behavior support (PBS). Fifty-four families who met the criteria of (a) having a child with a developmental disability, (b) whose child displayed serious challenging behavior (e.g., aggression, self-injury,…

What Works Clearinghouse Quick Review: "Transfer Incentives for High-Performing Teachers: Final Results from a Multisite Experiment"

ERIC Educational Resources Information Center

What Works Clearinghouse, 2014

2014-01-01

"Transfer Incentives for High-Performing Teachers: Final Results from a Multisite Experiment," examined the impact of the Talent Transfer Initiative (TTI) on both student achievement and teacher retention in 10 school districts across seven states. The initiative gave bonuses to high-performing teachers for them to transfer to and stay…

Latent Variable Regression 4-Level Hierarchical Model Using Multisite Multiple-Cohorts Longitudinal Data. CRESST Report 801

ERIC Educational Resources Information Center

Choi, Kilchan

2011-01-01

This report explores a new latent variable regression 4-level hierarchical model for monitoring school performance over time using multisite multiple-cohorts longitudinal data. This kind of data set has a 4-level hierarchical structure: time-series observation nested within students who are nested within different cohorts of students. These…

Multi-Sited Ethnography and the Field of Educational Research

ERIC Educational Resources Information Center

Pierides, Dean

2010-01-01

This paper responds to the challenge of how educational research might be practised in a contemporary world that is no longer necessarily organised by nearness and unity. Focusing on ethnography, it argues for what a multi-sited imaginary contributes to research in the field of education. By giving prominence to the notion of multi-sited…

Collaborative Imaginaries and Multi-Sited Ethnography: Space-Time Dimensions of Engagement in an Afterschool Science Programme for Girls

ERIC Educational Resources Information Center

Rahm, Jrene

2012-01-01

Temporal and spatial configurations that constitute learning and identity work across practices have been little explored in studies of science literacy development. Grounded in multi-sited ethnography, this paper explores diverse girls' engagement with and identity work in science locally, inside a newsletter activity in an afterschool programme…

Documenting the Impact of Multisite Evaluations on the Science, Technology, Engineering, and Mathematics Field

ERIC Educational Resources Information Center

Roseland, Denise; Greenseid, Lija O.; Volkov, Boris B.; Lawrenz, Frances

2011-01-01

This chapter discusses the impact that four multisite National Science Foundation (NSF) evaluations had on the broader field of science, technology, engineering, and mathematics education and evaluation. Three approaches were used to investigate the broader impact of these evaluations on the field: (a) a citation analysis, (b) an on-line survey,…

Multi-Site Ethnography, Hypermedia and the Productive Hazards of Digital Methods: A Struggle for Liveness

ERIC Educational Resources Information Center

Gallagher, Kathleen; Freeman, Barry

2011-01-01

This article explores the possibilities and frustrations of using digital methods in a multi-sited ethnographic research project. The project, "Urban School Performances: The interplay, through live and digital drama, of local-global knowledge about student engagement", is a study of youth and teachers in drama classrooms in contexts of…

The Peculiar Institution?: A Multisite Case Study of Athletic Department Cultures at Small Christian Institutions

ERIC Educational Resources Information Center

Ramseyer, Paul Robert

2017-01-01

This multisite case study explored the perceptions of athletic department members of the important factors of organizational cultures in athletic departments at small Christian institutions in the Midwestern United States. The study comprised of site visits to three small Christian institutions in the Midwestern United States. At each site, six…

A Qualitative Multi-Site Case Study: Examining Principals' Leadership Styles and School Performance

ERIC Educational Resources Information Center

Preyear, Loukisha

2015-01-01

The purpose of this qualitative multi-site case study was to explore the impact of principals' leadership styles on student academic achievement in a high-poverty low-performing school district in Louisiana. A total of 17 participants, principals and teachers, from this school district were used in this study. Data source triangulation of…

A Multisite Cluster Randomized Field Trial of Open Court Reading

ERIC Educational Resources Information Center

Borman, Geoffrey D.; Dowling, N. Maritza; Schneck, Carrie

2008-01-01

In this article, the authors report achievement outcomes of a multisite cluster randomized field trial of Open Court Reading 2005 (OCR), a K-6 literacy curriculum published by SRA/McGraw-Hill. The participants are 49 first-grade through fifth-grade classrooms from predominantly minority and poor contexts across the nation. Blocking by grade level…

Reliability of instruments in a cooperative, multisite study: employment intervention demonstration program.

PubMed

Salyers, M P; McHugo, G J; Cook, J A; Razzano, L A; Drake, R E; Mueser, K T

2001-09-01

Reliability of well-known instruments was examined in 202 people with severe mental illness participating in a multisite vocational study. We examined interrater reliability of the Positive and Negative Syndrome Scale (PANSS) and the internal consistency and test-retest reliability of the PANSS, the Rosenberg Self-Esteem Scale, the Medical Outcomes Study Short Form-36 (SF-36), and the Quality of Life Interview. Most scales had good levels of reliability, with intraclass correlation coefficients (ICCs) and coefficient alphas above .70. However, the SF-36 scales were generally less stable over time, particularly Social Functioning (ICC = .55). Test-retest reliability was lower among less educated respondents and among ethnic minorities. We recommend close monitoring of psychometric issues in future multisite studies.

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry.

PubMed

Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J

2006-11-01

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.

Mass spectrometric approaches for the identification and quantification of reactive carbonyl species protein adducts.

PubMed

Colzani, Mara; Aldini, Giancarlo; Carini, Marina

2013-10-30

Our current knowledge of the occurrence of proteins covalently modified by reactive carbonyl species (RCS) generated by lipid peroxidation indicates their involvement as pathogenic factors associated with several chronic degenerative diseases. Proteomics and mass spectrometry (MS) in the last decade have played a fundamental role in this context, allowing the demonstration of the formation of RCS-protein adducts in vitro and in vivo under different experimental conditions. In conjunction with functional and computational studies, MS has been widely applied in vitro to study the stoichiometry of the protein-RCS adduct formation, and, by identifying the site(s) of modification, to elucidate the molecular mechanisms of protein carbonylation and the physiologic impact of such modification on protein function. This review will provide an update of the MS methods commonly used in detecting and characterizing protein modification by RCS generated by lipid peroxidation, among which 4-hydroxy-trans-2-nonenal and acrolein represent the most studied and cytotoxic compounds. Research in this field, employing state-of-the-art MS, is rapidly and continuously evolving, owing also to the development of suitable derivatization and enrichment procedures enabling the improve MS detectability of RCS-protein adducts in complex biological matrices. By considering the emerging role of RCS in several human diseases, unequivocal analytical approaches by MS are needed to provide levels of intermediate diagnostic biomarkers for human diseases. This review focuses also on the different MS-based approaches so far developed for RCS-protein adduct quantification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

The etiology of human age-related cataract. Proteins don't last forever.

PubMed

Truscott, Roger J W; Friedrich, Michael G

2016-01-01

It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification. Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses. The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues. Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis of Cryptococcus humicola response to aluminum stress.

PubMed

Zhang, Jingjing; Zhang, Lei; Qiu, Jinkui; Nian, Hongjuan

2015-10-01

Cryptococcus humicola is a highly aluminum (Al) tolerant yeast strain isolated from a tea field. Here the relative changes of protein expression in C. humicola undergoing aluminum stress were analyzed to understand the genetic basis of aluminum tolerance. In this work, iTRAQ-based (isobaric tags for relative and absolute quantification) quantitative proteomic technology was used to detect statistically significant proteins associated with the response to aluminum stress. A total of 625 proteins were identified and were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, energy production and conversion, and amino acid transport and metabolism. Of these proteins, 59 exhibited differential expression during aluminum stress. Twenty-nine proteins up-regulated by aluminum were mainly involved in translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover and chaperones, and lipid transport and metabolism. Thirty proteins down-regulated by aluminum were mainly associated with energy transport and metabolism, translation/ribosomal structure and biogenesis, posttranslational modification/protein turnover/chaperones, and lipid transport and metabolism. The potential functions of some proteins in aluminum tolerance are discussed. These functional changes may be beneficial for cells to protect themselves from aluminum toxic conditions. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

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

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

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

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

PubMed

Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

2016-08-01

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA

PubMed Central

Toh, Seok-Ming; Xiong, Liqun; Bae, Taeok; Mankin, Alexander S.

2008-01-01

A2503 in 23S rRNA of the Gram-negative bacterium Escherichia coli is located in a functionally important region of the ribosome, at the entrance to the nascent peptide exit tunnel. In E. coli, and likely in other species, this adenosine residue is post-transcriptionally modified to m2A. The enzyme responsible for this modification was previously unknown. We identified E. coli protein YfgB, which belongs to the radical SAM enzyme superfamily, as the methyltransferase that modifies A2503 of 23S rRNA to m2A. Inactivation of the yfgB gene in E. coli led to the loss of modification at nucleotide A2503 of 23S rRNA as revealed by primer extension analysis and thin layer chromatography. The A2503 modification was restored when YfgB protein was expressed in the yfgB knockout strain. A similar protein was shown to catalyze post-transcriptional modification of A2503 in 23S rRNA in Gram-positive Staphylococcus aureus. The yfgB knockout strain loses in competition with wild type in a co-growth experiment, indicating functional importance of A2503 modification. The location of A2503 in the exit tunnel suggests its possible involvement in interaction with the nascent peptide and raises the possibility that its post-transcriptional modification may influence such an interaction. PMID:18025251

Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

PubMed

Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

2009-06-01

Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a potential regulatory role. Through a wider analysis of other poorly characterized DNA-modifying enzymes we also show that the phage Mu Mom-like proteins, which catalyze the N6-carbamoylmethylation of adenines, are also linked to diverse families of bacterial transposases, suggesting that DNA modification by transposable elements might have a more general presence than previously appreciated. Among the other families of 2-oxoglutarate- and iron(II)-dependent dioxygenases identified in this study, one which is found in algae, is predicted to mainly comprise of RNA-modifying enzymes and shows a striking diversity in protein domain architectures suggesting the presence of RNA modifications with possibly unique adaptive roles. The results presented here are likely to provide the means for future investigation of unexpected epigenetic modifications, such as hydroxymethyl cytosine, that could profoundly impact our understanding of gene regulation and processes such as DNA demethylation.

Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function.

PubMed Central

Edge, Albert S B

2003-01-01

The alteration of proteins by post-translational modifications, including phosphorylation, sulphation, processing by proteolysis, lipid attachment and glycosylation, gives rise to a broad range of molecules that can have an identical underlying protein core. An understanding of glycosylation of proteins is important in clarifying the nature of the numerous variants observed and in determining the biological roles of these modifications. Deglycosylation with TFMS (trifluoromethanesulphonic acid) [Edge, Faltynek, Hof, Reichert, and Weber, (1981) Anal. Biochem. 118, 131-137] has been used extensively to remove carbohydrate from glycoproteins, while leaving the protein backbone intact. Glycosylated proteins from animals, plants, fungi and bacteria have been deglycosylated with TFMS, and the most extensively studied types of carbohydrate chains in mammals, the N-linked, O-linked and glycosaminoglycan chains, are all removed by this procedure. The method is based on the finding that linkages between sugars are sensitive to cleavage by TFMS, whereas the peptide bond is stable and is not broken, even with prolonged deglycosylation. The relative susceptibility of individual sugars in glycosidic linkage varies with the substituents at C-2 and the occurrence of amido and acetyl groups, but even the most stable sugars are removed under conditions that are sufficiently mild to prevent scission of peptide bonds. The post-translational modifications of proteins have been shown to be required for diverse biological functions, and selective procedures to remove these modifications play an important role in the elucidation of protein structure and function. PMID:12974674

The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures.

PubMed

Bayart, Caroline; Peronin, Sébastien; Jean, Elisa; Paladino, Joseph; Talaga, Philippe; Borgne, Marc Le

2017-06-01

Aldehyde detoxification is a process used to convert toxin into toxoid for vaccine applications. In the case of tetanus toxin (TT), formaldehyde is used to obtain the tetanus toxoid (TTd), which is used either for the tetanus vaccine or as carrier protein in conjugate vaccines. Several studies have already been conducted to better understand the exact mechanism of this detoxification. Those studies led to the identification of a number of formaldehyde-induced modifications on lab scale TTd samples. To obtain greater insights of the changes induced by formaldehyde, we used three industrial TTd batches to identify repeatable modifications in the detoxification process. Our strategy was to combine seven analytical tools to map these changes. Mass spectrometry (MS), colorimetric test and amino acid analysis (AAA) were used to study modifications on amino acids. SDS-PAGE, asymmetric flow field flow fractionation (AF4), fluorescence spectroscopy and circular dichroism (CD) were used to study formaldehyde modifications on the whole protein structure. We identified 41 formaldehyde-induced modifications across the 1315 amino acid primary sequence of TT. Of these, five modifications on lysine residues were repeatable across TTd batches. Changes in protein conformation were also observed using SDS-PAGE, AF4 and CD techniques. Each analytical tool brought a piece of information regarding formaldehyde induced-modifications, and all together, these methods provided a comprehensive overview of the structural changes that occurred with detoxification. These results could be the first step leading to site-directed TT mutagenesis studies that may enable the production of a non-toxic equivalent protein without using formaldehyde. Copyright © 2017 Elsevier B.V. All rights reserved.

Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice

PubMed Central

Shearn, Colin T.; Fritz, Kristofer S.; Shearn, Alisabeth H.; Saba, Laura M.; Mercer, Kelly E.; Engi, Bridgette; Galligan, James J.; Zimniak, Piotr; Orlicky, David J.; Ronis, Martin J.; Petersen, Dennis R.

2015-01-01

Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4−/− mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4−/− mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4−/− mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4−/− mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4−/− PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins. PMID:26654979

Toxicological evaluation of proteins introduced into food crops

PubMed Central

Kough, John; Herouet-Guicheney, Corinne; Jez, Joseph M.

2013-01-01

This manuscript focuses on the toxicological evaluation of proteins introduced into GM crops to impart desired traits. In many cases, introduced proteins can be shown to have a history of safe use. Where modifications have been made to proteins, experience has shown that it is highly unlikely that modification of amino acid sequences can make a non-toxic protein toxic. Moreover, if the modified protein still retains its biological function, and this function is found in related proteins that have a history of safe use (HOSU) in food, and the exposure level is similar to functionally related proteins, then the modified protein could also be considered to be “as-safe-as” those that have a HOSU. Within nature, there can be considerable evolutionary changes in the amino acid sequence of proteins within the same family, yet these proteins share the same biological function. In general, food crops such as maize, soy, rice, canola etc. are subjected to a variety of processing conditions to generate different food products. Processing conditions such as cooking, modification of pH conditions, and mechanical shearing can often denature proteins in these crops resulting in a loss of functional activity. These same processing conditions can also markedly lower human dietary exposure to (functionally active) proteins. Safety testing of an introduced protein could be indicated if its biological function was not adequately characterized and/or it was shown to be structurally/functionally related to proteins that are known to be toxic to mammals. PMID:24164515

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

PubMed Central

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

2005-01-01

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

The Role of Protein-Mineral Interactions for Protein Adsorption or Fragmentation

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P.; Washton, N.; Kleber, M.

2014-12-01

Soil exo-enzymes (EE) are proteins with the capability to catalyze the depolymerization of soil organic matter (SOM). SOM must be disassembled by EEs in order to be transported through the microbial cell wall and become metabolized. One factor determining an EE's functionality is their affinity to mineral surfaces found in the soil. Our goal was to establish the range of protein modifications, either chemical or structural, as the protein becomes associated with mineral surfaces. We hypothesized that pedogenic oxides would generate more extensive chemical alterations to the protein structure than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We used 1H 15N Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance Spectroscopy (HSQC NMR) to observe structural modifications in the unadsorbed Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite and birnessite. Solid state NMR was used to observe the structural modifications of Gb1 while adsorbed onto kaolinite and montmorillonite. Preliminary results in the HSQC NMR spectra observed no changes in the native conformation of Gb1 when allowed to interact with goethite and kaolinite while birnessite induced strong structural modification of Gb1 at an acidic pH. Our results suggest that not all mineral surfaces in soil act as sorbents for EEs and changes in their catalytic activity upon adsorption to minerals surfaces may not just be an indication of conformational changes but of fragmentation of the protein itself.

Characterization of Surface Modification of Polyethersulfone Membrane

USDA-ARS?s Scientific Manuscript database

Surface modification of polyethersulfone (PES) membrane surface using UV/ozone-treated grafting and interfacial polymerization on membrane surface was investigated in order to improve the resistance of membrane surface to protein adsorption. These methods of surface modification were compared in te...

Research staff training in a multisite randomized clinical trial: Methods and recommendations from the Stimulant Reduction Intervention using Dosed Exercise (STRIDE) trial.

PubMed

Walker, Robrina; Morris, David W; Greer, Tracy L; Trivedi, Madhukar H

2014-01-01

Descriptions of and recommendations for meeting the challenges of training research staff for multisite studies are limited despite the recognized importance of training on trial outcomes. The STRIDE (STimulant Reduction Intervention using Dosed Exercise) study is a multisite randomized clinical trial that was conducted at nine addiction treatment programs across the United States within the National Drug Abuse Treatment Clinical Trials Network (CTN) and evaluated the addition of exercise to addiction treatment as usual (TAU), compared to health education added to TAU, for individuals with stimulant abuse or dependence. Research staff administered a variety of measures that required a range of interviewing, technical, and clinical skills. In order to address the absence of information on how research staff are trained for multisite clinical studies, the current manuscript describes the conceptual process of training and certifying research assistants for STRIDE. Training was conducted using a three-stage process to allow staff sufficient time for distributive learning, practice, and calibration leading up to implementation of this complex study. Training was successfully implemented with staff across nine sites. Staff demonstrated evidence of study and procedural knowledge via quizzes and skill demonstration on six measures requiring certification. Overall, while the majority of staff had little to no experience in the six measures, all research assistants demonstrated ability to correctly and reliably administer the measures throughout the study. Practical recommendations are provided for training research staff and are particularly applicable to the challenges encountered with large, multisite trials.

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

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

PubMed

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

2015-01-01

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

Awareness of Diagnosis and Knowledge of HPV in Women Patients: Data from a Multi-Site Study

ERIC Educational Resources Information Center

McCree, Donna Hubbard; Daley, Ellen M.; Gorbach, Pamina; Hamm, Robert M.; Sharpe, Patricia A.; Brandt, Heather M.; McFarlane, Mary; Kerndt, Peter; McDermott, Robert J.; Perrin, Karen M.; St. Lawrence, Janet S.

2010-01-01

Background: Persistent infection with high-risk types of human papillomavirus (HPV) is associated with cervical and other anogenital cancers. Purpose: This paper reports results of awareness of an HPV diagnosis and HPV knowledge from a multi-site study of HPV knowledge, attitudes and behavior, and the impact of an HPV diagnosis on women and their…

An Examination of Diversity within Three Southeastern Academic Libraries: A Mixed-Methods, Multi-Site Study

ERIC Educational Resources Information Center

Shaffer, Christopher A.

2014-01-01

The purpose of this study was to determine the extent to which three academic libraries in the Southeastern United States could be considered diverse. This was a multi-site, mixed methods study. It examined the climate and culture of the libraries, which was assessed through two methods; the first, through survey responses from full-time faculty…

The Setting-up of Multi-Site School Collaboratives: The Benefits of This Organizational Reform in Terms of Networking Opportunities and Their Effects

ERIC Educational Resources Information Center

Mifsud, Denise

2015-01-01

This article, which is set within the Maltese education scenario of unfolding decentralization through the setting-up of multi-site school collaboratives (legally termed "colleges") via a policy mandate, explores a particular aspect of this reform--that of "networking". This is examined in terms of the potential for…

A Hierarchical Modeling Approach to Data Analysis and Study Design in a Multi-Site Experimental fMRI Study

ERIC Educational Resources Information Center

Zhou, Bo; Konstorum, Anna; Duong, Thao; Tieu, Kinh H.; Wells, William M.; Brown, Gregory G.; Stern, Hal S.; Shahbaba, Babak

2013-01-01

We propose a hierarchical Bayesian model for analyzing multi-site experimental fMRI studies. Our method takes the hierarchical structure of the data (subjects are nested within sites, and there are multiple observations per subject) into account and allows for modeling between-site variation. Using posterior predictive model checking and model…

WWC Review of the Report "Transfer Incentives for High-Performing Teachers: Final Results from a Multisite Randomized Experiment." What Works Clearinghouse Single Study Review

ERIC Educational Resources Information Center

What Works Clearinghouse, 2015

2015-01-01

For the 2013 study, "Transfer Incentives for High-Performing Teachers: Final Results from a Multisite Randomized Experiment," researchers examined the impact of the Talent Transfer Initiative (TTI) on students' reading and mathematics achievement in 10 school districts. The TTI enabled principals of low-performing schools to provide…

How Much Do the Effects of Education and Training Programs Vary across Sites? Evidence from Past Multisite Randomized Trials

ERIC Educational Resources Information Center

Weiss, Michael J.; Bloom, Howard S.; Verbitsky-Savitz, Natalya; Gupta, Himani; Vigil, Alma E.; Cullinan, Daniel N.

2017-01-01

Multisite trials, in which individuals are randomly assigned to alternative treatment arms within sites, offer an excellent opportunity to estimate the cross-site average effect of treatment assignment (intent to treat or ITT) "and" the amount by which this impact varies across sites. Although both of these statistics are substantively…

Bias and Bias Correction in Multi-Site Instrumental Variables Analysis of Heterogeneous Mediator Effects

ERIC Educational Resources Information Center

Reardon, Sean F.; Unlu, Faith; Zhu, Pei; Bloom, Howard

2013-01-01

We explore the use of instrumental variables (IV) analysis with a multi-site randomized trial to estimate the effect of a mediating variable on an outcome in cases where it can be assumed that the observed mediator is the only mechanism linking treatment assignment to outcomes, as assumption known in the instrumental variables literature as the…

Bias and Bias Correction in Multisite Instrumental Variables Analysis of Heterogeneous Mediator Effects

ERIC Educational Resources Information Center

Reardon, Sean F.; Unlu, Fatih; Zhu, Pei; Bloom, Howard S.

2014-01-01

We explore the use of instrumental variables (IV) analysis with a multisite randomized trial to estimate the effect of a mediating variable on an outcome in cases where it can be assumed that the observed mediator is the only mechanism linking treatment assignment to outcomes, an assumption known in the IV literature as the exclusion restriction.…

Replication and Comparison of the Newly Proposed ADOS-2, Module 4 Algorithm in ASD without ID: A Multi-Site Study

ERIC Educational Resources Information Center

Pugliese, Cara E.; Kenworthy, Lauren; Bal, Vanessa Hus; Wallace, Gregory L.; Yerys, Benjamin E.; Maddox, Brenna B.; White, Susan W.; Popal, Haroon; Armour, Anna Chelsea; Miller, Judith; Herrington, John D.; Schultz, Robert T.; Martin, Alex; Anthony, Laura Gutermuth

2015-01-01

Recent updates have been proposed to the Autism Diagnostic Observation Schedule-2 Module 4 diagnostic algorithm. This new algorithm, however, has not yet been validated in an independent sample without intellectual disability (ID). This multi-site study compared the original and revised algorithms in individuals with ASD without ID. The revised…

Sensitivity of the Modified Children's Yale-Brown Obsessive Compulsive Scale to Detect Change: Results from Two Multi-Site Trials

ERIC Educational Resources Information Center

Scahill, Lawrence; Sukhodolsky, Denis G.; Anderberg, Emily; Dimitropoulos, Anastasia; Dziura, James; Aman, Michael G.; McCracken, James; Tierney, Elaine; Hallett, Victoria; Katz, Karol; Vitiello, Benedetto; McDougle, Christopher

2016-01-01

Repetitive behavior is a core feature of autism spectrum disorder. We used 8-week data from two federally funded, multi-site, randomized trials with risperidone conducted by the Research Units on Pediatric Psychopharmacology Autism Network to evaluate the sensitivity of the Children's Yale-Brown Obsessive Compulsive Scale modified for autism…

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

NASA Astrophysics Data System (ADS)

Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

2015-11-01

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications

PubMed Central

2018-01-01

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described. PMID:29849913

Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

PubMed Central

Nuriel, Tal; Deeb, Ruba S.; Hajjar, David P.; Gross, Steven S.

2008-01-01

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues. PMID:18554526

Progress in centralised ethics review processes: Implications for multi-site health evaluations.

PubMed

Prosser, Brenton; Davey, Rachel; Gibson, Diane

2015-04-01

Increasingly, public sector programmes respond to complex social problems that intersect specific fields and individual disciplines. Such responses result in multi-site initiatives that can span nations, jurisdictions, sectors and organisations. The rigorous evaluation of public sector programmes is now a baseline expectation. For evaluations of large and complex multi-site programme initiatives, the processes of ethics review can present a significant challenge. However in recent years, there have been new developments in centralised ethics review processes in many nations. This paper provides the case study of an evaluation of a national, inter-jurisdictional, cross-sector, aged care health initiative and its encounters with Australian centralised ethics review processes. Specifically, the paper considers progress against the key themes of a previous five-year, five nation study (Fitzgerald and Phillips, 2006), which found that centralised ethics review processes would save time, money and effort, as well as contribute to more equitable workloads for researchers and evaluators. The paper concludes with insights for those charged with refining centralised ethics review processes, as well as recommendations for future evaluators of complex multi-site programme initiatives. Copyright © 2015 Elsevier Ltd. All rights reserved.

Redox proteomics of tomato in response to Pseudomonas syringae infection

PubMed Central

Balmant, Kelly Mayrink; Parker, Jennifer; Yoo, Mi-Jeong; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2015-01-01

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation–reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses. PMID:26504582

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes.

PubMed

Nady, Norhan

2016-04-18

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled "green surface modification". This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers-ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)-is presented.

Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1

DOE PAGES

Leon, Deborah R.; Ytterberg, A. Jimmy; Boontheung, Pinmanee; ...

2015-03-05

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

A Robust Analytical Approach for the Identification of Specific Protein Carbonylation Sites: Metal-Catalyzed Oxidations of Human Serum Albumin

PubMed Central

Ugur, Zafer; Gronert, Scott

2017-01-01

The formation of protein carbonyls in the metal-catalyzed oxidation of human serum albumin (HSA) is characterized using a new analytical approach that involves tagging the modification site with multiple hydrazide reagents. Protein carbonyl formation at lysine and arginine residues was catalyzed with copper and iron ions, and the resulting oxidation patterns in HSA are contrasted. A total of 18 modification sites were identified with iron ion catalysis and 14 with copper ion catalysis. However, with the more stringent requirement of identification with at least two tagging reagents, the number of validated modification sites drops to 10 for iron and 9 for copper. Of the 14 total validated sites, there were only five in common for the two metal ions. The results illustrate the value of using multiple tagging agents and highlight the selective and specific nature of metal-catalyzed protein oxidations. PMID:28303033

Enrichment and separation techniques for large-scale proteomics analysis of the protein post-translational modifications.

PubMed

Huang, Junfeng; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

2014-11-06

Comprehensive analysis of the post-translational modifications (PTMs) on proteins at proteome level is crucial to elucidate the regulatory mechanisms of various biological processes. In the past decades, thanks to the development of specific PTM enrichment techniques and efficient multidimensional liquid chromatography (LC) separation strategy, the identification of protein PTMs have made tremendous progress. A huge number of modification sites for some major protein PTMs have been identified by proteomics analysis. In this review, we first introduced the recent progresses of PTM enrichment methods for the analysis of several major PTMs including phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and oxidation/reduction status. We then briefly summarized the challenges for PTM enrichment. Finally, we introduced the fractionation and separation techniques for efficient separation of PTM peptides in large-scale PTM analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell signaling, post-translational protein modifications and NMR spectroscopy

PubMed Central

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

2016-01-01

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

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.

High Prevalence of Echocardiographic Abnormalities among HIV-infected Persons in the Era of Highly Active Antiretroviral Therapy.

PubMed

Mondy, Kristin E; Gottdiener, John; Overton, E Turner; Henry, Keith; Bush, Tim; Conley, Lois; Hammer, John; Carpenter, Charles C; Kojic, Erna; Patel, Pragna; Brooks, John T

2011-02-01

in the era of highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV)-infected persons have higher cardiovascular disease risk. Little is known about asymptomatic abnormalities in cardiac structure and function in this population. the Study to Understand the Natural History of HIV/AIDS in the Era of Effective Therapy (SUN Study) is a prospective, observational, multi-site cohort of 656 HIV-infected participants who underwent baseline echocardiography during 2004-2006. We examined prevalence of and factors associated with left ventricular systolic dysfunction (LVSD), diastolic dysfunction (DD), pulmonary hypertension (PHTN), left ventricular hypertrophy (LVH), and left atrial enlargement (LAE). participant characteristics were as follows: median age, 41 years; 24% women; 29% non-Hispanic black; 73% receiving HAART; and median CD4+ cell count, 462 cells/μL. Among evaluable participants, 18% had LVSD, 26% had DD, 57% had PHTN (right ventricular pressure >30 mm Hg), 6.5% had LVH, and 40% had LAE. In multivariate analyses, significant factors (P < .05) associated with LVSD were history of MI, elevated highly sensitive C-reactive protein (hsCRP) level, and current tobacco smoking; for DD, elevated hsCRP level and hypertension; for PHTN, current use of ritonavir; for LVH, hypertension, diabetes, non-white race, female sex with elevated body mass index, calculated as the weight in kilograms divided by the square of height in meters, of ≥ 25, elevated hsCRP level, and current use of abacavir; for LAE, hypertension and recent marijuana use. in this large contemporary HIV cohort, the prevalence of subclinical functional and structural cardiac abnormalities was greater than expected for age. Abnormalities were mostly associated with expected and often modifiable risks. Lifestyle modification should become a greater priority in the management of chronic HIV disease.

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 m/r SEBT: development of a functional screening tool for dance educators.

PubMed

Wilson, Margaret; Batson, Glenna

2014-12-01

Dance screenings provide direct and indirect data bearing on a dancer's readiness to undertake rigorous physical training. Rarely, however, are dance teachers able to translate results from these screenings into practical technical knowledge. In this article, an example of a preseason assessment tool is presented that translates scientific findings into useful information for dance teachers conducting auditions. Designed as a baseline assessment of the dancer during auditioning, the m/r SEBT tool helps teachers stratify technical levels, identify injury risk, and consequently assist with immediate and appropriate recommendations for supplemental training and//or follow-up with a medical professional. The tool evolved out of more than 3 years of collaborative, multisite research utilizing the Star Excursion Balance Test (SEBT) as a dynamic test of balance. Modifications were made to render the test more dance-specific and to increase balance challenges. Within the 3-year period, more than 100 dancers were tested in four sites, two in the United States and two in the United Kingdom. Despite the relatively large collective sample size, neither the original SEBT nor its modifications (m/r SEBT) held robust face or content validity as balance screens. What did emerge, however, were qualitative criteria that the authors organized into a feasible assessment tool for preseason auditions. While this tool awaits further validation, its current evolution helps serve as a bridge between dance teachers' clinical and practical knowledge.

Implementation Considerations for Multisite Clinical Trials with Cognitive Neuroscience Tasks

PubMed Central

Keefe, Richard S. E.; Harvey, Philip D.

2008-01-01

Multisite clinical trials aimed at cognitive enhancement across various neuropsychiatric conditions have employed standard neuropsychological tests as outcome measures. While these tests have enjoyed wide clinical use and have proven reliable and predictive of functional disability, a number of implementation challenges have arisen when these tests are used in clinical trials. These issues are likely to be magnified in future studies when cognitive neuroscience (CN) procedures are explored in these trials, because in their current forms CN procedures are less standardized and more difficult to teach and monitor. For multisite trials, we anticipate that the most challenging issues will include assuring tester competence, monitoring tester performance, specific challenges with complex assessment methods, and having resources available for adequate monitoring of data quality. Suggestions for overcoming these implementation challenges are offered. PMID:18495645

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease

PubMed Central

Ying, Peng; Serife, Ayaz-Guner; Deyang, Yu; Ying, Ge

2014-01-01

Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed. PMID:24945106

Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation

PubMed Central

Kovacs, Izabella; Lindermayr, Christian

2013-01-01

Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation. PMID:23717319

Epigenetic modifications: An important mechanism in diabetic disturbances.

PubMed

Rorbach-Dolata, Anna; Kubis, Adriana; Piwowar, Agnieszka

2017-11-29

In the search for explanations of diabetes pathomechanisms, especially the development of its vascular complications (micro- and macrovascular ), although current, good metabolic control of diabetes, attention was drawn to the role of epigenetic inheritance associated with epigenetic modifications of histone proteins and DNA in hyperglycemia conditions. This study showed the significant role of DNA methylation and histone epigenetic modifications (a different nature and a different degree) in the transmission of information that is not connected with gene inheritance but concerns the persistent changes induced by hyperglycemia..Attention was paid to the role of DNA methylation of pancreatic cells in the pathogenesis of type 1 diabetes, but also type 2. The important role of DNA methylation changes in a so-called intrauterine growth restriction (IUGR) as reason of subsequent development of diabetes was particularly emphasized. In the pathogenesis of type 2 diabetes and its complications, especially microvascular complications, the greatest share and importance of epigenetic modifications on mitochondrial DNA metylation are the most important. The multidirectionality Complicaand complexity of epigenetic modifications of histone proteins indicate their importance in the development of diabetic disturbances. An especially important role is attributed to methylation and acetylation of histone proteins, in particular on arginine and lysine, whose changes occur most frequently. Moreover, epigenetic modifications of the enzymes, especially methylases, responsible for these processes are the underlying. It has been indicated that the identification of epigenetic differences within the DNA or histone proteins may be a useful prognostic biomarker of susceptibility to the disease development in the future. Moreover, they may become a potential target for future therapeutic interventions for clinical disorders in diabetes.

Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.

PubMed

Stroylova, Yulia Y; Semenyuk, Pavel I; Asriyantz, Regina A; Gaillard, Cedric; Haertlé, Thomas; Muronetz, Vladimir I

2014-09-01

The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes. © 2014 Wiley Periodicals, Inc.

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

PubMed

Grasso, Giuseppe; Axelsen, Paul H

2017-01-01

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

Methods for evaluating the Caenorhabditis elegans dauer state: standard dauer-formation assay using synthetic daumones and proteomic analysis of O-GlcNAc modifications.

PubMed

Lee, Jeeyong; Kim, Kwang-Youl; Joo, Hyoe-Jin; Kim, Heekyeong; Jeong, Pan-Young; Paik, Young-Ki

2011-01-01

The dauer state is a non-feeding, alternative L3 state characterized by a number of distinctive metabolic and morphological changes. There are many naturally occurring dauer-inducing pheromones, termed daumones, that have been suggested by some to exhibit differences in dauer-inducing activity. Here, we have established a standard dauer-formation assay that uses synthetic daumones 1, 2, and 3, the three major daumones. To analyze the proteome of Caenorhabditis elegans in the dauer state, we focused on O-GlcNAc modification, a cytosolic modification of proteins that is known to interact either competitively or synergistically with protein phosphorylation. Protein O-GlcNAc modification is an important biological process in cells that can ensure the timely response to extracellular stimuli, such as daumone, and maintain cellular homeostasis. Establishing a standard method for assaying dauer formation using different synthetic daumones, and using differences in O-GlcNAcylated proteins during the dauer state to analyze the dauer proteome will lead to a better understanding of dauer biology of C. elegans in the context of animal longevity and adaptation under harsh environments. Copyright © 2011 Elsevier Inc. All rights reserved.

The story of protein arginine methylation: characterization, regulation, and function.

PubMed

Peng, Chao; Wong, Catherine Cl

2017-02-01

Arginine methylation is an important post-translational modification (PTM) in cells, which is catalyzed by a group of protein arginine methyltransferases (PRMTs). It plays significant roles in diverse cellular processes and various diseases. Misregulation and aberrant expression of PRMTs can provide potential biomarkers and therapeutic targets for drug discovery. Areas covered: Herein, we review the arginine methylation literature and summarize the methodologies for the characterization of this modification, as well as describe the recent insights into arginine methyltransferases and their biological functions in diseases. Expert commentary: Benefits from the enzyme-based large-scale screening approach, the novel affinity enrichment strategies, arginine methylated protein family is the focus of attention. Although a number of arginine methyltransferases and related substrates are identified, the catalytic mechanism of different types of PRMTs remains unclear and few related demethylases are characterized. Novel functional studies continuously reveal the importance of this modification in the cell cycle and diseases. A deeper understanding of arginine methylated proteins, modification sites, and their mechanisms of regulation is needed to explore their role in life processes, especially their relationship with diseases, thus accelerating the generation of potent, selective, cell-penetrant drug candidates.

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

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

Kou, Qiang; Zhu, Binhai; Wu, Si

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

Interracial Contact and Racial Constancy: A Multi-Site Study of Racial Intergroup Bias in 3-5 Year Old Anglo-British Children

ERIC Educational Resources Information Center

Rutland, Adam; Cameron, Lindsey; Bennett, Laura; Ferrell, Jennifer

2005-01-01

This paper examined the influence of interracial contact and racial constancy on the racial intergroup bias of young Anglo-British children. This multi-site study was conducted in areas of Great Britain that varied in terms of racial diversity. The study also investigated whether preschool children express bias on positive, but not negative,…

The Study to Explore Early Development (SEED): A Multisite Epidemiologic Study of Autism by the Centers for Autism and Developmental Disabilities Research and Epidemiology (CADDRE) Network

ERIC Educational Resources Information Center

Schendel, Diana E.; DiGuiseppi, Carolyn; Croen, Lisa A.; Fallin, M. Daniele; Reed, Philip L.; Schieve, Laura A.; Wiggins, Lisa D.; Daniels, Julie; Grether, Judith; Levy, Susan E.; Miller, Lisa; Newschaffer, Craig; Pinto-Martin, Jennifer; Robinson, Cordelia; Windham, Gayle C.; Alexander, Aimee; Aylsworth, Arthur S.; Bernal, Pilar; Bonner, Joseph D.; Blaskey, Lisa; Bradley, Chyrise; Collins, Jack; Ferretti, Casara J.; Farzadegan, Homayoon; Giarelli, Ellen; Harvey, Marques; Hepburn, Susan; Herr, Matthew; Kaparich, Kristina; Landa, Rebecca; Lee, Li-Ching; Levenseller, Brooke; Meyerer, Stacey; Rahbar, Mohammad H.; Ratchford, Andria; Reynolds, Ann; Rosenberg, Steven; Rusyniak, Julie; Shapira, Stuart K.; Smith, Karen; Souders, Margaret; Thompson, Patrick Aaron; Young, Lisa; Yeargin-Allsopp, Marshalyn

2012-01-01

The Study to Explore Early Development (SEED), a multisite investigation addressing knowledge gaps in autism phenotype and etiology, aims to: (1) characterize the autism behavioral phenotype and associated developmental, medical, and behavioral conditions and (2) investigate genetic and environmental risks with emphasis on immunologic, hormonal,…

The Challenges and Benefits of Employing a Mobile Research Fellow to Facilitate Team Work on a Large, Interdisciplinary, Multi-Sited Project

ERIC Educational Resources Information Center

Sugden, Fraser; Punch, Samantha

2014-01-01

Over the last few years research funding has increasingly moved in favour of large, multi-partner, interdisciplinary and multi-site research projects. This article explores the benefits and challenges of employing a full-time research fellow to work across multiple field sites, with all the local research teams, on an international,…

Sources of Site Differences in the Efficacy of a Multisite Clinical Trial: The Treatment of SSRI-Resistant Depression in Adolescents

ERIC Educational Resources Information Center

Spirito, Anthony; Abebe, Kaleab Z.; Iyengar, Satish; Brent, David; Vitiello, Benedetto; Clarke, Gregory; Wagner, Karen Dineen; Asarnow, Joan; Emslie, Graham; Keller, Martin

2009-01-01

Site differences in treatment outcomes are not often highlighted when the results of multisite randomized clinical trials (MRCTs) are reported. The primary analyses of a 6-site MRCT, the Treatment of SSRI-Resistant Depression in Adolescents (TORDIA) study, showed substantial variation by site in the performance of a medication-only condition and a…

Year One Results from the Multisite Randomized Evaluation of the i3 Scale-Up of Reading Recovery

ERIC Educational Resources Information Center

May, Henry; Gray, Abigail; Sirinides, Philip; Goldsworthy, Heather; Armijo, Michael; Sam, Cecile; Gillespie, Jessica N.; Tognatta, Namrata

2015-01-01

Reading Recovery (RR) is a short-term, one-to-one intervention designed to help the lowest achieving readers in first grade. This article presents first-year results from the multisite randomized controlled trial (RCT) and implementation study under the $55 million Investing in Innovation (i3) Scale-Up Project. For the 2011-2012 school year, the…

Distortions in Distributions of Impact Estimates in Multi-Site Trials: The Central Limit Theorem Is Not Your Friend

ERIC Educational Resources Information Center

May, Henry

2014-01-01

Interest in variation in program impacts--How big is it? What might explain it?--has inspired recent work on the analysis of data from multi-site experiments. One critical aspect of this problem involves the use of random or fixed effect estimates to visualize the distribution of impact estimates across a sample of sites. Unfortunately, unless the…

MULTI-SITE EVALUATIONS OF CANDIDATE METHODOLOGIES FOR DETERMINING COARSE PARTICULATE (PM 10-2.5) CONCENTRATIONS: AUGUST 2005 UPDATED REPORT REGARDING SECOND-GENERATION AND NEW PM 10-2.5 SAMPLERS

EPA Science Inventory

Multi-site field studies were conducted to evaluate the performance of sampling methods for measuring the coarse fraction of PM10 (PM10 2.5) in ambient air. The field studies involved the use of both time-integrated filter-based and direct continuous methods. Despite operationa...

Multisite HPV16/18 Vaccine Efficacy Against Cervical, Anal, and Oral HPV Infection

PubMed Central

Kreimer, Aimée R.; Schiffman, Mark; Herrero, Rolando; Wacholder, Sholom; Rodriguez, Ana Cecilia; Lowy, Douglas R.; Porras, Carolina; Schiller, John T.; Quint, Wim; Jimenez, Silvia; Safaeian, Mahboobeh; Struijk, Linda; Schussler, John; Hildesheim, Allan; Gonzalez, Paula

2016-01-01

Background: Previous Costa Rica Vaccine Trial (CVT) reports separately demonstrated vaccine efficacy against HPV16 and HPV18 (HPV16/18) infections at the cervical, anal, and oral regions; however, the combined overall multisite efficacy (protection at all three sites) and vaccine efficacy among women infected with HPV16 or HPV18 prior to vaccination are less known. Methods: Women age 18 to 25 years from the CVT were randomly assigned to the HPV16/18 vaccine (Cervarix) or a hepatitis A vaccine. Cervical, oral, and anal specimens were collected at the four-year follow-up visit from 4186 women. Multisite and single-site vaccine efficacies (VEs) and 95% confidence intervals (CIs) were computed for one-time detection of point prevalent HPV16/18 in the cervical, anal, and oral regions four years after vaccination. All statistical tests were two-sided. Results: The multisite woman-level vaccine efficacy was highest among “naïve” women (HPV16/18 seronegative and cervical HPV high-risk DNA negative at vaccination) (vaccine efficacy = 83.5%, 95% CI = 72.1% to 90.8%). Multisite woman-level vaccine efficacy was also demonstrated among women with evidence of a pre-enrollment HPV16 or HPV18 infection (seropositive for HPV16 and/or HPV18 but cervical HPV16/18 DNA negative at vaccination) (vaccine efficacy = 57.8%, 95% CI = 34.4% to 73.4%), but not in those with cervical HPV16 and/or HPV18 DNA at vaccination (anal/oral HPV16/18 VE = 25.3%, 95% CI = -40.4% to 61.1%). Concordant HPV16/18 infections at two or three sites were also less common in HPV16/18-infected women in the HPV vaccine vs control arm (7.4% vs 30.4%, P < .001). Conclusions: This study found high multisite vaccine efficacy among “naïve” women and also suggests the vaccine may provide protection against HPV16/18 infections at one or more anatomic sites among some women infected with these types prior to HPV16/18 vaccination. PMID:26467666

Using an Engineered Protein Model to Constrain Protein-Mineral Interactions

NASA Astrophysics Data System (ADS)

Chacon, S. S.; Reardon, P. N.; Washton, N.; Kleber, M.

2015-12-01

Exoenzymes are proteins that can catalyze the depolymerization of soil organic matter (SOM). Proteins can also be an important source of organic N for microorganisms, but must be fragmented into small peptides in order to be transported through their membranes. An exoenzyme's affinity to mineral surfaces found in soil affects their capacity to degrade SOM or other proteins. Our goal was to determine the range of modifications on proteins when they interact with a mineral surface. We hypothesized that pedogenic oxides would fragment or promote greater chemical modifications to a protein than phyllosilicates. A well-characterized protein proxy (Gb1, IEP 4.0, 6.2 kDA) was adsorbed onto functionally different mineral surfaces (goethite, montmorillonite, kaolinite and birnesite) at pH 5 and pH 7. We then generated three engineered proxies of Gb1 by inserting either negatively charged, positively charged or aromatic amino acids into the second loop. We used liquid chromatography coupled with a mass spectrometer (LC-MS/MS) and solution-state Heteronuclear Single Quantum Coherence Spectroscopy Nuclear Magnetic Resonance (HSQC NMR) to observe modifications to Gb1 that was allowed to equilibrate during the adsorption process for kaolinite, goethite, birnessite, and montmorillonite. We also used Helium Ion Microscopy (HIM) to determine which surface archetypes Gb1 preferentially adsorbed to as a function of the mineral type. The three engineered proxies were used to determine how variation of the amino acid sequence affects a protein interaction with a mineral surface. Preliminary results in the LC-MS/MS indicate that birnessite hydrolytically fragments Gb1 into polypeptides. Our results suggest that not all mineral surfaces in soil may act as sorbents for EEs and that chemical modification of their structure should also be considered as an explanation for decrease in EE activity. Our results also indicate an abiotic pathway for the turnover of proteins, although its relative contribution to protein turnover in soil must be further investigated.

Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

PubMed

Wanigasekara, Maheshika S K; Chowdhury, Saiful M

2016-09-07

Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-03-01

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

Human Cells as Platform to Produce Gamma-Carboxylated Proteins.

PubMed

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

2018-01-01

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

Tripping Up Trp: Modification of Protein Tryptophan Residues by Reactive Oxygen Species, Modes of Detection, and Biological Consequences

PubMed Central

Ehrenshaft, Marilyn; Deterding, Leesa J.; Mason, Ronald P.

2015-01-01

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes. PMID:26393422

Redox Proteomics: A Key Tool for New Insights into Protein Modification with Relevance to Disease.

PubMed

Butterfield, D Allan; Perluigi, Marzia

2017-03-01

Oxidatively modified proteins are characterized by elevations in protein-resident carbonyls or 3-nitrotyrosine, measures of protein oxidation, or protein bound reactive alkenals such as 4-hydroxy-2-nonenal, a measure of lipid peroxidation. Oxidatively modified proteins nearly always have altered structure and function. Redox proteomics is that branch of proteomics used to identify oxidized proteins and determine the extent and location of oxidative modifications in the proteomes of interest. This technique nearly always employs mass spectrometry as the major platform to achieve the goals of identifying the target proteins. Once identified, oxidatively modified proteins can be placed in specific molecular pathways to provide insights into protein oxidation and human disease. Both original research and review articles are included in this Forum on Redox Proteomics. The topics related to redox proteomics range from basic chemistry of sulfur radical-induced redox modifications in proteins, to the thiol secretome and inflammatory network, to reversible thiol oxidation in proteomes, to the role of glutamine synthetase in peripheral and central environments on inflammation and insulin resistance, to bioanalytical aspects of tyrosine nitrated proteins, to protein oxidation in human smokers and models thereof, and to Alzheimer disease, including articles on the brain ubiquitinylome and the "triangle of death" composed of oxidatively modified proteins involved in energy metabolism, mammalian target of rampamycin activation, and the proteostasis network. This Forum on Redox Proteomics is both timely and a critically important resource to highlight one of the key tools needed to better understand protein structure and function in oxidative environments in health and disease. Antioxid. Redox Signal. 26, 277-279.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

PubMed Central

Naz, Anam; Obaid, Ayesha; Awan, Faryal M.; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells. PMID:28932213

Solid state, thermal synthesis of site-specific protein-boron cluster conjugates and their physicochemical and biochemical properties.

PubMed

Goszczyński, Tomasz M; Kowalski, Konrad; Leśnikowski, Zbigniew J; Boratyński, Janusz

2015-02-01

Boron clusters represent a vast family of boron-rich compounds with extraordinary properties that provide the opportunity of exploitation in different areas of chemistry and biology. In addition, boron clusters are clinically used in boron neutron capture therapy (BNCT) of tumors. In this paper, a novel, in solid state (solvent free), thermal method for protein modification with boron clusters has been proposed. The method is based on a cyclic ether ring opening in oxonium adduct of cyclic ether and a boron cluster with nucleophilic centers of the protein. Lysozyme was used as the model protein, and the physicochemical and biological properties of the obtained conjugates were characterized. The main residues of modification were identified as arginine-128 and threonine-51. No significant changes in the secondary or tertiary structures of the protein after tethering of the boron cluster were found using mass spectrometry and circular dichroism measurements. However, some changes in the intermolecular interactions and hydrodynamic and catalytic properties were observed. To the best of our knowledge, we have described the first example of an application of cyclic ether ring opening in the oxonium adducts of a boron cluster for protein modification. In addition, a distinctive feature of the proposed approach is performing the reaction in solid state and at elevated temperature. The proposed methodology provides a new route to protein modification with boron clusters and extends the range of innovative molecules available for biological and medical testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma.

PubMed

Naz, Anam; Obaid, Ayesha; Awan, Faryal M; Ikram, Aqsa; Ahmad, Jamil; Ali, Amjad

2017-01-01

Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

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

PubMed

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

2009-01-01

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

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA.

PubMed

Kinsella, B T; Erdman, R A; Maltese, W A

1991-05-25

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

PubMed

Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

2017-03-02

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Multisite rainfall downscaling and disaggregation in a tropical urban area

NASA Astrophysics Data System (ADS)

Lu, Y.; Qin, X. S.

2014-02-01

A systematic downscaling-disaggregation study was conducted over Singapore Island, with an aim to generate high spatial and temporal resolution rainfall data under future climate-change conditions. The study consisted of two major components. The first part was to perform an inter-comparison of various alternatives of downscaling and disaggregation methods based on observed data. This included (i) single-site generalized linear model (GLM) plus K-nearest neighbor (KNN) (S-G-K) vs. multisite GLM (M-G) for spatial downscaling, (ii) HYETOS vs. KNN for single-site disaggregation, and (iii) KNN vs. MuDRain (Multivariate Rainfall Disaggregation tool) for multisite disaggregation. The results revealed that, for multisite downscaling, M-G performs better than S-G-K in covering the observed data with a lower RMSE value; for single-site disaggregation, KNN could better keep the basic statistics (i.e. standard deviation, lag-1 autocorrelation and probability of wet hour) than HYETOS; for multisite disaggregation, MuDRain outperformed KNN in fitting interstation correlations. In the second part of the study, an integrated downscaling-disaggregation framework based on M-G, KNN, and MuDRain was used to generate hourly rainfall at multiple sites. The results indicated that the downscaled and disaggregated rainfall data based on multiple ensembles from HadCM3 for the period from 1980 to 2010 could well cover the observed mean rainfall amount and extreme data, and also reasonably keep the spatial correlations both at daily and hourly timescales. The framework was also used to project future rainfall conditions under HadCM3 SRES A2 and B2 scenarios. It was indicated that the annual rainfall amount could reduce up to 5% at the end of this century, but the rainfall of wet season and extreme hourly rainfall could notably increase.

Quantification of source impact to PM using three-dimensional weighted factor model analysis on multi-site data

NASA Astrophysics Data System (ADS)

Shi, Guoliang; Peng, Xing; Huangfu, Yanqi; Wang, Wei; Xu, Jiao; Tian, Yingze; Feng, Yinchang; Ivey, Cesunica E.; Russell, Armistead G.

2017-07-01

Source apportionment technologies are used to understand the impacts of important sources of particulate matter (PM) air quality, and are widely used for both scientific studies and air quality management. Generally, receptor models apportion speciated PM data from a single sampling site. With the development of large scale monitoring networks, PM speciation are observed at multiple sites in an urban area. For these situations, the models should account for three factors, or dimensions, of the PM, including the chemical species concentrations, sampling periods and sampling site information, suggesting the potential power of a three-dimensional source apportionment approach. However, the principle of three-dimensional Parallel Factor Analysis (Ordinary PARAFAC) model does not always work well in real environmental situations for multi-site receptor datasets. In this work, a new three-way receptor model, called "multi-site three way factor analysis" model is proposed to deal with the multi-site receptor datasets. Synthetic datasets were developed and introduced into the new model to test its performance. Average absolute error (AAE, between estimated and true contributions) for extracted sources were all less than 50%. Additionally, three-dimensional ambient datasets from a Chinese mega-city, Chengdu, were analyzed using this new model to assess the application. Four factors are extracted by the multi-site WFA3 model: secondary source have the highest contributions (64.73 and 56.24 μg/m3), followed by vehicular exhaust (30.13 and 33.60 μg/m3), crustal dust (26.12 and 29.99 μg/m3) and coal combustion (10.73 and 14.83 μg/m3). The model was also compared to PMF, with general agreement, though PMF suggested a lower crustal contribution.

Multisite accelerometry for sleep and wake classification in children.

PubMed

Lamprecht, Marnie L; Bradley, Andrew P; Tran, Tommy; Boynton, Alison; Terrill, Philip I

2015-01-01

Actigraphy is a useful alternative to the gold standard polysomnogram for non-invasively measuring sleep and wakefulness. However, it is unable to accurately assess sleep fragmentation due to its inability to differentiate restless sleep from wakefulness and quiet wake from sleep. This presents significant limitations in the assessment of sleep-related breathing disorders where sleep fragmentation is a common symptom. We propose that this limitation may be caused by hardware constraints and movement representation techniques. Our objective was to determine if multisite tri-axial accelerometry improves sleep and wake classification. Twenty-four patients aged 6-15 years (median: 8 years, 16 male) underwent a diagnostic polysomnogram while simultaneously recording motion from the left wrist and index fingertip, upper thorax and left ankle and great toe using a custom accelerometry system. Movement was quantified using several features and two feature selection techniques were employed to select optimal features for restricted feature set sizes. A heuristic was also applied to identify movements during restless sleep. The sleep and wake classification performance was then assessed and validated against the manually scored polysomnogram using discriminant analysis. Tri-axial accelerometry measured at the wrist significantly improved the wake detection when compared to uni-axial accelerometry (specificity at 85% sensitivity: 71.3(14.2)% versus 55.2(24.7)%, p < 0.01). Multisite accelerometry significantly improved the performance when compared to the single wrist placement (specificity at 85% sensitivity: 82.1(12.5)% versus 71.3(14.2)%, p < 0.05). Our results indicate that multisite accelerometry offers a significant performance benefit which could be further improved by analysing movement in raw multisite accelerometry data.

Planning and conducting a multi-institutional project on fatigue.

PubMed

Nail, L M; Barsevick, A M; Meek, P M; Beck, S L; Jones, L S; Walker, B L; Whitmer, K R; Schwartz, A L; Stephen, S; King, M E

1998-09-01

To describe the process used in proposal development and study implementation for a complex multisite project on cancer treatment-related fatigue (CRF), identify strategies used to manage the project, and provide recommendations for teams planning multisite research. Information derived from project team meeting records, correspondence, proposals, and personal recollection. The project was built on preexisting relationships among the three site investigators who then built a team including faculty, research coordinators, staff nurses, and students. Study sites had a range of organizational models, and the proposal was designed to capitalize on the organizational and resource strengths of each setting. Three team members drawn from outside oncology nursing provided expertise in measurement and experience with fatigue in other populations. Planning meetings were critical to the success of the project. Conference calls, fax technology, and electronic mail were used for communication. Flexibility was important in managing crises and shifting responsibility for specific components of the work. The team documented and evaluated the process used for multisite research, completed a major instrumentation study, and developed a cognitive-behavioral intervention for CRF. Accomplishments during the one-year planning grant exceeded initial expectations. The process of conducting multisite research is complex, especially when the starting point is a planning grant with specific research protocols to be developed and implemented over one year. Explicit planning for decision-making processes to be used throughout the project, acknowledging the differences among the study settings and planning the protocols to capitalize upon those differences, and recruiting a strong research team that included a member with planning grant and team-building expertise were essential elements for success. Specific recommendations for others planning multisite research are related to team-building, team membership, communication, behavioral norms, role flexibility, resources, feedback, problem management, and shared recognition.

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

A targeted mass spectrometry-based approach for the identification and characterization of proteins containing α-aminoadipic and γ-glutamic semialdehyde residues

PubMed Central

Chavez, Juan D.; Bisson, William H.

2011-01-01

The site-specific identification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues in proteins is reported. Semialdehydic protein modifications result from the metal-catalyzed oxidation of Lys or Arg and Pro residues, respectively. Most of the analytical methods for the analysis of protein carbonylation measure change to the global level of carbonylation and fail to provide details regarding protein identity, site, and chemical nature of the carbonylation. In this work, we used a targeted approach, which combines chemical labeling, enrichment, and tandem mass spectrometric analysis, for the site-specific identification of AAS and GGS sites in proteins. The approach is applied to in vitro oxidized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and an untreated biological sample, namely cardiac mitochondrial proteins. The analysis of GAPDH resulted in the site-specific identification of two AAA and four GGS residues. Computational evaluation of the identified AAS and GGS sites in GAPDH indicated that these sites are located in flexible regions, show high solvent accessibility values, and are in proximity with possible metal ion binding sites. The targeted proteomic analysis of semialdehydic modifications in cardiac mitochondria yielded nine AAS modification sites which were unambiguously assigned to distinct lysine residues in the following proteins: ATP/ATP translocase isoforms 1 and 2, ubiquinol cytochrome-c reductase core protein 2, and ATP synthase α-subunit. PMID:20957471

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

PubMed

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

2018-06-01

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

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

PubMed

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

2013-03-01

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

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

PubMed Central

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

2013-01-01

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

Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns.

PubMed

Craig, Jeffrey M; Earle, Elizabeth; Canham, Paul; Wong, Lee H; Anderson, Melissa; Choo, K H Andy

2003-12-01

We have examined the metaphase chromosomal localization of 15 proteins that have previously been described as involved in mammalian chromatin modification and/or transcriptional modulation. Immunofluorescence data indicate that all the proteins localize to human and mouse centromeres, a neocentromere, and the active centromere of a dicentric chromosome, with six of these proteins (Sin3A, PCAF, MYST, MBD2, ORC2, P300/CBP) being demonstrated at mammalian centromeres for the first time. Most of these proteins fall into two distinct chromosomal distribution patterns: (a) kinetochore-associated proteins (Sin3A, PCAF, MYST and BAF180), which colocalize with metaphase kinetochores, but not any of the pericentric and other major heterochromatic regions; and (b) heterochromatin-associated proteins (MeCP2, MBD1, MBD2, ATRX, HP1alpha, HDAC1, HDAC2, DNMT1 and DNMT3b), which colocalize with centromeric/pericentric heterochromatin and all other major heterochromatic sites. A heterogeneous third group (c) consists of the origin recognition complex subunit ORC2 and the histone acetyltransferase P300/CBP, which associate generally with kinetochores in humans and centromeric/pericentric heterochromatin in mouse, with some minor differences in localization. These observations indicate an extensive sharing of protein components involved in chromatin modification at gene loci, centromeres and various chromosomal heterochromatic landmarks. The definition of distinct patterns of chromosomal distribution for these proteins provides a useful basis for the further investigation of the broad-ranging roles of these proteins.

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

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

PubMed

Wu, Meiye; Singh, Anup K

2014-12-01

Cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation of a kinase cascade that culminates in induction of messenger RNA (mRNA) and noncoding microRNA (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 posttranslational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for posttranslational modifications. Since we know that cells in populations behave heterogeneously,(1) 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 Technology Brief, we describe our automated microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and posttranslational modifications in single intact cells with >95% reduction in reagent requirement in under 8 h. © 2014 Society for Laboratory Automation and Screening.

Measuring protein-bound glutathioine (PSSG): Critical correction for cytosolic glutathione species

USDA-ARS?s Scientific Manuscript database

Introduction: Protein glutathionylation is gaining recognition as an important posttranslational protein modification. The common first step in measuring protein glutathionylation is the denaturation and precipitation of protein away from soluble, millimolar quantities of glutathione (GSH) and glut...

Proton adsorption onto alumina: extension of multisite complexation (MUSIC) theory

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

Nagashima, K.; Blum, F.D.

1999-09-01

The adsorption isotherm of protons onto a commercial {gamma}-alumina sample was determined in aqueous nitric acid with sodium nitrate as a background electrolyte. Three discrete regions could be discerned in the log-log plots of the proton isotherm determined at the solution pH 5 to 2. The multisite complexation (MUSIC) model was modified to analyze the simultaneous adsorption of protons onto various kinds of surface species.

Creative Entextualizations of Discourses about Race in Multi-Sited Discursive Practices in the Brazilian "Periphery"

ERIC Educational Resources Information Center

Guimarães, Thayse Figueira; Moita-Lopes, Luiz Paulo

2017-01-01

This paper focuses on Luan's race performances both on the web and in classroom interaction. Luan is a black young man, who identifies himself as gay. The study is part of a multi-sited ethnographic piece of research on a group of high-school students in the state sector, in the "periphery" of a town on the Rio de Janeiro State north…

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

PubMed

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

2017-08-17

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

Crosslinking Protein Glutathionylation Mediated by O2-Arylated Bis-Diazeniumdiolate “Double JS-K”

PubMed Central

Holland, Ryan J.; Maciag, Anna E.; Kumar, Varun; Shi, Lei; Saavedra, Joseph E.; Prud’homme, Robert K.; Chakrapani, Harinath; Keefer, Larry K.

2012-01-01

Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, e.g., by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semi-quantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein’s thiol group are covalently bound via a crosslinking agent, rather than through a disulfide bond. This form of thiol crosslinking has been shown to occur and confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them. PMID:23106594

Supervision of Facilitators in a Multisite Study: Goals, Process, and Outcomes

PubMed Central

2010-01-01

Objective To describe the aims, implementation, and desired outcomes of facilitator supervision for both interventions (treatment and control) in Project Eban and to present the Eban Theoretical Framework for Supervision that guided the facilitators’ supervision. The qualifications and training of supervisors and facilitators are also described. Design This article provides a detailed description of supervision in a multisite behavioral intervention trial. The Eban Theoretical Framework for Supervision is guided by 3 theories: cognitive behavior therapy, the Life-long Model of Supervision, and “Empowering supervisees to empower others: a culturally responsive supervision model.” Methods Supervision is based on the Eban Theoretical Framework for Supervision, which provides guidelines for implementing both interventions using goals, process, and outcomes. Results Because of effective supervision, the interventions were implemented with fidelity to the protocol and were standard across the multiple sites. Conclusions Supervision of facilitators is a crucial aspect of multisite intervention research quality assurance. It provides them with expert advice, optimizes the effectiveness of facilitators, and increases adherence to the protocol across multiple sites. Based on the experience in this trial, some of the challenges that arise when conducting a multisite randomized control trial and how they can be handled by implementing the Eban Theoretical Framework for Supervision are described. PMID:18724192

The value and potential of multisited ethnography for science education research: a review of Jrène Rahm's Science in the Making at the Margin

NASA Astrophysics Data System (ADS)

Beeman-Cadwallader, Nicole

2012-03-01

Rahm sought to illuminate how children and youth make meaning of science in three out-of-school time programs, and the identity work that is done through the trajectory of their youth toward their young adulthood. Through using multisited ethnography, she asserts that we can learn more about what the youth say about their science learning and science literacy development in these programs. She has revealed through her rich data the potential value of multisited ethnography for science education research. A similar theme of insider/outsider status to science emerged in each of the three sites. The nature of the data collected in Rahm's multisited ethnography allowed for the unveiling of the similarities in this theme, but also made evident the nuanced ways that this theme presented itself differently across the three sites. The additional potential came through in the varied data Rahm could have delved into and did not. A unique characteristic of ethnographic research, that is not necessarily present in other research methods, is that a wide array of varied data is collected. An analysis of documents, images, and observational field notes, which go beyond interview data, may provide deeper understandings previously untouched in science education.

Use of personal digital assistants for data collection in a multi-site AIDS stigma study in rural south Nyanza, Kenya.

PubMed

Onono, M A; Carraher, N; Cohen, R C; Bukusi, E A; Turan, J M

2011-09-01

To describe the development, cost effectiveness and implementation of a PDA based electronic system to collect, verify and manage data from a multi-site study on HIV/AIDS stigma and pregnancy in a rural, resource-poor area. We worked within a large prevention of mother-to-child-transmission (PMTCT) program in nine rural health facilities to implement a PDA-based data collection system and to study the feasibility of its use in a multisite HIV research study in rural Kenya. The PDAs were programmed for collecting screening and eligibility data, and responses to structured interviews on HIV/AIDS stigma and violence in three local languages. Between November 2007 and December 2008, nine PDAs were used by Clinic and Community Health Assistants to enrol 1,270 participants on to the PMTCT program. Successes included: capacity-building of interviewers, low cost of implementation, quick turnaround time of data entry with good data quality, and convenience. Our study demonstrated the feasibility of utilizing PDAs for data collection in a multi-site observational study on HIV/AIDS stigma conducted in remote rural health facilities in Kenya. However, appropriate and frequent data backup protocols need to be established and paper forms are still needed as backup tools in resource-poor settings.

Site‐Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

PubMed Central

Kuan, Seah Ling; Wang, Tao

2016-01-01

Abstract The synthetic transformation of polypeptides with molecular accuracy holds great promise for providing functional and structural diversity beyond the proteome. Consequently, the last decade has seen an exponential growth of site‐directed chemistry to install additional features into peptides and proteins even inside living cells. The disulfide rebridging strategy has emerged as a powerful tool for site‐selective modifications since most proteins contain disulfide bonds. In this Review, we present the chemical design, advantages and limitations of the disulfide rebridging reagents, while summarizing their relevance for synthetic customization of functional protein bioconjugates, as well as the resultant impact and advancement for biomedical applications. PMID:27778400

Glycation of antibodies: Modification, methods and potential effects on biological functions.

PubMed

Wei, Bingchuan; Berning, Kelsey; Quan, Cynthia; Zhang, Yonghua Taylor

Glycation is an important protein modification that could potentially affect bioactivity and molecular stability, and glycation of therapeutic proteins such as monoclonal antibodies should be well characterized. Glycated protein could undergo further degradation into advance glycation end (AGE) products. Here, we review the root cause of glycation during the manufacturing, storage and in vivo circulation of therapeutic antibodies, and the current analytical methods used to detect and characterize glycation and AGEs, including boronate affinity chromatography, charge-based methods, liquid chromatography-mass spectrometry and colorimetric assay. The biological effects of therapeutic protein glycation and AGEs, which ranged from no affect to loss of activity, are also discussed.

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.

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

[The effect of hydrophobic surface properties of protein on its resistance to denaturation by organic solvents (using modified alpha-chymotrypsin as an example].

PubMed

Kudriashova, E V; Belova, A B; Vinogradov, A A; Mozhaev, V V

1994-03-01

Catalytic activity of covalently modified alpha-chymotrypsin in water/cosolvent solutions was investigated. The stability of chymotrypsin increases upon modification with hydrophilic reagents, such as glyceraldehyde, pyrometallic and succinic anhydrides, and glucosamine. Correlation was observed between the protein's stability in organic solvents and the degree of hydrophilization of the protein's surface. The protein is the more stable, the higher are the modification degree and the hydrophilicity of the modifying residue. At a certain critical hydrophilization degree of chymotrypsin a limit of stability is achieved. The stabilization effect can be accounted for by the fact that the interaction between water molecules on the surface and protein's functional groups become stronger in the hydrophilized protein.

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.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS)

NASA Astrophysics Data System (ADS)

Badgett, Majors J.; Boyes, Barry; Orlando, Ron

2017-05-01

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS).

PubMed

Badgett, Majors J; Boyes, Barry; Orlando, Ron

2017-05-01

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts. Furthermore, coefficients describing the extent of the hydrophilicity of these modifications have been derived and were incorporated into a previously made peptide retention prediction model that is capable of predicting the retention times of peptides with and without these modifications. Graphical Abstract ᅟ.

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

PubMed

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

2015-07-15

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

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.

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

DOE PAGES

Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.; ...

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 identifiedmore » 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.« less

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

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

Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.

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 identifiedmore » 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.« less

Prokaryotic Ubiquitin-Like Protein Modification

PubMed Central

Maupin-Furlow, Julie A.

2016-01-01

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers. PMID:24995873

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

Sharma, Seema; Simpson, David C.; Tolic, Nikola

We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction.more » Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.« less

O-linked N-acetylglucosamine (O-GlcNAc) protein modification is increased in the cartilage of patients with knee osteoarthritis.

PubMed

Tardio, L; Andrés-Bergós, J; Zachara, N E; Larrañaga-Vera, A; Rodriguez-Villar, C; Herrero-Beaumont, G; Largo, R

2014-02-01

There is increasing evidence that the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins plays an important role in cell signaling pathways. In chondrocytes, accumulation of O-GlcNAc-modified proteins induces hypertrophic differentiation. Osteoarthritis (OA) is characterized by cartilage degradation, and hypertrophic-like changes in hyaline chondrocytes. However, the mechanisms responsible for these changes have not been described. Our aim was to study whether O-GlcNAcylation and the enzymes responsible for this modification are dysregulated in the cartilage of patients with knee OA and whether interleukin-1 could induce these modifications in cultured human OA chondrocytes (HOC). Human cartilage was obtained from patients with knee OA and from age and sex-matched healthy donors. HOC were cultured and stimulated with the catabolic cytokine IL-1α. Global protein O-GlcNAcylation and the synthesis of the key enzymes responsible for this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), were assessed by western blot. OA was associated with a 4-fold increase in the global O-GlcNAcylation in the cartilage. OA cartilage showed a re-distribution of the OGT and OGA isoforms, with a net increase in the presence of both enzymes, in comparison to healthy cartilage. In HOC, IL-1α stimulation rapidly increased O-GlcNAcylation and OGT and OGA synthesis. Our results indicate that a proinflammatory milieu could favor the accumulation of O-GlcNAcylated proteins in OA cartilage, together with the dysregulation of the enzymes responsible for this modification. The increase in O-GlcNAcylation could be responsible, at least partially, for the re-expression of hypertrophic differentiation markers that have been observed in OA. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study.

PubMed

Islam, Sidra; Moinuddin; Mir, Abdul Rouf; Raghav, Alok; Habib, Safia; Alam, Khursheed; Ali, Asif

2017-09-12

Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-07-01

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

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.

Evolution of a Histone H4-K16 Acetyl-Specific DNA Aptamer

PubMed Central

Williams, Berea A. R.; Lin, Liyun; Lindsay, Stuart M.; Chaput, John C.

2009-01-01

We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a Kd of 21 nM, and discriminates against both the non-acetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody, but shows significantly greater specificity (15-fold versus 2,400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications. PMID:19385619

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Modification of Silk Fibroin Using Diazonium Coupling Chemistry and the Effects on hMSC Proliferation and Differentiation

PubMed Central

Murphy, Amanda R.; John, Peter St.; Kaplan, David L.

2009-01-01

A simple chemical modification method using diazonium coupling chemistry was developed to tailor the structure and hydrophilicity of silk fibroin protein. The extent of modification using several aniline derivatives was characterized using UV/vis and 1H NMR spectroscopy, and the resulting protein structure was analyzed with ATR-FTIR spectroscopy. Introduction of hydrophobic functional groups facilitated rapid conversion of the protein from a random coil to a β-sheet structure, while addition of hydrophilic groups inhibited this process. hMSCs were grown on these modified silks to assess the biocompatibility of these materials. The hydrophilicity of the silk derivatives was found to affect the growth rate and morphology, but hMSCs were able to attach, proliferate and differentiate into an osteogenic lineage on all of the silk derivatives. PMID:18417206

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2016-02-15

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

In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

PubMed

Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

2006-01-01

Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

Posttranslational modification of Birch and Ragweed allergen proteins by common gas phase pollutants, NO2 and O3

NASA Astrophysics Data System (ADS)

Mahmood, M. A.; Pope, F.; Bloss, W.

2015-12-01

The global incidence of hay fever has been rising for decades, however, the underlying reasons behind this rise remain unclear. It is hypothesized that exposure of pollen to common gas phase pollutants, such as nitrogen dioxide (NO2) and ozone (O3), increases the allergenicity of the pollen and thus increases hay fever incidence. Since atmospheric pollutants tend to have greater concentrations within urban areas (in particular NO2) the hypothesis suggests that greater allergenicity should occur in urban areas. Indeed, several studies do suggest higher hay fever incidence within urban areas compared to rural areas. Previous published work suggests a link between increased allergies with changes in the chemical composition of the pollen protein via posttranslational modification of the protein. This study investigates the posttranslational modification of two highly allergenic pollen species (Birch and Ragweed) that are common in Europe. Within the laboratory, we expose pollen grains to atmospherically relevant exposures of gas phase NO2, O3 and other common gas phase oxidants under a range of environmentally relevant conditions. The effects of the environmentally relevant exposures on the biochemistry of the pollen grains were probed using a proteomic approach (liquid chromatography coupled ultra-high resolution spectrometer). Our findings indicate the interaction between gas phase pollutants and pollen cause protein specific modifications; in particular, nitration occurs upon tyrosine residues and nitrosylation on cysteine residues. Possibly, these modifications may affect the immune response of the pollen protein, which may suggest a possible reason for increased allergies in reaction to such biologically altered protein. The laboratory-derived results will be supported with a time series analysis of asthma incidence rates for the London area, which take into account the pollen count, and pollutant concentrations. The implications of the results will be discussed in terms of better planning of city infrastructure. In particular, the relevance of the results upon urban tree planting schemes will be put into context.

How Many Interviews Are Enough to Identify Metathemes in Multisited and Cross-Cultural Research? Another Perspective on Guest, Bunce, and Johnson's (2006) Landmark Study

ERIC Educational Resources Information Center

Hagaman, Ashley K.; Wutich, Amber

2017-01-01

There is much debate over the number of interviews needed to reach data saturation for themes and metathemes in qualitative research. The primary purpose of this study is to determine the number of interviews needed to reach data saturation for metathemes in multisited and cross-cultural research. The analysis is based on a cross-cultural study on…

Influence of reactive species on the modification of biomolecules generated from the soft plasma

NASA Astrophysics Data System (ADS)

Attri, Pankaj; Kumar, Naresh; Park, Ji Hoon; Yadav, Dharmendra Kumar; Choi, Sooho; Uhm, Han S.; Kim, In Tae; Choi, Eun Ha; Lee, Weontae

2015-02-01

Plasma medicine is an upcoming research area that has attracted the scientists to explore more deeply the utility of plasma. So, apart from the treating biomaterials and tissues with plasma, we have studied the effect of soft plasma with different feeding gases such as Air, N2 and Ar on modification of biomolecules. Hence, in this work we have used the soft plasma on biomolecules such as proteins ((Hemoglobin (Hb) and Myoglobin (Mb)), calf thymus DNA and amino acids. The structural changes or structural modification of proteins and DNA have been studied using circular dichroism (CD), fluorescence spectroscopy, protein oxidation test, gel electrophoresis, UV-vis spectroscopy, dynamic light scattering (DLS) and 1D NMR, while Liquid Chromatograph/Capillary Electrophoresis-Mass Spectrometer (LC/CE-MS) based on qualitative and quantitative bio-analysis have been used to study the modification of amino acids. Further, the thermal analysis of the protein has been studied with differential scanning calorimetry (DSC) and CD. Additionally, we have performed docking studies of H2O2 with Hb and Mb, which reveals that H2O2 molecules preferably attack the amino acids near heme group. We have also shown that N2 gas plasma has strong deformation action on biomolecules and compared to other gases plasma.

From Controlled Trial to Community Adoption: The Multisite Translational Community Trial

PubMed Central

Murimi, Mary; Gonzalez, Anjelica; Njike, Valentine; Green, Lawrence W.

2011-01-01

Methods for translating the findings of controlled trials, such as the Diabetes Prevention Program, into real-world community application have not been clearly defined. A standardized research methodology for making and evaluating such a transition is needed. We introduce the multisite translational community trial (mTCT) as the research analog to the multisite randomized controlled trial. The mTCT is adapted to incorporate the principles and practices of community-based participatory research and the increased relevance and generalizability gained from diverse community settings. The mTCT is a tool designed to bridge the gap between what a clinical trial demonstrates can work in principle and what is needed to make it workable and effective in real-world settings. Its utility could be put to the test, in particular with practice-based research networks such as the Prevention Research Centers. PMID:21680935

Analytic hierarchy process-based approach for selecting a Pareto-optimal solution of a multi-objective, multi-site supply-chain planning problem

NASA Astrophysics Data System (ADS)

Ayadi, Omar; Felfel, Houssem; Masmoudi, Faouzi

2017-07-01

The current manufacturing environment has changed from traditional single-plant to multi-site supply chain where multiple plants are serving customer demands. In this article, a tactical multi-objective, multi-period, multi-product, multi-site supply-chain planning problem is proposed. A corresponding optimization model aiming to simultaneously minimize the total cost, maximize product quality and maximize the customer satisfaction demand level is developed. The proposed solution approach yields to a front of Pareto-optimal solutions that represents the trade-offs among the different objectives. Subsequently, the analytic hierarchy process method is applied to select the best Pareto-optimal solution according to the preferences of the decision maker. The robustness of the solutions and the proposed approach are discussed based on a sensitivity analysis and an application to a real case from the textile and apparel industry.

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen*

PubMed Central

Hill, Ryan C.; Wither, Matthew J.; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C.

2015-01-01

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757

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

Genome Modification Technologies and Their Applications in Avian Species.

PubMed

Lee, Hong Jo; Kim, Young Min; Ono, Tamao; Han, Jae Yong

2017-10-26

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology.

Genome Modification Technologies and Their Applications in Avian Species

PubMed Central

Lee, Hong Jo; Kim, Young Min; Ono, Tamao

2017-01-01

The rapid development of genome modification technology has provided many great benefits in diverse areas of research and industry. Genome modification technologies have also been actively used in a variety of research areas and fields of industry in avian species. Transgenic technologies such as lentiviral systems and piggyBac transposition have been used to produce transgenic birds for diverse purposes. In recent years, newly developed programmable genome editing tools such as transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) have also been successfully adopted in avian systems with primordial germ cell (PGC)-mediated genome modification. These genome modification technologies are expected to be applied to practical uses beyond system development itself. The technologies could be used to enhance economic traits in poultry such as acquiring a disease resistance or producing functional proteins in eggs. Furthermore, novel avian models of human diseases or embryonic development could also be established for research purposes. In this review, we discuss diverse genome modification technologies used in avian species, and future applications of avian biotechnology. PMID:29072628

Redox proteomics for the assessment of redox-related posttranslational regulation in plants.

PubMed

Mock, Hans-Peter; Dietz, Karl-Josef

2016-08-01

The methodological developments of in vivo and in vitro protein labeling and subsequent detection enable sensitive and specific detection of redox modifications. Such methods are presently applied to diverse cells and tissues, subproteomes and developmental as well as environmental conditions. The chloroplast proteome is particularly suitable for such kind of studies, because redox regulation of chloroplast proteins is well established, many plastid proteins are abundant, redox network components have been inventoried in great depth, and functional consequences explored. Thus the repertoire of redox-related posttranslational modifications on the one hand side and their abundance on the other pose a challenge for the near future to understand their contribution to physiological regulation. The various posttranslational redox modifications are introduced, followed by a description of the available proteomics methods. The significance of the redox-related posttranslational modification is exemplarily worked out using established examples from photosynthesis. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. Copyright © 2016. Published by Elsevier B.V.

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.

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes

PubMed Central

Nady, Norhan

2016-01-01

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled “green surface modification”. This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers—ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)—is presented. PMID:27096873

Protein kinesis: The dynamics of protein trafficking and stability

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

NONE

The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

PubMed Central

Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

2017-01-01

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

Ceruloplasmin inhibits carbonyl formation in endogenous proteins in phorbol myristate acetate (PMA)-stimulated neutrophils

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

Krsek-Staples, J.; Webster, R.O.

1991-03-11

The respiratory burst of stimulated neutrophils can cause oxidative modifications of endogenous neutrophil proteins as measured by increased carbonyl formation. Ceruloplasmin is an acute phase protein and may act as an antioxidant during inflammation. Therefore, the role of ceruloplasmin in preventing oxidative damage of endogenous neutrophil proteins was investigated. Protein carbonyl content was determined spectrophotometrically using 2,4-dinitrophenylhydrazine. Ceruloplasmin, at a concentration present during inflammation significantly inhibited carbonyl formation in endogenous proteins of PMA-stimulated neutrophils. In order to determine if oxidative damage was occurring to the ceruloplasmin upon incubation with stimulated neutrophils, carbonyl formation in the ceruloplasmin in the presence andmore » absence of stimulated neutrophils. This data suggests that ceruloplasmin may play a role in regulating oxidative damage to proteins and that ceruloplasmin itself may act as a target for these modifications.« less

Carbonylation of milk powder proteins as a consequence of processing conditions.

PubMed

Fenaille, François; Parisod, Véronique; Tabet, Jean-Claude; Guy, Philippe A

2005-08-01

During industrial treatments, milk proteins could be oxidatively modified, thus leading to the formation of modified/oxidised amino acid residues. The apparition of such modified residues may contribute to the formation of new immunologically reactive structures. Some of these adducts could, in an advanced stage, lead to cross-linked protein species whose proteolytic susceptibility would be drastically decreased. Such protein species, that are resistant to digestion, could also constitute major food allergens. Therefore, these oxidative protein modifications tend to increase the natural allergenicity of milk proteins. For these reasons, monitoring milk protein oxidative modifications could be very useful regarding both product quality and allergenicity issues. In the present paper, we highlight, using different analytical approaches, the preferential carbonylation of beta-lactoglobulin (beta-Lg) during industrial treatments of milk. This result is particularly interesting since native beta-Lg represents one of the major milk allergens.

The genetic incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast.

PubMed

Supekova, Lubica; Zambaldo, Claudio; Choi, Seihyun; Lim, Reyna; Luo, Xiaozhou; Kazane, Stephanie A; Young, Travis S; Schultz, Peter G

2018-05-15

The noncanonical amino acid p-azidomethyl-l-phenylalanine can be genetically incorporated into proteins in bacteria, and has been used both as a spectroscopic probe and for the selective modification of proteins by alkynes using click chemistry. Here we report identification of Escherichia coli tyrosyl tRNA synthetase mutants that allow incorporation of p-azidomethyl-l-phenylalanine into proteins in yeast. When expressed together with the cognate E. coli tRNA CUA Tyr , the new mutant tyrosyl tRNA synthetases directed robust incorporation of p-azidomethyl-l-phenylalanine into a model protein, human superoxide dismutase, in response to the UAG amber nonsense codon. Mass spectrometry analysis of purified superoxide dismutase proteins confirmed the efficient site-specific incorporation of p-azidomethyl-l-phenylalanine. This work provides an additional tool for the selective modification of proteins in eukaryotic cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cell Signalling Through Covalent Modification and Allostery

NASA Astrophysics Data System (ADS)

Johnson, Louise N.

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases catalyze the transfer of the γ-phosphate of ATP to a serine, threonine or tyrosine residue in protein substrates. This covalent modification allows activation or inhibition of enzyme activity, creates recognition sites for other proteins and promotes order/disorder or disorder/order transitions. These properties regulate ­signalling pathways and cellular processes that mediate metabolism, transcription, cell cycle progression, differentiation, cytoskeleton arrangement and cell movement, apoptosis, intercellular communication, and neuronal and immunological functions. In this lecture I shall review the structural consequences of protein phosphorylation using our work on glycogen phosphorylase and the cell cycle cyclin dependent protein kinases as illustrations. Regulation of protein phosphorylation may be disrupted in the diseased state and protein kinases have become high profile targets for drug development. To date there are 11 compounds that have been approved for clinical use in the treatment of cancer.

Strategies for analysing and improving the expression and quality of recombinant proteins made in mammalian cells.

PubMed

Jenkins, Nigel; Meleady, Paula; Tyther, Raymond; Murphy, Lisa

2009-05-06

The production of monoclonal antibodies and other recombinant proteins is one of the highest growth areas in the pharmaceutical industry. Mammalian cells are used to manufacture the majority of biotherapeutics, largely due to their ability to perform complex post-translational modifications. Although significant progress has been made recently in improving product yields and protein quality, many challenges still lie ahead to achieve consistently high yields while avoiding potentially damaging protein modifications. The present review first considers the strategies used to analyse and improve recombinant protein expression of industrial cell lines, with an emphasis on proteomic technologies. Next, cellular and environmental influences on protein production and quality are examined, and strategies for improvements in product yield and quality are reviewed. The analytical techniques required to detect these protein changes are also described, together with prospects for assay improvements.

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.

Sulfur Radical-Induced Redox Modifications in Proteins: Analysis and Mechanistic Aspects.

PubMed

Schöneich, Christian

2017-03-10

The sulfur-containing amino acids cysteine (Cys) and methionine (Met) are prominent protein targets of redox modification during conditions of oxidative stress. Here, two-electron pathways have received widespread attention, in part due to their role in signaling processes. However, Cys and Met are equally prone to one-electron pathways, generating intermediary radicals and/or radial ions. These radicals/radical ions can generate various reaction products that are not commonly monitored in redox proteomic studies, but they may be relevant for the fate of proteins during oxidative stress. Recent Advances: Time-resolved kinetic studies and product analysis have expanded our mechanistic understanding of radical reaction pathways of sulfur-containing amino acids. These reactions are now studied in some detail for Met and Cys in proteins, and homocysteine (Hcy) chemically linked to proteins, and the role of protein radical reactions in physiological processes is evolving. Radical-derived products from Cys, Hcy, and Met can react with additional amino acids in proteins, leading to secondary protein modifications, which are potentially remote from initial points of radical attack. These products may contain intra- and intermolecular cross-links, which may lead to protein aggregation. Protein sequence and conformation will have a significant impact on the formation of such products, and a thorough understanding of reaction mechanisms and specifically how protein structure influences reaction pathways will be critical for identification and characterization of novel reaction products. Future studies must evaluate the biological significance of novel reaction products that are derived from radical reactions of sulfur-containing amino acids. Antioxid. Redox Signal. 26, 388-405.

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

PubMed

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

2016-02-01

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

Automated selected reaction monitoring software for accurate label-free protein quantification.

PubMed

Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

2012-07-06

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

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.

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

PubMed

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

2011-11-01

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

Ornithine is a novel amino acid and a marker of arginine damage by oxoaldehydes in senescent proteins.

PubMed

Sell, David R; Monnier, Vincent M

2005-06-01

Long-lived proteins undergo age-related postsynthetic modifications by glycation and advanced glycation end products (AGEs), which destabilize them by altering their conformation and charge. It was accidentally discovered that ornithine (orn) increased with age in acid hydrolyzates of human skin collagen and lens crystallins which led us to investigate the source of orn. Here, we detected such modifications of orn in these proteins. Acid hydrolysis of arginine (arg)-base AGE standards produced orn at different yields. The data provide unequivocal evidence for the in vivo formation of orn and its own AGEs in aging proteins, and suggest that arg-based AGEs serve as precursors of orn.

Artificial enzymes with protein scaffolds: structural design and modification.

PubMed

Matsuo, Takashi; Hirota, Shun

2014-10-15

Recent development in biochemical experiment techniques and bioinformatics has enabled us to create a variety of artificial biocatalysts with protein scaffolds (namely 'artificial enzymes'). The construction methods of these catalysts include genetic mutation, chemical modification using synthetic molecules and/or a combination of these methods. Designed evolution strategy based on the structural information of host proteins has become more and more popular as an effective approach to construct artificial protein-based biocatalysts with desired reactivities. From the viewpoint of application of artificial enzymes for organic synthesis, recently constructed artificial enzymes mediating oxidation, reduction and C-C bond formation/cleavage are introduced in this review article. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction.

PubMed

Saha, Ranajay; Rakshit, Surajit; Pal, Samir Kumar

2013-11-01

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo-Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady-state and picosecond-resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N-dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo-Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β-CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature-dependent circular dichroism studies explore the change in the secondary structure of Apo-Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.

Amides are novel protein modifications formed by physiological sugars.

PubMed

Glomb, M A; Pfahler, C

2001-11-09

The Maillard reaction, or nonenzymatic browning, proceeds in vivo, and the resulting protein modifications (advanced glycation end products) have been associated with various pathologies. Despite intensive research only very few structures have been established in vivo. We report here for the first time N(6)-[2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl]lysine (GOLA) and N(6)-glycoloyllysine (GALA) as prototypes for novel amide protein modifications produced by reducing sugars. Their identity was confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with N(alpha)-t-butoxycarbonyl-lysine showed that glyoxal and glycolaldehyde are immediate precursors, and reaction pathways are directly linked to N(epsilon)-carboxymethyllysine via glyoxal-imine structures. GOLA, the amide cross-link, and 1,3-bis(5-amino-5-carboxypentyl)imidazolium salt (GOLD), the imidazolium cross-link, share a common intermediate. The ratio of GOLA to GOLD is greater when glyoxal levels are low at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose and lysine depends directly upon oxidation. With the advanced glycation end product inhibitors aminoguanidine and pyridoxamine we were able to dissect oxidative fragmentation of the Amadori product as a second mechanism of GOLA formation exactly coinciding with N(epsilon)-carboxymethyllysine synthesis. In contrast, the formation of GALA appears to depend solely upon glyoxal-imines. After enzymatic hydrolysis GOLA was found at 66 pmol/mg of brunescent lens protein. This suggests amide protein modifications as important markers of pathophysiological processes.

Integrating ecosystems measurements from multiple eddy-covariance sites to a simple model of ecosystem process - Are there possibilities for a uniform model calibration?

NASA Astrophysics Data System (ADS)

Minunno, Francesco; Peltoniemi, Mikko; Launiainen, Samuli; Mäkelä, Annikki

2014-05-01

Biogeochemical models quantify the material and energy flux exchanges between biosphere, atmosphere and soil, however there is still considerable uncertainty underpinning model structure and parametrization. The increasing availability of data from of multiple sources provides useful information for model calibration and validation at different space and time scales. We calibrated a simplified ecosystem process model PRELES to data from multiple sites. In this work we had the following objective: to compare a multi-site calibration and site-specific calibrations, in order to test if PRELES is a model of general applicability, and to test how well one parameterization can predict ecosystem fluxes. Model calibration and evaluation were carried out by the means of the Bayesian method; Bayesian calibration (BC) and Bayesian model comparison (BMC) were used to quantify the uncertainty in model parameters and model structure. Evapotranspiration (ET) and gross primary production (GPP) measurements collected in 9 sites of Finland and Sweden were used in the study; half dataset was used for model calibrations and half for the comparative analyses. 10 BCs were performed; the model was independently calibrated for each of the nine sites (site-specific calibrations) and a multi-site calibration was achieved using the data from all the sites in one BC. Then 9 BMCs were carried out, one for each site, using output from the multi-site and the site-specific versions of PRELES. Similar estimates were obtained for the parameters at which model outputs are most sensitive. Not surprisingly, the joint posterior distribution achieved through the multi-site calibration was characterized by lower uncertainty, because more data were involved in the calibration process. No significant differences were encountered in the prediction of the multi-site and site-specific versions of PRELES, and after BMC, we concluded that the model can be reliably used at regional scale to simulate carbon and water fluxes of Boreal forests. Despite being a simple model, PRELES provided good estimates of GPP and ET; only for one site PRELES multi-site version underestimated water fluxes. Our study implies convergence of GPP and water processes in boreal zone to the extent that their plausible prediction is possible with a simple model using global parameterization.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

PubMed

Yu, Xiaobo; LaBaer, Joshua

2015-05-01

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Improving membrane protein expression by optimizing integration efficiency

PubMed Central

2017-01-01

The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. PMID:28918393

Tunable and reversible drug control of protein production via a self-excising degron.

PubMed

Chung, Hokyung K; Jacobs, Conor L; Huo, Yunwen; Yang, Jin; Krumm, Stefanie A; Plemper, Richard K; Tsien, Roger Y; Lin, Michael Z

2015-09-01

An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe small molecule-assisted shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, resulting in the production of an untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized copies of the protein. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto an RNA virus for which no licensed inhibitors exist. As SMASh does not require the permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh involves only a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

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

Mechanism-based strategies for protein thermostabilization.

PubMed

Mozhaev, V V

1993-03-01

Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.

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.

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

PubMed Central

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

2017-01-01

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

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

Histones: Controlling Tumor Signaling Circuitry

PubMed Central

Martins, Manoela D.; Castilho, Rogerio M.

2014-01-01

Epigenetic modifications constitute the next frontier in tumor biology research. Post-translation modification of histones dynamically influences gene expression independent of alterations to the DNA sequence. These mechanisms are often mediated by histone linkers or by proteins associated with the recruitment of DNA-binding proteins, HDAC I and II interacting proteins and transcriptional activators, coactivators or corepressors. Early evidence suggested that histones and their modifiers are involved in sophisticated processes that modulate tumor behavior and cellular phenotype. In this review, we discuss how recent discoveries about chromatin modifications, particularly histone acetylation, are shaping our knowledge of cell biology and our understanding of the molecular circuitry governing tumor progression and consider whether recent insights may extend to novel therapeutic approaches. Furthermore, we discuss the latest oncogenomic findings in Head and Neck Squamous Cell Carcinoma (HNSCC) from studies using Next Generation Sequencing (NGS) technology and highlight the impact of mutations identified in histones and their modifiers. PMID:25177526

Multiperiod planning tool for multisite pig production systems.

PubMed

Nadal-Roig, E; Plà, L M

2014-09-01

This paper presents a multiperiod planning tool for multisite pig production systems based on Linear Programming (LP). The aim of the model is to help pig managers of multisite systems in making short-term decisions (mainly related to pig transfers between farms and batch management in fattening units) and mid-term or long-term decisions (according to company targets and expansion strategy). The model skeleton follows the structure of a three-site system that can be adapted to any multisite system present in the modern pig industry. There are three basic phases, namely, piglet production, rearing pigs, and fattening. Each phase involves a different set of farms; therefore, transportation between farms and delivering of pigs to the abattoir are under consideration. The model maximizes the total gross margin calculated from the income of sales to the abattoir and the production costs over the time horizon considered. Production cost depends on each type of farm involved in the process. Parameters like number of farms per phase and distance, farm capacity, reproduction management policies, feeding and veterinary expenses, and transportation costs are taken into account. The model also provides a schedule of transfers between farms in terms of animals to be transported and number of trucks involved. The use of the model is illustrated with a case study based on a real instance of a company located in Catalonia (Spain).

Suppression of protein adsorption on a charged phospholipid polymer interface.

PubMed

Xu, Yan; Takai, Madoka; Ishihara, Kazuhiko

2009-02-09

High capability of a charged interface to suppress adsorption of both anionic and cationic proteins was reported. The interface was covalently constructed on quartz by modifying with an anionic phospholipid copolymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-potassium 3-methacryloyloxypropyl sulfonate (PMPS)-co-3-methacryloxypropyl trimethoxysilane (MPTMSi)) (PMBSSi). The PMBSSi interfaces were very hydrophilic and homogeneous and could function effectively for a long time even under long-term fluidic working conditions. The PMBSSi density on the interface, which was controllable by adjusting the PMBSSi concentration of the modification solution, affected the surface properties, including the surface contact angle, the surface roughness, and the surface zeta-potential. When a PMBSSi modification was applied, the adsorption of various proteins (isoelectric point varying from 1.0 to 11.0) on quartz was reduced to at least 87% in amount, despite the various electrical natures these proteins have. The protein adsorption behavior on the PMBSSi interface depended more on the PMBSSi density than on the surface charge. The PMBSSi modification had a stable impact on the surface, not only at the physiologic ionic strength, but also over a range of the ionic strength, suggesting that electrostatic interactions do not dominate the behavior of protein adsorption to the PMBSSi surface.

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.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast.

PubMed

Klassen, Roland; Ciftci, Akif; Funk, Johanna; Bruch, Alexander; Butter, Falk; Schaffrath, Raffael

2016-12-15

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm 5 s 2 U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct 6 A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct 6 A37 or Ψ38/39 and mcm 5 U34 or s 2 U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNA Lys UUU or tRNA Gln UUG , respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm 5 U34 or s 2 U34, and this defect can be rescued by overexpression of tRNA Gln UUG Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification and characterization of ana o 3 modifications on arginine-111 residue in heated cashew nuts

USDA-ARS?s Scientific Manuscript database

Heating foods can alter the physical, chemical, and biological characteristics of the proteins we consume. Raw and roasted cashew nut extracts were evaluated for allergen modifications by mass-spectrometry. We did not identify modifications on Ana o 1 or Ana o 2, but we observed two independent mo...

Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

PubMed

Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

2016-03-16

Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

Validating a dance-specific screening test for balance: preliminary results from multisite testing.

PubMed

Batson, Glenna

2010-09-01

Few dance-specific screening tools adequately capture balance. The aim of this study was to administer and modify the Star Excursion Balance Test (oSEBT) to examine its utility as a balance screen for dancers. The oSEBT involves standing on one leg while lightly targeting with the opposite foot to the farthest distance along eight spokes of a star-shaped grid. This task simulates dance in the spatial pattern and movement quality of the gesturing limb. The oSEBT was validated for distance on athletes with history of ankle sprain. Thirty-three dancers (age 20.1 +/- 1.4 yrs) participated from two contemporary dance conservatories (UK and US), with or without a history of lower extremity injury. Dancers were verbally instructed (without physical demonstration) to execute the oSEBT and four modifications (mSEBT): timed (speed), timed with cognitive interference (answering questions aloud), and sensory disadvantaging (foam mat). Stepping strategies were tracked and performance strategies video-recorded. Unlike the oSEBT results, distances reached were not significant statistically (p = 0.05) or descriptively (i.e., shorter) for either group. Performance styles varied widely, despite sample homogeneity and instructions to control for strategy. Descriptive analysis of mSEBT showed an increased number of near-falls and decreased timing on the injured limb. Dancers appeared to employ variable strategies to keep balance during this test. Quantitative analysis is warranted to define balance strategies for further validation of SEBT modifications to determine its utility as a balance screening tool.

User acceptance of observation and response charts with a track and trigger system: a multisite staff survey.

PubMed

Elliott, Doug; Allen, Emily; McKinley, Sharon; Perry, Lin; Duffield, Christine; Fry, Margaret; Gallagher, Robyn; Iedema, Rick; Roche, Michael

2016-08-01

To examine user acceptance with a new format of charts for recording observations and as a prompt for responding to episodes of clinical deterioration in adult medical-surgical patients. Improving recognition and response to clinical deterioration remains a challenge for acute healthcare institutions globally. Five chart templates were developed in Australia, combining human factors design principles with a track and trigger system for escalation of care. Two chart templates were previously tested in simulations, but none had been evaluated in clinical practice. Prospective multisite survey of user acceptance of the charts in practice. New observation and response charts were trialled in parallel with existing charts for 24 hours across 36 adult acute medical-surgical wards, covering 108 shifts, in five Australian states. Surveys were completed by 477 staff respondents, with open-ended comments and narrative from short informal feedback groups providing elaboration and context of user experiences. Respondents were broadly supportive of the chart format and content for monitoring patients, and as a prompt for escalating care. Some concerns were noted for chart size and style, use of ranges to graph vital signs and with specific human factors design features. Information and training issues were identified to improve usability and adherence to chart guidelines and to support improved detection and response for patients with clinical deterioration. This initial evaluation demonstrated that the charts were perceived as appropriate for documenting observations and as a prompt to detect clinical deterioration. Further evaluation after some minor modifications to the chart is recommended. Explicit training on the principles and rationale of human factors chart design, use of embedded change management strategies and addressing practical issues will improve authentic engagement, staff acceptance and adoption by all clinical users when implementing a similar observation and response chart into practice. © 2016 John Wiley & Sons Ltd.

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.

Genetic and pharmacological suppression of oncogenic mutations in RAS genes of yeast and humans

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

Schafer, W.R.; Sterne, R.; Thorner, J.

1989-07-28

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast-a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay.more » The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations. 50 refs., 3 figs., 3 tabs.« less

Lysine Methylation of Nuclear Co-repressor Receptor Interacting Protein 140

PubMed Central

Huq, MD Mostaqul; Ha, Sung Gil; Barcelona, Helene; Wei, Li-Na

2009-01-01

Receptor interacting protein 140 (RIP140) undergoes extensive posttranslational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its sub-cellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg240, Arg650, and Arg948 suppresses the repressive activity of RIP140. Here we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys591, Lys653, and Lys757 are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bi-directionally regulate the functionality of a non-histone protein. PMID:19216533

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.

PubMed

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-11-01

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

PubMed Central

Mininno, Morgane; Brugière, Sabine; Gilgen, Annabelle; Ma, Sheng; Mazzoleni, Meryl; Gigarel, Océane; Martin-Laffon, Jacqueline; Ferro, Myriam; Ravanel, Stéphane

2014-01-01

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology. PMID:24748391

An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins

PubMed Central

Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

2017-01-01

The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for different diseases, which may help to identify the proteins that may serve as markers for diagnostics as well as targets for development of new therapeutic potential. PMID:28469466

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells*

PubMed Central

Galisson, Frederic; Mahrouche, Louiza; Courcelles, Mathieu; Bonneil, Eric; Meloche, Sylvain; Chelbi-Alix, Mounira K.; Thibault, Pierre

2011-01-01

The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. PMID:21098080

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

PubMed

Ekman, Martin; Tollbäck, Petter; Bergman, Birgitta

2008-01-01

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

Nanoparticles for Protein Sensing in Primary Containers: Interaction Analysis and Application.

PubMed

Pérez Medina Martínez, Víctor; Espinosa-de la Garza, Carlos E; Méndez-Silva, Diego A; Bolívar-Vichido, Mariana; Flores-Ortiz, Luis F; Pérez, Néstor O

2018-05-01

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network.

PubMed

Tomikawa, Chie; Yokogawa, Takashi; Kanai, Tamotsu; Hori, Hiroyuki

2010-01-01

N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.

Assessment of test methods for evaluating effectiveness of cleaning flexible endoscopes.

PubMed

Washburn, Rebecca E; Pietsch, Jennifer J

2018-06-01

Strict adherence to each step of reprocessing is imperative to removing potentially infectious agents. Multiple methods for verifying proper reprocessing exist; however, each presents challenges and limitations, and best practice within the industry has not been established. Our goal was to evaluate endoscope cleaning verification tests with particular interest in the evaluation of the manual cleaning step. The results of the cleaning verification tests were compared with microbial culturing to see if a positive cleaning verification test would be predictive of microbial growth. This study was conducted at 2 high-volume endoscopy units within a multisite health care system. Each of the 90 endoscopes were tested for adenosine triphosphate, protein, microbial growth via agar plate, and rapid gram-negative culture via assay. The endoscopes were tested in 3 locations: the instrument channel, control knob, and elevator mechanism. This analysis showed substantial level of agreement between protein detection postmanual cleaning and protein detection post-high-level disinfection at the control head for scopes sampled sequentially. This study suggests that if protein is detected postmanual cleaning, there is a significant likelihood that protein will also be detected post-high-level disinfection. It also infers that a cleaning verification test is not predictive of microbial growth. Copyright © 2018 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2017-10-01

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

Protein modeling and molecular dynamics simulation of the two novel surfactant proteins SP-G and SP-H.

PubMed

Rausch, Felix; Schicht, Martin; Bräuer, Lars; Paulsen, Friedrich; Brandt, Wolfgang

2014-11-01

Surfactant proteins are well known from the human lung where they are responsible for the stability and flexibility of the pulmonary surfactant system. They are able to influence the surface tension of the gas-liquid interface specifically by directly interacting with single lipids. This work describes the generation of reliable protein structure models to support the experimental characterization of two novel putative surfactant proteins called SP-G and SP-H. The obtained protein models were complemented by predicted posttranslational modifications and placed in a lipid model system mimicking the pulmonary surface. Molecular dynamics simulations of these protein-lipid systems showed the stability of the protein models and the formation of interactions between protein surface and lipid head groups on an atomic scale. Thereby, interaction interface and strength seem to be dependent on orientation and posttranslational modification of the protein. The here presented modeling was fundamental for experimental localization studies and the simulations showed that SP-G and SP-H are theoretically able to interact with lipid systems and thus are members of the surfactant protein family.

Safety and Clinical Activity of Pembrolizumab and Multisite Stereotactic Body Radiotherapy in Patients With Advanced Solid Tumors.

PubMed

Luke, Jason J; Lemons, Jeffrey M; Karrison, Theodore G; Pitroda, Sean P; Melotek, James M; Zha, Yuanyuan; Al-Hallaq, Hania A; Arina, Ainhoa; Khodarev, Nikolai N; Janisch, Linda; Chang, Paul; Patel, Jyoti D; Fleming, Gini F; Moroney, John; Sharma, Manish R; White, Julia R; Ratain, Mark J; Gajewski, Thomas F; Weichselbaum, Ralph R; Chmura, Steven J

2018-02-13

Purpose Stereotactic body radiotherapy (SBRT) may stimulate innate and adaptive immunity to augment immunotherapy response. Multisite SBRT is an emerging paradigm for treating metastatic disease. Anti-PD-1-treatment outcomes may be improved with lower disease burden. In this context, we conducted a phase I study to evaluate the safety of pembrolizumab with multisite SBRT in patients with metastatic solid tumors. Patients and Methods Patients progressing on standard treatment received SBRT to two to four metastases. Not all metastases were targeted, and metastases > 65 mL were partially irradiated. SBRT dosing varied by site and ranged from 30 to 50 Gy in three to five fractions with predefined dose de-escalation if excess dose-limiting toxicities were observed. Pembrolizumab was initiated within 7 days after completion of SBRT. Pre- and post-SBRT biopsy specimens were analyzed in a subset of patients to quantify interferon-γ-induced gene expression. Results A total of 79 patients were enrolled; three patients did not receive any treatment and three patients only received SBRT. Patients included in the analysis were treated with SBRT and at least one cycle of pembrolizumab. Most (94.5%) of patients received SBRT to two metastases. Median follow-up for toxicity was 5.5 months (interquartile range, 3.3 to 8.1 months). Six patients experienced dose-limiting toxicities with no radiation dose reductions. In the 68 patients with imaging follow-up, the overall objective response rate was 13.2%. Median overall survival was 9.6 months (95% CI, 6.5 months to undetermined) and median progression-free survival was 3.1 months (95% CI, 2.9 to 3.4 months). Expression of interferon-γ-associated genes from post-SBRT tumor biopsy specimens significantly correlated with nonirradiated tumor response. Conclusion Multisite SBRT followed by pembrolizumab was well tolerated with acceptable toxicity. Additional studies exploring the clinical benefit and predictive biomarkers of combined multisite SBRT and PD-1-directed immunotherapy are warranted.

Structures of ribonucleoprotein particle modification enzymes

PubMed Central

Liang, Bo; Li, Hong

2016-01-01

Small nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo. PMID:21108865

The effect of high pressure on the functional properties of pork myofibrillar proteins.

PubMed

Grossi, Alberto; Olsen, Karsten; Bolumar, Tomas; Rinnan, Åsmund; Øgendal, Lars H; Orlien, Vibeke

2016-04-01

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments. Copyright © 2015 Elsevier Ltd. All rights reserved.

Systematic discovery of Xist RNA binding proteins

PubMed Central

Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

2015-01-01

Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri

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

Prochnik, Simon E.; Umen, James; Nedelcu, Aurora

2010-07-01

Analysis of the Volvox carteri genome reveals that this green alga's increased organismal complexity and multicellularity are associated with modifications in protein families shared with its unicellular ancestor, and not with large-scale innovations in protein coding capacity. The multicellular green alga Volvox carteri and its morphologically diverse close relatives (the volvocine algae) are uniquely suited for investigating the evolution of multicellularity and development. We sequenced the 138 Mb genome of V. carteri and compared its {approx}14,500 predicted proteins to those of its unicellular relative, Chlamydomonas reinhardtii. Despite fundamental differences in organismal complexity and life history, the two species have similarmore » protein-coding potentials, and few species-specific protein-coding gene predictions. Interestingly, volvocine algal-specific proteins are enriched in Volvox, including those associated with an expanded and highly compartmentalized extracellular matrix. Our analysis shows that increases in organismal complexity can be associated with modifications of lineage-specific proteins rather than large-scale invention of protein-coding capacity.« less

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lynham, Steven; Garcia-Manyes, Sergi

2016-08-01

The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.

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.

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

PubMed Central

Bragulla, Hermann H; Homberger, Dominique G

2009-01-01

Historically, the term ‘keratin’ stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament-associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament-forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament-forming proteins that were extracted from the living layers of the epidermis were grouped as ‘prekeratins’ or ‘cytokeratins’. Currently, the term ‘keratin’ covers all intermediate filament-forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non-keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non-modified epidermis is a keratinized stratified epithelium, and that all other stratified and non-stratified epithelia are non-keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered. PMID:19422428

Efficient Site-Specific Labeling of Proteins via Cysteines

PubMed Central

Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

2011-01-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

Efficient site-specific labeling of proteins via cysteines.

PubMed

Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

2008-03-01

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.

PubMed

Lin, Hong-Yu; Haegele, Joseph A; Disare, Michael T; Lin, Qishan; Aye, Yimon

2015-05-20

Despite the known propensity of small-molecule electrophiles to react with numerous cysteine-active proteins, biological actions of individual signal inducers have emerged to be chemotype-specific. To pinpoint and quantify the impacts of modifying one target out of the whole proteome, we develop a target-protein-personalized "electrophile toolbox" with which specific intracellular targets can be selectively modified at a precise time by specific reactive signals. This general methodology, T-REX (targetable reactive electrophiles and oxidants), is established by (1) constructing a platform that can deliver a range of electronic and sterically different bioactive lipid-derived signaling electrophiles to specific proteins in cells; (2) probing the kinetics of targeted delivery concept, which revealed that targeting efficiency in cells is largely driven by initial on-rate of alkylation; and (3) evaluating the consequences of protein-target- and small-molecule-signal-specific modifications on the strength of downstream signaling. These data show that T-REX allows quantitative interrogations into the extent to which the Nrf2 transcription factor-dependent antioxidant response element (ARE) signaling is activated by selective electrophilic modifications on Keap1 protein, one of several redox-sensitive regulators of the Nrf2-ARE axis. The results document Keap1 as a promiscuous electrophile-responsive sensor able to respond with similar efficiencies to discrete electrophilic signals, promoting comparable strength of Nrf2-ARE induction. T-REX is also able to elicit cell activation in cases in which whole-cell electrophile flooding fails to stimulate ARE induction prior to causing cytotoxicity. The platform presents a previously unavailable opportunity to elucidate the functional consequences of small-molecule-signal- and protein-target-specific electrophilic modifications in an otherwise unaffected cellular background.

Modifications in small nuclear RNAs and their roles in spliceosome assembly and function.

PubMed

Bohnsack, Markus T; Sloan, Katherine E

2018-06-01

Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2'-O-methylated nucleotides, which are largely introduced by small Cajal body-specific-RNPs. Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNAs splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.

Effects of tRNA modification on translational accuracy depend on intrinsic codon-anticodon strength.

PubMed

Manickam, Nandini; Joshi, Kartikeya; Bhatt, Monika J; Farabaugh, Philip J

2016-02-29

Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10(-6) per codon but is codon-specific with some error frequencies above 10(-3) per codon. Here we test the effect on error frequency of blocking post-transcriptional modifications of the anticodon loops of four tRNAs in Escherichia coli. We find two types of responses to removing modification. Blocking modification of tRNA(UUC)(Glu) and tRNA(QUC)(Asp) increases errors, suggesting that the modifications act at least in part to maintain accuracy. Blocking even identical modifications of tRNA(UUU)(Lys) and tRNA(QUA)(Tyr) has the opposite effect of decreasing errors. One explanation could be that the modifications play opposite roles in modulating misreading by the two classes of tRNAs. Given available evidence that modifications help preorder the anticodon to allow it to recognize the codons, however, the simpler explanation is that unmodified 'weak' tRNAs decode too inefficiently to compete against cognate tRNAs that normally decode target codons, which would reduce the frequency of misreading. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

PubMed

Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

2017-05-01

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Ubiquitin modifications

PubMed Central

Swatek, Kirby N; Komander, David

2016-01-01

Protein ubiquitination is a dynamic multifaceted post-translational modification involved in nearly all aspects of eukaryotic biology. Once attached to a substrate, the 76-amino acid protein ubiquitin is subjected to further modifications, creating a multitude of distinct signals with distinct cellular outcomes, referred to as the 'ubiquitin code'. Ubiquitin can be ubiquitinated on seven lysine (Lys) residues or on the N-terminus, leading to polyubiquitin chains that can encompass complex topologies. Alternatively or in addition, ubiquitin Lys residues can be modified by ubiquitin-like molecules (such as SUMO or NEDD8). Finally, ubiquitin can also be acetylated on Lys, or phosphorylated on Ser, Thr or Tyr residues, and each modification has the potential to dramatically alter the signaling outcome. While the number of distinctly modified ubiquitin species in cells is mind-boggling, much progress has been made to characterize the roles of distinct ubiquitin modifications, and many enzymes and receptors have been identified that create, recognize or remove these ubiquitin modifications. We here provide an overview of the various ubiquitin modifications present in cells, and highlight recent progress on ubiquitin chain biology. We then discuss the recent findings in the field of ubiquitin acetylation and phosphorylation, with a focus on Ser65-phosphorylation and its role in mitophagy and Parkin activation. PMID:27012465

Studying Catabolism of Protein ADP-Ribosylation.

PubMed

Palazzo, Luca; James, Dominic I; Waddell, Ian D; Ahel, Ivan

2017-01-01

Protein ADP-ribosylation is a conserved posttranslational modification that regulates many major cellular functions, such as DNA repair, transcription, translation, signal transduction, stress response, cell division, aging, and cell death. Protein ADP-ribosyl transferases catalyze the transfer of an ADP-ribose (ADPr) group from the β-nicotinamide adenine dinucleotide (β-NAD + ) cofactor onto a specific target protein with the subsequent release of nicotinamide. ADP-ribosylation leads to changes in protein structure, function, stability, and localization, thus defining the appropriate cellular response. Signaling processes that are mediated by modifications need to be finely tuned and eventually silenced and one of the ways to achieve this is through the action of enzymes that remove (reverse) protein ADP-ribosylation in a timely fashion such as PARG, TARG1, MACROD1, and MACROD2. Here, we describe several basic methods used to study the enzymatic activity of de-ADP-ribosylating enzymes.

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.

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

PubMed Central

Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

2015-01-01

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

Emerging roles of lysine methylation on non-histone proteins.

PubMed

Zhang, Xi; Huang, Yaling; Shi, Xiaobing

2015-11-01

Lysine methylation is a common posttranslational modification (PTM) of histones that is important for the epigenetic regulation of transcription and chromatin in eukaryotes. Increasing evidence demonstrates that in addition to histones, lysine methylation also occurs on various non-histone proteins, especially transcription- and chromatin-regulating proteins. In this review, we will briefly describe the histone lysine methyltransferases (KMTs) that have a broad spectrum of non-histone substrates. We will use p53 and nuclear receptors, especially estrogen receptor alpha, as examples to discuss the dynamic nature of non-histone protein lysine methylation, the writers, erasers, and readers of these modifications, and the crosstalk between lysine methylation and other PTMs in regulating the functions of the modified proteins. Understanding the roles of lysine methylation in normal cells and during development will shed light on the complex biology of diseases associated with the dysregulation of lysine methylation on both histones and non-histone proteins.

Use of hydrostatic pressure for modulation of protein chemical modification and enzymatic selectivity.

PubMed

Makarov, Alexey A; Helmy, Roy; Joyce, Leo; Reibarkh, Mikhail; Maust, Mathew; Ren, Sumei; Mergelsberg, Ingrid; Welch, Christopher J

2016-05-11

Using hydrostatic pressure to induce protein conformational changes can be a powerful tool for altering the availability of protein reactive sites and for changing the selectivity of enzymatic reactions. Using a pressure apparatus, it has been demonstrated that hydrostatic pressure can be used to modulate the reactivity of lysine residues of the protein ubiquitin with a water-soluble amine-specific homobifunctional coupling agent. Fewer reactive lysine residues were observed when the reaction was carried out under elevated pressure of 3 kbar, consistent with a pressure-induced conformational change of ubiquitin that results in fewer exposed lysine residues. Additionally, modulation of the stereoselectivity of an enzymatic transamination reaction was observed at elevated hydrostatic pressure. In one case, the minor diasteromeric product formed at atmospheric pressure became the major product at elevated pressure. Such pressure-induced alterations of protein reactivity may provide an important new tool for enzymatic reactions and the chemical modification of proteins.

The equilibrium assumption is valid for the kinetic treatment of most time-dependent protein-modification reactions.

PubMed Central

Brocklehurst, K

1979-01-01

To facilitate mechanistic interpretation of the kinetics of time-dependent inhibition of enzymes and of similar protein modification reactions, it is important to know when the equilibrium assumption may be applied to the model: formula: (see text). The conventional criterion of quasi-equilibrium, k + 2 less than k-1, is not always easy to assess, particularly when k + 2 cannot be separately determined. It is demonstrated that the condition k + 2 less than k-1 is necessarily true, however, when the value of the apparent second-order rate constant for the modification reaction is much smaller than the value of k + 1. Since k + 1 is commonly at least 10(7)M-1.S-1 for substrates, it is probable that the equilibrium assumption may be properly applied to most irreversible inhibitions and modification reactions. PMID:518556

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Tang, Yisi; Wang, Huiyuan; Xu, Qin; Li, Yaping; Li, Feng; Huang, Yongzhuo

2017-01-01

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation. PMID:27914267

Translating Current Bioanalytical Techniques for Studying Corona Activity.

PubMed

Wang, Chunming; Wang, Zhenzhen; Dong, Lei

2018-07-01

The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces. Copyright © 2018 Elsevier Ltd. All rights reserved.

DNA sequence transfer between two high-cysteine chorion gene families in the silkmoth Bombyx mori.

PubMed Central

Iatrou, K; Tsitilou, S G; Kafatos, F C

1984-01-01

We have previously shown that one type of high-cysteine silkmoth chorion protein (Hc-A) has evolved from the A family of chorion proteins by radical modifications of the NH2-terminal and COOH-terminal polypeptide arms: most of the arm sequences have been deleted, while short cysteine- and glycine-containing repeats have expanded into long arrays. Strikingly similar modifications of the arms have led to the evolution of a second type of high-cysteine protein (Hc-B) from the B family of chorion proteins. It appears that the parallel evolution of these high-cysteine-encoding gene families has not been entirely independent: examination of 3' untranslated regions shows evidence of information transfer between the two families. PMID:6589605

Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge.

PubMed

Ottaviani, Enzo; Accorsi, Alice; Rigillo, Giovanna; Malagoli, Davide; Blom, Joan M C; Tascedda, Fabio

2013-11-06

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications. © 2013 Published by Elsevier B.V.

BuD, a helix–loop–helix DNA-binding domain for genome modification

PubMed Central

Stella, Stefano; Molina, Rafael; López-Méndez, Blanca; Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza; Campos-Olivas, Ramon; Duchateau, Phillippe; Montoya, Guillermo

2014-01-01

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. PMID:25004980

R Factor-Controlled Restriction and Modification of Deoxyribonucleic Acid: Restriction Mutants

PubMed Central

Yoshimori, Robert; Roulland-Dussoix, Daisy; Boyer, Herbert W.

1972-01-01

Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase. PMID:4565538

Quantification of protein carbonylation.

PubMed

Wehr, Nancy B; Levine, Rodney L

2013-01-01

Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is most often measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent 2,4 dinitrophenylhydrazine (DNPH). We present protocols for the derivatization and quantification of protein carbonylation with these two methods, including a newly described dot blot with greatly increased sensitivity.

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.

Gel-based methods in redox proteomics.

PubMed

Charles, Rebecca; Jayawardhana, Tamani; Eaton, Philip

2014-02-01

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification. This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated. A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein. By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Copyright © 2013 Elsevier B.V. All rights reserved.

Change of the isoelectric point of hemoglobin at the air/water interface probed by the orientational flip-flop of water molecules.

PubMed

Devineau, Stéphanie; Inoue, Ken-Ichi; Kusaka, Ryoji; Urashima, Shu-Hei; Nihonyanagi, Satoshi; Baigl, Damien; Tsuneshige, Antonio; Tahara, Tahei

2017-04-19

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Im χ (2) spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

PubMed

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection.

PubMed

Pérez, José de Jesús; Udeshi, Namrata D; Shabanowitz, Jeffrey; Ciordia, Sergio; Juárez, Silvia; Scott, Cheryl L; Olszewski, Neil E; Hunt, Donald F; García, Juan Antonio

2013-08-01

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation of Elp1 by Hrr25 Is Required for Elongator-Dependent tRNA Modification in Yeast

PubMed Central

Abdel-Fattah, Wael; Jablonowski, Daniel; Di Santo, Rachael; Thüring, Kathrin L.; Scheidt, Viktor; Hammermeister, Alexander; ten Have, Sara; Helm, Mark; Schaffrath, Raffael; Stark, Michael J. R.

2015-01-01

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase. PMID:25569479

Modifications of wheat flour proteins during in vitro digestion of bread dough, crumb, and crust: an electrophoretic and immunological study.

PubMed

Pasini, G; Simonato, B; Giannattasio, M; Peruffo, A D; Curioni, A

2001-05-01

The proteins of wheat flour have several biological activities that can affect human health and physiology when wheat-based foods are consumed. The modifications of bread crumb and crust proteins during an in vitro peptic/pancreatic digestion process were studied by electrophoresis and immunoblotting with polyclonal antibodies specific for single proteins or groups of homologous proteins of the wheat flour, and the results were compared to those obtained for an unheated dough sample. The results show that baking affects the extent of proteolysis and the immunological and physicochemical features of the digestion products in relation to the level of the heat treatment. Therefore, the results concerning the digestion of the unheated wheat flour or dough are not representative of what happens when baked products enter the human digestive tract.

A bioinformatics-based overview of protein Lys-Ne-acetylation

USDA-ARS?s Scientific Manuscript database

Among posttranslational modifications, there are some conceptual similarities between Lys-N'-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. T...

A double stain for total and oxidized proteins from two-dimensional fingerprints.

PubMed

Talent, J M; Kong, Y; Gracy, R W

1998-10-01

Oxidative modification of proteins plays a major role in the etiology of aging and age-related diseases. For example, in Alzheimer's disease, although evidence points to oxidation of proteins as a causative factor in loss of cognitive abilities, it is not known which specific proteins of the brain are most susceptible to these modifications. Thus, it is of interest to identify the specific proteins which are susceptible to oxidation in vivo. Two-dimensional protein fingerprint methods offer the analytical potential for resolution of thousands of individual proteins from tissues, and the oxidized proteins can be visualized with immunological probes. Sensitive methods permit recovery and sufficient amino acid sequencing to identify these proteins. However, for such analyses it is essential to simultaneously analyze both protein content and level of oxidation. We have evaluated several approaches, identified the sources of artifacts and interferences, and developed a double-staining procedure that allows visualization and quantitation of total protein patterns as well as the specific oxidized proteins from two-dimensional protein fingerprints. The method has been applied to cells grown in culture and to tissue extracts from young and old animals. Copyright 1998 Academic Press.

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.

[Methods of quantitative proteomics].

PubMed

Kopylov, A T; Zgoda, V G

2007-01-01

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

S-Nitrosothiol measurements in biological systems⋄

PubMed Central

Gow, Andrew; Doctor, Allan; Mannick, Joan; Gaston, Benjamin

2007-01-01

S-Nitrosothiol (SNO) cysteine modifications are regulated signaling reactions that dramatically affect, and are affected by, protein conformation. The lability of the S-NO bond can make SNO-modified proteins cumbersome to measure accurately. Here, we review methodologies for detecting SNO modifications in biology. There are three caveats. 1) Many assays for biological SNOs are used near the limit of detection: standard curves must be in the biologically relevant concentration range. 2) The assays that are most reliable are those that modify SNO protein or peptide chemistry the least. 3) Each result should be quantitatively validated using more than one assay. Improved assays are needed and are in development. PMID:17379583

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins.

PubMed

Piscitelli, Alessandra; Cicatiello, Paola; Gravagnuolo, Alfredo Maria; Sorrentino, Ilaria; Pezzella, Cinzia; Giardina, Paola

2017-06-26

Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

Patterns of disclosure among youth who are HIV-positive: a multisite study.

PubMed

Dempsey, Allison G; MacDonell, Karen E; Naar-King, Sylvie; Lau, Chuen-Yen

2012-03-01

Disclosure of serostatus is critical in preventing the transmission of HIV among youth. The purpose of this exploratory study was to describe serostatus disclosure in a multisite study of youth living with HIV. This study investigated serostatus disclosure and its relationship to unprotected sex among 146 youth participating in a multisite study of young people living with HIV who were sexually active within the past 3 months. Forty percent of participants reported a sexual relationship with a partner to whom they had not disclosed their serostatus. Participants with multiple sexual partners were less likely to disclose their serostatus than those with one partner. Disclosure was more frequent when the serostatus of the sexual partner was known. Disclosure was not associated with unprotected sex. Prevention initiatives should focus on both disclosure and condom use in this high-risk population, particularly for youth with multiple sexual partners. Copyright © 2012 Society for Adolescent Health and Medicine. All rights reserved.

Transmission Heterogeneity and Autoinoculation in a Multisite Infection Model of HPV

PubMed Central

Brouwer, Andrew F.; Meza, Rafael; Eisenberg, Marisa C.

2015-01-01

The human papillomavirus (HPV) is sexually transmitted and can infect oral, genital, and anal sites in the human epithelium. Here, we develop a multisite transmission model that includes autoinoculation, to study HPV and other multisite diseases. Under a homogeneous-contacts assumption, we analyze the basic reproduction number R0, as well as type and target reproduction numbers, for a two-site model. In particular, we find that R0 occupies a space between taking the maximum of next generation matrix terms for same site transmission and taking the geometric average of cross-site transmission terms in such a way that heterogeneity in the same-site transmission rates increases R0 while heterogeneity in the cross-site transmission decreases it. Additionally, autoinoculation adds considerable complexity to the form of R0. We extend this analysis to a heterosexual population, which additionally yields dynamics analogous to those of vector–host models. We also examine how these issues of heterogeneity may affect disease control, using type and target reproduction numbers. PMID:26518265

The opportunities and challenges of multi-site evaluations: lessons from the jail diversion and trauma recovery national cross-site evaluation.

PubMed

Stainbrook, Kristin; Penney, Darby; Elwyn, Laura

2015-06-01

Multi-site evaluations, particularly of federally funded service programs, pose a special set of challenges for program evaluation. Not only are there contextual differences related to project location, there are often relatively few programmatic requirements, which results in variations in program models, target populations and services. The Jail Diversion and Trauma Recovery-Priority to Veterans (JDTR) National Cross-Site Evaluation was tasked with conducting a multi-site evaluation of thirteen grantee programs that varied along multiple domains. This article describes the use of a mixed methods evaluation design to understand the jail diversion programs and client outcomes for veterans with trauma, mental health and/or substance use problems. We discuss the challenges encountered in evaluating diverse programs, the benefits of the evaluation in the face of these challenges, and offer lessons learned for other evaluators undertaking this type of evaluation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of a continuous multisite accelerometry system for studying movements during sleep.

PubMed

Terrill, Philip I; Mason, David G; Wilson, Stephen J

2010-01-01

Actigraphy has proven to be a useful tool in the assessment of circadian rhythms, and more recently in the automatic staging of sleep and wake states. Whilst accuracy of commercial systems appears good over 24 hour periods, the sensitivity of detecting wake during time in bed is poor. One possible explanation for these poor results is the technical limitations of currently available commercial actigraphs. In particular, raw data is generally not available to the user. Instead, activity counts for each epoch (typically between 10-60 secs) are calculated using various algorithms, from which sleep state is identified. Consequently morphologically different movements observed during sleep and wake states may not be detected as such. In this paper, the development of a continuous multisite, accelerometry system (CMAS) is described. Initial results, comparing data collected using a commercial actigraph (Actiwatch- Mini Motionlogger), and the continuous multisite accelerometry system are presented. The CMAS is able to differentiate brief movement "twitches" from postural changes.

Destabilized bioluminescent proteins

DOEpatents

Allen, Michael S [Knoxville, TN; Rakesh, Gupta [New Delhi, IN; Gary, Sayler S [Blaine, TN

2007-07-31

Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

Structure modification and functionality of whey proteins: quantitative structure-activity relationship approach.

PubMed

Nakai, S; Li-Chan, E

1985-10-01

According to the original idea of quantitative structure-activity relationship, electric, hydrophobic, and structural parameters should be taken into consideration for elucidating functionality. Changes in these parameters are reflected in the property of protein solubility upon modification of whey proteins by heating. Although solubility is itself a functional property, it has been utilized to explain other functionalities of proteins. However, better correlations were obtained when hydrophobic parameters of the proteins were used in conjunction with solubility. Various treatments reported in the literature were applied to whey protein concentrate in an attempt to obtain whipping and gelling properties similar to those of egg white. Mapping simplex optimization was used to search for the best results. Improvement in whipping properties by pepsin hydrolysis may have been due to higher protein solubility, and good gelling properties resulting from polyphosphate treatment may have been due to an increase in exposable hydrophobicity. However, the results of angel food cake making were still unsatisfactory.

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.

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

Cellular Binding of Anionic Nanoparticles is Inhibited by Serum Proteins Independent of Nanoparticle Composition.

PubMed

Fleischer, Candace C; Kumar, Umesh; Payne, Christine K

2013-09-01

Nanoparticles used in biological applications encounter a complex mixture of extracellular proteins. Adsorption of these proteins on the nanoparticle surface results in the formation of a "protein corona," which can dominate the interaction of the nanoparticle with the cellular environment. The goal of this research was to determine how nanoparticle composition and surface modification affect the cellular binding of protein-nanoparticle complexes. We examined the cellular binding of a collection of commonly used anionic nanoparticles: quantum dots, colloidal gold nanoparticles, and low-density lipoprotein particles, in the presence and absence of extracellular proteins. These experiments have the advantage of comparing different nanoparticles under identical conditions. Using a combination of fluorescence and dark field microscopy, flow cytometry, and spectroscopy, we find that cellular binding of these anionic nanoparticles is inhibited by serum proteins independent of nanoparticle composition or surface modification. We expect these results will aid in the design of nanoparticles for in vivo applications.

A two-step enzymatic modification method to reduce immuno-reactivity of milk proteins.

PubMed

Damodaran, Srinivasan; Li, Yan

2017-12-15

A two-step enzymatic approach to reduce immuno-reactivity of whey protein isolate and casein has been studied. The method involves partial hydrolysis of proteins with proteases, followed by repolymerization with microbial transglutaminase. Whey protein isolate partially hydrolyzed with chymotrypsin, trypsin, or thermolysin retained about 80%, 30%, and 20% of the original immuno-reactivity, respectively. Upon repolymerization the immuno-reactivity decreased to 45%, 35%, and 5%, respectively. The immuno-reactivity of hydrolyzed and repolymerized casein was negligible compared to native casein. The repolymerized products were partially resistant to in vitro digestion. Peptides released during digestion of repolymerized thermolysin-whey protein hydrolysate had less than 5% immuno-reactivity, whereas those of whey protein control exhibited a sinusoidal immuno-reactivity ranging from 5 to 20%. Peptides released during digestion of repolymerized thermolysin-casein hydrolysates had no immuno-reactivity. These results indicated that it is possible to produce hypoallergenic milk protein products using the two-step enzymatic modification method involving thermolysin and transglutaminase. Copyright © 2017. Published by Elsevier Ltd.

Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

PubMed

Luo, Xiaoting; Wu, Jinzi; Jin, Zhen; Yan, Liang-Jun

2017-02-02

Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

PRMT7, a new protein arginine methyltransferase that synthesizes symmetric dimethylarginine.

PubMed

Lee, Jin-Hyung; Cook, Jeffry R; Yang, Zhi-Hong; Mirochnitchenko, Olga; Gunderson, Samuel I; Felix, Arthur M; Herth, Nicole; Hoffmann, Ralf; Pestka, Sidney

2005-02-04

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

PubMed

Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

2016-01-01

A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective efficacy of adenoviral based malaria vaccines.

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

PubMed

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

2018-04-22

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

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