Separomics applied to the proteomics and peptidomics of low-abundance proteins: choice of methods and challenges - a review

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Maria Cristina Baracat-Pereira

2012-01-01

Full Text Available The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes.

Workshop proceedings: challenges and opportunities in evaluating protein allergenicity across biotechnology industries.

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Stagg, Nicola J; Ghantous, Hanan N; Ladics, Gregory S; House, Robert V; Gendel, Steven M; Hastings, Kenneth L

2013-01-01

A workshop entitled "Challenges and Opportunities in Evaluating Protein Allergenicity across Biotechnology Industries" was held at the 51st Annual Meeting of the Society of Toxicology (SOT) in San Francisco, California. The workshop was sponsored by the Biotechnology Specialty Section of SOT and was designed to present the science-based approaches used in biotechnology industries to evaluate and regulate protein allergenicity. A panel of experts from industry and government highlighted the allergenicity testing requirements and research in the agricultural, pharmaceutical/biopharma, and vaccine biotechnology industries and addressed challenges and opportunities for advancing the science of protein allergenicity. The main learning from the workshop was that immunoglobulin E-mediated allergenicity of biotechnology-derived products is difficult to assess without human data. The approaches currently being used to evaluate potential for allergenicity across biotechnology industries are very different and range from bioinformatics, in vitro serology, in vivo animal testing, in vitro and in vivo functional assays, and "biosimilar" assessments (ie, biotherapeutic equivalents to innovator products). The challenge remains with regard to the different or lack of regulatory requirements for allergenicity testing across industries, but the novel approaches being used with bioinformatics and biosimilars may lead to opportunities in the future to collaborate across biotechnology industries.

Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge.

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Huang, Jingwei; Zhang, Zhenchao; Li, Menghui; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2015-10-01

E. maxima is one of the seven species of Eimeria that infects chicken. Until now, only a few antigenic genes of E. maxima have been reported. In the present study, the immune protective effects against E. maxima challenge of recombinant protein and DNA vaccine encoding EmMIC2 were evaluated. Two-week-old chickens were randomly divided into five groups. The experimental group of chickens was immunized with 100 μg DNA vaccine pVAX1-MIC2 or 200 μg rEmMIC2 protein while the control group of chickens was injected with pVAX1 plasmid or sterile PBS. The results showed that the anti-EmMIC2 antibody titers of both rEmMIC2 protein and pVAX1-MIC2 groups were significantly higher as compared to PBS and pVAX1 control (Pmaxima challenge and it could be an effective antigen candidate for the development of new vaccines against E. maxima. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Practical aspects of NMR signal assignment in larger and challenging proteins

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Frueh, Dominique P.

2014-01-01

NMR has matured into a technique routinely employed for studying proteins in near physiological conditions. However, applications to larger proteins are impeded by the complexity of the various correlation maps necessary to assign NMR signals. This article reviews the data analysis techniques traditionally employed for resonance assignment and describes alternative protocols necessary for overcoming challenges in large protein spectra. In particular, simultaneous analysis of multiple spectra may help overcome ambiguities or may reveal correlations in an indirect manner. Similarly, visualization of orthogonal planes in a multidimensional spectrum can provide alternative assignment procedures. We describe examples of such strategies for assignment of backbone, methyl, and nOe resonances. We describe experimental aspects of data acquisition for the related experiments and provide guidelines for preliminary studies. Focus is placed on large folded monomeric proteins and examples are provided for 37, 48, 53, and 81 kDa proteins. PMID:24534088

Separomics applied to the proteomics and peptidomics of low-abundance proteins: Choice of methods and challenges – A review

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Baracat-Pereira, Maria Cristina; de Oliveira Barbosa, Meire; Magalhães, Marcos Jorge; Carrijo, Lanna Clicia; Games, Patrícia Dias; Almeida, Hebréia Oliveira; Sena Netto, José Fabiano; Pereira, Matheus Rodrigues; de Barros, Everaldo Gonçalves

2012-01-01

The enrichment and isolation of proteins are considered limiting steps in proteomic studies. Identification of proteins whose expression is transient, those that are of low-abundance, and of natural peptides not described in databases, is still a great challenge. Plant extracts are in general complex, and contaminants interfere with the identification of proteins involved in important physiological processes, such as plant defense against pathogens. This review discusses the challenges and strategies of separomics applied to the identification of low-abundance proteins and peptides in plants, especially in plants challenged by pathogens. Separomics is described as a group of methodological strategies for the separation of protein molecules for proteomics. Several tools have been used to remove highly abundant proteins from samples and also non-protein contaminants. The use of chromatographic techniques, the partition of the proteome into subproteomes, and an effort to isolate proteins in their native form have allowed the isolation and identification of rare proteins involved in different processes. PMID:22802713

Delivery of Therapeutic Proteins Using Electrospun Fibers-Recent Developments and Current Challenges.

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Seif, Salem; Planz, Viktoria; Windbergs, Maike

2017-10-01

Proteins play a vital role within the human body by regulating various functions and even serving as structural constituent of many body parts. In this context, protein-based therapeutics have attracted a lot of attention in the last few decades as potential treatment of different diseases. Due to the steadily increasing interest in protein-based therapeutics, different dosage forms were investigated for delivering such complex macromolecules to the human body. Here, electrospun fibers hold a great potential for embedding proteins without structural damage and for controlled release of the protein for therapeutic applications. This review provides a comprehensive overview of the current state of protein-based carrier systems using electrospun fibers, with special emphasis on discussing their potential and key challenges in developing such therapeutic strategies, along with a prospective view of anticipated future directions. © 2017 Deutsche Pharmazeutische Gesellschaft.

ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets.

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Yumerefendi, Hayretin; Tarendeau, Franck; Mas, Philippe J; Hart, Darren J

2010-10-01

Expression of sufficient quantities of soluble protein for structural biology and other applications is often a very difficult task, especially when multimilligram quantities are required. In order to improve yield, solubility or crystallisability of a protein, it is common to subclone shorter genetic constructs corresponding to single- or multi-domain fragments. However, it is not always clear where domain boundaries are located, especially when working on novel targets with little or no sequence similarity to other proteins. Several methods have been described employing aspects of directed evolution to the recombinant expression of challenging proteins. These combine the construction of a random library of genetic constructs of a target with a screening or selection process to identify solubly expressing protein fragments. Here we review several datasets from the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technology to provide a view on its capabilities. Firstly, we demonstrate how it functions using the well-characterised NF-kappaB p50 transcription factor as a model system. Secondly, application of ESPRIT to the challenging PB2 subunit of influenza polymerase has led to several novel atomic resolution structures; here we present an overview of the screening phase of that project. Thirdly, analysis of the human kinase TBK1 is presented to show how the ESPRIT technology rapidly addresses the compatibility of challenging targets with the Escherichia coli expression system.

Mass Spectrometry-Based Methods for Identifying Oxidized Proteins in Disease: Advances and Challenges

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Ivan Verrastro

2015-04-01

Full Text Available Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease.

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

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Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Science.gov (United States)

Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Directory of Open Access Journals (Sweden)

Krishnamurthy Konduru

Full Text Available Ebola virus (EBOV, a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data

Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges

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Meili Gao

2012-01-01

Full Text Available Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed.

The challenges in and importance of analysing protein structure and physical stability in complex formulations

DEFF Research Database (Denmark)

Jorgensen, L.; Jensen, Minna Grønning; Roest, N.

2013-01-01

In this review several analytical challenges that may be encountered during protein formulation development of complex formulations are discussed through recent examples. These examples show how selected advanced biophysical methods can greatly increase our understanding of the system under...

Detection and quantification of proteins and cells by use of elemental mass spectrometry: progress and challenges.

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Yan, Xiaowen; Yang, Limin; Wang, Qiuquan

2013-07-01

Much progress has been made in identification of the proteins in proteomes, and quantification of these proteins has attracted much interest. In addition to popular tandem mass spectrometric methods based on soft ionization, inductively coupled plasma mass spectrometry (ICPMS), a typical example of mass spectrometry based on hard ionization, usually used for analysis of elements, has unique advantages in absolute quantification of proteins by determination of an element with a definite stoichiometry in a protein or attached to the protein. In this Trends article, we briefly describe state-of-the-art ICPMS-based methods for quantification of proteins, emphasizing protein-labeling and element-tagging strategies developed on the basis of chemically selective reactions and/or biospecific interactions. Recent progress from protein to cell quantification by use of ICPMS is also discussed, and the possibilities and challenges of ICPMS-based protein quantification for universal, selective, or targeted quantification of proteins and cells in a biological sample are also discussed critically. We believe ICPMS-based protein quantification will become ever more important in targeted quantitative proteomics and bioanalysis in the near future.

Protein lipoxidation: Detection strategies and challenges

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Giancarlo Aldini

2015-08-01

Full Text Available Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Protein Biomarkers for Early Detection of Pancreatic Ductal Adenocarcinoma: Progress and Challenges.

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Root, Alex; Allen, Peter; Tempst, Paul; Yu, Kenneth

2018-03-07

Approximately 75% of patients with pancreatic ductal adenocarcinoma are diagnosed with advanced cancer, which cannot be safely resected. The most commonly used biomarker CA19-9 has inadequate sensitivity and specificity for early detection, which we define as Stage I/II cancers. Therefore, progress in next-generation biomarkers is greatly needed. Recent reports have validated a number of biomarkers, including combination assays of proteins and DNA mutations; however, the history of translating promising biomarkers to clinical utility suggests that several major hurdles require careful consideration by the medical community. The first set of challenges involves nominating and verifying biomarkers. Candidate biomarkers need to discriminate disease from benign controls with high sensitivity and specificity for an intended use, which we describe as a two-tiered strategy of identifying and screening high-risk patients. Community-wide efforts to share samples, data, and analysis methods have been beneficial and progress meeting this challenge has been achieved. The second set of challenges is assay optimization and validating biomarkers. After initial candidate validation, assays need to be refined into accurate, cost-effective, highly reproducible, and multiplexed targeted panels and then validated in large cohorts. To move the most promising candidates forward, ideally, biomarker panels, head-to-head comparisons, meta-analysis, and assessment in independent data sets might mitigate risk of failure. Much more investment is needed to overcome these challenges. The third challenge is achieving clinical translation. To moonshot an early detection test to the clinic requires a large clinical trial and organizational, regulatory, and entrepreneurial know-how. Additional factors, such as imaging technologies, will likely need to improve concomitant with molecular biomarker development. The magnitude of the clinical translational challenge is uncertain, but interdisciplinary

Protein Biomarkers for Early Detection of Pancreatic Ductal Adenocarcinoma: Progress and Challenges

Directory of Open Access Journals (Sweden)

Alex Root

2018-03-01

Full Text Available Approximately 75% of patients with pancreatic ductal adenocarcinoma are diagnosed with advanced cancer, which cannot be safely resected. The most commonly used biomarker CA19-9 has inadequate sensitivity and specificity for early detection, which we define as Stage I/II cancers. Therefore, progress in next-generation biomarkers is greatly needed. Recent reports have validated a number of biomarkers, including combination assays of proteins and DNA mutations; however, the history of translating promising biomarkers to clinical utility suggests that several major hurdles require careful consideration by the medical community. The first set of challenges involves nominating and verifying biomarkers. Candidate biomarkers need to discriminate disease from benign controls with high sensitivity and specificity for an intended use, which we describe as a two-tiered strategy of identifying and screening high-risk patients. Community-wide efforts to share samples, data, and analysis methods have been beneficial and progress meeting this challenge has been achieved. The second set of challenges is assay optimization and validating biomarkers. After initial candidate validation, assays need to be refined into accurate, cost-effective, highly reproducible, and multiplexed targeted panels and then validated in large cohorts. To move the most promising candidates forward, ideally, biomarker panels, head-to-head comparisons, meta-analysis, and assessment in independent data sets might mitigate risk of failure. Much more investment is needed to overcome these challenges. The third challenge is achieving clinical translation. To moonshot an early detection test to the clinic requires a large clinical trial and organizational, regulatory, and entrepreneurial know-how. Additional factors, such as imaging technologies, will likely need to improve concomitant with molecular biomarker development. The magnitude of the clinical translational challenge is uncertain, but

Annotation and Curation of Uncharacterized proteins- Challenges

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Johny eIjaq

2015-03-01

Full Text Available Hypothetical Proteins are the proteins that are predicted to be expressed from an open reading frame (ORF, constituting a substantial fraction of proteomes in both prokaryotes and eukaryotes. Genome projects have led to the identification of many therapeutic targets, the putative function of the protein and their interactions. In this review we have enlisted various methods. Annotation linked to structural and functional prediction of hypothetical proteins assist in the discovery of new structures and functions serving as markers and pharmacological targets for drug designing, discovery and screening. Mass spectrometry is an analytical technique for validating protein characterisation. Matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS is an efficient analytical method. Microarrays and Protein expression profiles help understanding the biological systems through a systems-wide study of proteins and their interactions with other proteins and non-proteinaceous molecules to control complex processes in cells and tissues and even whole organism. Next generation sequencing technology accelerates multiple areas of genomics research.

Intramammary Immunization of Pregnant Mice with Staphylococcal Protein A Reduces the Post-Challenge Mammary Gland Bacterial Load but Not Pathology.

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Jully Gogoi-Tiwari

Full Text Available Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c or intramammary (i/mam routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05 higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05. There was significant reduction (p<0.05 in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion

A scored human protein-protein interaction network to catalyze genomic interpretation

DEFF Research Database (Denmark)

Li, Taibo; Wernersson, Rasmus; Hansen, Rasmus B

2017-01-01

Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap,......Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (In...

Addressing challenges of heterogeneous tumor treatment through bispecific protein-mediated pretargeted drug delivery.

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Yang, Qi; Parker, Christina L; McCallen, Justin D; Lai, Samuel K

2015-12-28

Tumors are frequently characterized by genomically and phenotypically distinct cancer cell subpopulations within the same tumor or between tumor lesions, a phenomenon termed tumor heterogeneity. These diverse cancer cell populations pose a major challenge to targeted delivery of diagnostic and/or therapeutic agents, as the conventional approach of conjugating individual ligands to nanoparticles is often unable to facilitate intracellular delivery to the full spectrum of cancer cells present in a given tumor lesion or patient. As a result, many cancers are only partially suppressed, leading to eventual tumor regrowth and/or the development of drug-resistant tumors. Pretargeting (multistep targeting) approaches involving the administration of 1) a cocktail of bispecific proteins that can collectively bind to the entirety of a mixed tumor population followed by 2) nanoparticles containing therapeutic and/or diagnostic agents that can bind to the bispecific proteins accumulated on the surface of target cells offer the potential to overcome many of the challenges associated with drug delivery to heterogeneous tumors. Despite its considerable success in improving the efficacy of radioimmunotherapy, the pretargeting strategy remains underexplored for a majority of nanoparticle therapeutic applications, especially for targeted delivery to heterogeneous tumors. In this review, we will present concepts in tumor heterogeneity, the shortcomings of conventional targeted systems, lessons learned from pretargeted radioimmunotherapy, and important considerations for harnessing the pretargeting strategy to improve nanoparticle delivery to heterogeneous tumors. Copyright © 2015 Elsevier B.V. All rights reserved.

Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans

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Tan, Li; Showalter, Allan M.; Egelund, Jack; Hernandez-Sanchez, Arianna; Doblin, Monika S.; Bacic, Antony

2012-01-01

Arabinogalactan-proteins (AGPs) are complex glycoconjugates that are commonly found at the cell surface and in secretions of plants. Their location and diversity of structures have made them attractive targets as modulators of plant development but definitive proof of their direct role(s) in biological processes remains elusive. Here we overview the current state of knowledge on AGPs, identify key challenges impeding progress in the field and propose approaches using modern bioinformatic, (bio)chemical, cell biological, molecular and genetic techniques that could be applied to redress these gaps in our knowledge. PMID:22754559

Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

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Ashutosh Kumar

2018-05-01

Full Text Available Exposure to (bisulfite (HSO3– and sulfite (SO32– has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bisulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–, peroxymonosulfate (–O3SOO., and especially the sulfate (SO4. – anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO, which has been shown to form protein radicals. Although formation of (bisulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bisulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bisulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bisulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bisulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bisulfite-derived protein radical formation and show the involvement of

Glutamate alleviates muscle protein loss by modulating TLR4, NODs, Akt/FOXO and mTOR signaling pathways in LPS-challenged piglets.

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Ping Kang

Full Text Available The experiment was conducted to study the effect of the glutamate (Glu on muscle protein loss through toll-like receptor 4 (TLR4, nucleotide-binding oligomerization domain proteins (NODs, Akt/Forkhead Box O (Akt/FOXO and mammalian target of rapamycin (mTOR signaling pathways in LPS-challenged piglets. Twenty-four weaned piglets were assigned into four treatments: (1 Control; (2 LPS+0% Glu; (3 LPS + 1.0% Glu; (4 LPS + 2.0% Glu. The experiment was lasted for 28 days. On d 28, the piglets in the LPS challenged groups were injected with LPS on 100 μg/kg body weight (BW, and the piglets in the control group were injected with the same volume of 0.9% NaCl solution. After 4 h LPS or saline injection, the piglets were slaughtered and the muscle samples were collected. Glu supplementation increased the protein/DNA ratio in gastrocnemius muscle, and the protein content in longissimus dorsi (LD muscle after LPS challenge (P<0.05. In addition, Glu supplementation decreased TLR4, IL-1 receptor-associated kinase (IRAK 1, receptor-interacting serine/threonine-protein kinase (RIPK 2, and nuclear factor-κB (NF-κB mRNA expression in gastrocnemius muscle (P<0.05, MyD88 mRNA expression in LD muscle, and FOXO1 mRNA expression in LD muscle (P<0.05. Moreover, Glu supplementation increased p-Akt/t-Akt ratio (P<0.05 in gastrocnemius muscle, and p-4EBP1/t-4EBP1 ratio in both gastrocnemius and LD muscles (P<0.05. Glu supplementation in the piglets' diets might be an effective strategy to alleviate LPS-induced muscle protein loss, which might be due to suppressing the mRNA expression of TLR4 and NODs signaling-related genes, and modulating Akt/FOXO and mTOR signaling pathways.

Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease

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Aims: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study,...

The Roles of Heat Shock Proteins 70 and 90 in Exopalaemon carinicauda After WSSV and Vibrio anguillarum Challenges

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Li, Jitao; Li, Jian; Duan, Yafei; Chen, Ping; Liu, Ping

2018-04-01

Heat shock proteins (HSPs), such as HSP70 and HSP90, are a suite of highly conserved proteins produced in all cellular organisms when they are exposed to stresses. In aquatic animals, they have been proved to play important roles in response to environmental pollutants and particularly in the non-specific immune responses to pathogen infections. In the present study, the expression profiles of HSP70 and HSP90 genes in hemocytes and hepatopancreas from the ridgetail white prawn Exopalaemon carinicauda infected with WSSV and Vibrio anguillarum were detected using reverse transcription polymerase chain reaction (RT-PCR). After WSSV challenge, the expression level of HSP 70 gene transcripts in the hemocytes and hepatopancreas increased to peak level at 6 h and 48 h, respectively. HSP90 gene transcripts in hemocytes and hepatopancreas were up-regulated significantly at 12 h and 6 h, respectively. During V. anguillarum challenge, the mRNA content of HSP70 gene in hemocytes and hepatopancreas increased significantly at 12 h and 6 h post-infection, respectively. The expression level of HSP90 gene both in hemocytes and hepatopancreas were up-regulated in the first 3 h. The expression patterns of HSP70 and HSP90 genes in hemocytes and hepatopancreas showed temporal and spatial differences after challenged with WSSV and V. anguillarum. The results suggested that HSPs might be involved in immune responses to pathogens challenge in E. carinicauda.

Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

DEFF Research Database (Denmark)

Nguyen, E.D.; Meiler, J.; Norn, C.

2013-01-01

screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone...... extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves...

A Neospora caninum vaccine using recombinant proteins fails to prevent foetal infection in pregnant cattle after experimental intravenous challenge.

Science.gov (United States)

Hecker, Yanina P; Cóceres, Verónica; Wilkowsky, Silvina E; Jaramillo Ortiz, José M; Morrell, Eleonora L; Verna, Andrea E; Ganuza, Agustina; Cano, Dora B; Lischinsky, Lilian; Angel, Sergio O; Zamorano, Patricia; Odeón, Anselmo C; Leunda, María R; Campero, Carlos M; Morein, Bror; Moore, Dadín P

2014-12-15

The aim of the present study was to evaluate the immunogenicity and protective efficacy of rNcSAG1, rNcHSP20 and rNcGRA7 recombinant proteins formulated with immune stimulating complexes (ISCOMs) in pregnant heifers against vertical transmission of Neospora caninum. Twelve pregnant heifers were divided into 3 groups of 4 heifers each, receiving different formulations before mating. Immunogens were administered twice subcutaneously: group A animals were inoculated with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) formulated with ISCOMs; group B animals received ISCOM-MATRIX (without antigen) and group C received sterile phosphate-buffered saline (PBS) only. The recombinant proteins were expressed in Escherichia coli and purified nickel resin. All groups were intravenously challenged with the NC-1 strain of N. caninum at Day 70 of gestation and dams slaughtered at week 17 of the experiment. Heifers from group A developed specific antibodies against rNcSAG1, rNcHSP20 and rNcGRA7 prior to the challenge. Following immunization, an statistically significant increase of antibodies against rNcSAG1 and rNcHSP20 in all animals of group A was detected compared to animals in groups B and C at weeks 5, 13 and 16 (P0.001). There were no differences in IFN-γ production among the experimental groups at any time point (P>0.05). Transplacental transmission was determined in all foetuses of groups A, B and C by Western blot, immunohistochemistry and nested PCR. This work showed that rNcSAG1, rNcHSP20 and rNcGRA7 proteins while immunogenic in cattle failed to prevent the foetal infection in pregnant cattle challenged at Day 70 of gestation. Copyright © 2014 Elsevier B.V. All rights reserved.

Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita

Directory of Open Access Journals (Sweden)

Jose T. A. Oliveira

2014-11-01

Full Text Available The root knot nematodes (RKN, Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31 inoculated with M. incognita (Race 3 in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4–6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, β-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.

A heterogeneous fleet vehicle routing model for solving the LPG distribution problem: A case study

International Nuclear Information System (INIS)

Onut, S; Kamber, M R; Altay, G

2014-01-01

Vehicle Routing Problem (VRP) is an important management problem in the field of distribution and logistics. In VRPs, routes from a distribution point to geographically distributed points are designed with minimum cost and considering customer demands. All points should be visited only once and by one vehicle in one route. Total demand in one route should not exceed the capacity of the vehicle that assigned to that route. VRPs are varied due to real life constraints related to vehicle types, number of depots, transportation conditions and time periods, etc. Heterogeneous fleet vehicle routing problem is a kind of VRP that vehicles have different capacity and costs. There are two types of vehicles in our problem. In this study, it is used the real world data and obtained from a company that operates in LPG sector in Turkey. An optimization model is established for planning daily routes and assigned vehicles. The model is solved by GAMS and optimal solution is found in a reasonable time

A heterogeneous fleet vehicle routing model for solving the LPG distribution problem: A case study

Science.gov (United States)

Onut, S.; Kamber, M. R.; Altay, G.

2014-03-01

Vehicle Routing Problem (VRP) is an important management problem in the field of distribution and logistics. In VRPs, routes from a distribution point to geographically distributed points are designed with minimum cost and considering customer demands. All points should be visited only once and by one vehicle in one route. Total demand in one route should not exceed the capacity of the vehicle that assigned to that route. VRPs are varied due to real life constraints related to vehicle types, number of depots, transportation conditions and time periods, etc. Heterogeneous fleet vehicle routing problem is a kind of VRP that vehicles have different capacity and costs. There are two types of vehicles in our problem. In this study, it is used the real world data and obtained from a company that operates in LPG sector in Turkey. An optimization model is established for planning daily routes and assigned vehicles. The model is solved by GAMS and optimal solution is found in a reasonable time.

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans.

Science.gov (United States)

Atzingen, Marina V; Gonçales, Amane P; de Morais, Zenaide M; Araújo, Eduardo R; De Brito, Thales; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-09-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Protein nanoparticles for therapeutic protein delivery.

Science.gov (United States)

Herrera Estrada, L P; Champion, J A

2015-06-01

Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

Comparison of Stem Map Developed from Crown Geometry Allometry Linked Census Data to Airborne and Terrestrial Lidar at Harvard Forest, MA

Science.gov (United States)

Sullivan, F.; Palace, M. W.; Ducey, M. J.; David, O.; Cook, B. D.; Lepine, L. C.

2014-12-01

Harvard Forest in Petersham, MA, USA is the location of one of the temperate forest plots established by the Center for Tropical Forest Science (CTFS) as a joint effort with Harvard Forest and the Smithsonian Institute's Forest Global Earth Observatory (ForestGEO) to characterize ecosystem processes and forest dynamics. Census of a 35 ha plot on Prospect Hill was completed during the winter of 2014 by researchers at Harvard Forest. Census data were collected according to CTFS protocol; measured variables included species, stem diameter, and relative X-Y locations. Airborne lidar data were collected over the censused plot using the high spatial resolution Goddard LiDAR, Hyperspectral, and Thermal sensor package (G-LiHT) during June 2012. As part of a separate study, 39 variable radius plots (VRPs) were randomly located and sampled within and throughout the Prospect Hill CTFS/ForestGEO plot during September and October 2013. On VRPs, biometric properties of trees were sampled, including species, stem diameter, total height, crown base height, crown radii, and relative location to plot centers using a 20 Basal Area Factor prism. In addition, a terrestrial-based lidar scanner was used to collect one lidar scan at plot center for 38 of the 39 VRPs. Leveraging allometric equations of crown geometry and tree height developed from 374 trees and 16 different species sampled on 39 VRPs, a 3-dimensional stem map will be created using the Harvard Forest ForestGEO Prospect Hill census. Vertical and horizontal structure of 3d field-based stem maps will be compared to terrestrial and airborne lidar scan data. Furthermore, to assess the quality of allometric equations, a 2d canopy height raster of the field-based stem map will be compared to a G-LiHT derived canopy height model for the 35 ha census plot. Our automated crown delineation methods will be applied to the 2d representation of the census stem map and the G-LiHT canopy height model. For future work related to this study

Algal Proteins: Extraction, Application, and Challenges Concerning Production

Directory of Open Access Journals (Sweden)

Stephen Bleakley

2017-04-01

Full Text Available Population growth combined with increasingly limited resources of arable land and fresh water has resulted in a need for alternative protein sources. Macroalgae (seaweed and microalgae are examples of under-exploited “crops”. Algae do not compete with traditional food crops for space and resources. This review details the characteristics of commonly consumed algae, as well as their potential for use as a protein source based on their protein quality, amino acid composition, and digestibility. Protein extraction methods applied to algae to date, including enzymatic hydrolysis, physical processes, and chemical extraction and novel methods such as ultrasound-assisted extraction, pulsed electric field, and microwave-assisted extraction are discussed. Moreover, existing protein enrichment methods used in the dairy industry and the potential of these methods to generate high value ingredients from algae, such as bioactive peptides and functional ingredients are discussed. Applications of algae in human nutrition, animal feed, and aquaculture are examined.

Algal Proteins: Extraction, Application, and Challenges Concerning Production

Science.gov (United States)

Bleakley, Stephen; Hayes, Maria

2017-01-01

Population growth combined with increasingly limited resources of arable land and fresh water has resulted in a need for alternative protein sources. Macroalgae (seaweed) and microalgae are examples of under-exploited “crops”. Algae do not compete with traditional food crops for space and resources. This review details the characteristics of commonly consumed algae, as well as their potential for use as a protein source based on their protein quality, amino acid composition, and digestibility. Protein extraction methods applied to algae to date, including enzymatic hydrolysis, physical processes, and chemical extraction and novel methods such as ultrasound-assisted extraction, pulsed electric field, and microwave-assisted extraction are discussed. Moreover, existing protein enrichment methods used in the dairy industry and the potential of these methods to generate high value ingredients from algae, such as bioactive peptides and functional ingredients are discussed. Applications of algae in human nutrition, animal feed, and aquaculture are examined. PMID:28445408

Ranking docking poses by graph matching of protein-ligand interactions: lessons learned from the D3R Grand Challenge 2

Science.gov (United States)

da Silva Figueiredo Celestino Gomes, Priscila; Da Silva, Franck; Bret, Guillaume; Rognan, Didier

2018-01-01

A novel docking challenge has been set by the Drug Design Data Resource (D3R) in order to predict the pose and affinity ranking of a set of Farnesoid X receptor (FXR) agonists, prior to the public release of their bound X-ray structures and potencies. In a first phase, 36 agonists were docked to 26 Protein Data Bank (PDB) structures of the FXR receptor, and next rescored using the in-house developed GRIM method. GRIM aligns protein-ligand interaction patterns of docked poses to those of available PDB templates for the target protein, and rescore poses by a graph matching method. In agreement with results obtained during the previous 2015 docking challenge, we clearly show that GRIM rescoring improves the overall quality of top-ranked poses by prioritizing interaction patterns already visited in the PDB. Importantly, this challenge enables us to refine the applicability domain of the method by better defining the conditions of its success. We notably show that rescoring apolar ligands in hydrophobic pockets leads to frequent GRIM failures. In the second phase, 102 FXR agonists were ranked by decreasing affinity according to the Gibbs free energy of the corresponding GRIM-selected poses, computed by the HYDE scoring function. Interestingly, this fast and simple rescoring scheme provided the third most accurate ranking method among 57 contributions. Although the obtained ranking is still unsuitable for hit to lead optimization, the GRIM-HYDE scoring scheme is accurate and fast enough to post-process virtual screening data.

Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

Science.gov (United States)

Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

2014-12-05

The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Scoring functions for protein-protein interactions.

Science.gov (United States)

Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

2013-12-01

The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

Directory of Open Access Journals (Sweden)

Scaloni Andrea

2007-08-01

Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

Whey protein concentrate enhances intestinal integrity and influences transforming growth factor-β1 and mitogen-activated protein kinase signalling pathways in piglets after lipopolysaccharide challenge.

Science.gov (United States)

Xiao, Kan; Jiao, Lefei; Cao, Shuting; Song, Zehe; Hu, Caihong; Han, Xinyan

2016-03-28

Whey protein concentrate (WPC) has been reported to have protective effects on the intestinal barrier. However, the molecular mechanisms involved are not fully elucidated. Transforming growth factor-β1 (TGF-β1) is an important component in the WPC, but whether TGF-β1 plays a role in these processes is not clear. The aim of this study was to investigate the protective effects of WPC on the intestinal epithelial barrier as well as whether TGF-β1 is involved in these protection processes in a piglet model after lipopolysaccharide (LPS) challenge. In total, eighteen weanling pigs were randomly allocated to one of the following three treatment groups: (1) non-challenged control and control diet; (2) LPS-challenged control and control diet; (3) LPS+5 %WPC diet. After 19 d of feeding with control or 5 %WPC diets, pigs were injected with LPS or saline. At 4 h after injection, pigs were killed to harvest jejunal samples. The results showed that WPC improved (Pprotein, phosphorylated-Smad2 expression and Smad4 and Smad7 mRNA expressions and decreased (Pprotein kinase signalling pathways.

Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

Science.gov (United States)

Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

2017-08-01

The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

Imaging protein-protein interactions in living cells

NARCIS (Netherlands)

Hink, M.A.; Bisseling, T.; Visser, A.J.W.G.

2002-01-01

The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide

Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine

NARCIS (Netherlands)

Wisselink, H.J.; Vecht, U.; Stockhofe Zurwieden, N.; Smith, H.E.

2001-01-01

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP EF ) was compared with the efficacy of a vaccine containing

Composition of Overlapping Protein-Protein and Protein-Ligand Interfaces.

Directory of Open Access Journals (Sweden)

Ruzianisra Mohamed

Full Text Available Protein-protein interactions (PPIs play a major role in many biological processes and they represent an important class of targets for therapeutic intervention. However, targeting PPIs is challenging because often no convenient natural substrates are available as starting point for small-molecule design. Here, we explored the characteristics of protein interfaces in five non-redundant datasets of 174 protein-protein (PP complexes, and 161 protein-ligand (PL complexes from the ABC database, 436 PP complexes, and 196 PL complexes from the PIBASE database and a dataset of 89 PL complexes from the Timbal database. In all cases, the small molecule ligands must bind at the respective PP interface. We observed similar amino acid frequencies in all three datasets. Remarkably, also the characteristics of PP contacts and overlapping PL contacts are highly similar.

A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces

NARCIS (Netherlands)

Melo, Rita; Fieldhouse, Robert; Melo, André; Correia, João D G; Cordeiro, Maria Natália D S; Gümüş, Zeynep H; Costa, Joaquim; Bonvin, Alexandre M J J; de Sousa Moreira, Irina

2016-01-01

Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS) in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML) techniques. Our model

Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

Science.gov (United States)

Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

2014-11-01

Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets

International Nuclear Information System (INIS)

Bellstedt, Peter; Seiboth, Thomas; Häfner, Sabine; Kutscha, Henriette; Ramachandran, Ramadurai; Görlach, Matthias

2013-01-01

NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ('scrambling”) of NH and CO groups in a standard Escherichia coli expression host is provided

Challenges in the Development of Functional Assays of Membrane Proteins

Directory of Open Access Journals (Sweden)

Sophie Demarche

2012-11-01

Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Functional aspects of protein flexibility

DEFF Research Database (Denmark)

Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

2009-01-01

this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. The present review addresses protein flexibility, focusing on protein-ligand interactions...

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015.

Science.gov (United States)

Deng, Nanjie; Flynn, William F; Xia, Junchao; Vijayan, R S K; Zhang, Baofeng; He, Peng; Mentes, Ahmet; Gallicchio, Emilio; Levy, Ronald M

2016-09-01

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate

Integrated cancer therapy combined radiotherapy and immunotherapy. The challenge of using Gc protein-derived macrophage activating factor (GcMAF) as a key molecule

International Nuclear Information System (INIS)

Uto, Yoshihiro; Hori, Hitoshi

2013-01-01

Radiation oncologists know the conflict between radiotherapy and immunotherapy, but now challenged trails of the integrative cancer therapies combined radiation therapy and various immunoreaction/immune therapies begin. We therefore review the recent results of basic research and clinical trial of the integrated cancer therapies which combined radiotherapy and various immune therapies/immunoreaction, and the challenged studies of combined use of radiotherapy and our developed cancer immunotherapy using serum GcMAF which is human serum containing Gc protein-derived macrophage activating factor (GcMAF). (author)

Combining random gene fission and rational gene fusion to discover near-infrared fluorescent protein fragments that report on protein-protein interactions.

Science.gov (United States)

Pandey, Naresh; Nobles, Christopher L; Zechiedrich, Lynn; Maresso, Anthony W; Silberg, Jonathan J

2015-05-15

Gene fission can convert monomeric proteins into two-piece catalysts, reporters, and transcription factors for systems and synthetic biology. However, some proteins can be challenging to fragment without disrupting function, such as near-infrared fluorescent protein (IFP). We describe a directed evolution strategy that can overcome this challenge by randomly fragmenting proteins and concomitantly fusing the protein fragments to pairs of proteins or peptides that associate. We used this method to create libraries that express fragmented IFP as fusions to a pair of associating peptides (IAAL-E3 and IAAL-K3) and proteins (CheA and CheY) and screened for fragmented IFP with detectable near-infrared fluorescence. Thirteen novel fragmented IFPs were identified, all of which arose from backbone fission proximal to the interdomain linker. Either the IAAL-E3 and IAAL-K3 peptides or CheA and CheY proteins could assist with IFP fragment complementation, although the IAAL-E3 and IAAL-K3 peptides consistently yielded higher fluorescence. These results demonstrate how random gene fission can be coupled to rational gene fusion to create libraries enriched in fragmented proteins with AND gate logic that is dependent upon a protein-protein interaction, and they suggest that these near-infrared fluorescent protein fragments will be suitable as reporters for pairs of promoters and protein-protein interactions within whole animals.

Empirical investigations into full-text protein interaction Article Categorization Task (ACT) in the BioCreative II.5 Challenge.

Science.gov (United States)

Lan, Man; Su, Jian

2010-01-01

The selection of protein interaction documents is one important application for biology research and has a direct impact on the quality of downstream BioNLP applications, i.e., information extraction and retrieval, summarization, QA, etc. The BioCreative II.5 Challenge Article Categorization task (ACT) involves doing a binary text classification to determine whether a given structured full-text article contains protein interaction information. This may be the first attempt at classification of full-text protein interaction documents in wide community. In this paper, we compare and evaluate the effectiveness of different section types in full-text articles for text classification. Moreover, in practice, the less number of true-positive samples results in unstable performance and unreliable classifier trained on it. Previous research on learning with skewed class distributions has altered the class distribution using oversampling and downsampling. We also investigate the skewed protein interaction classification and analyze the effect of various issues related to the choice of external sources, oversampling training sets, classifiers, etc. We report on the various factors above to show that 1) a full-text biomedical article contains a wealth of scientific information important to users that may not be completely represented by abstracts and/or keywords, which improves the accuracy performance of classification and 2) reinforcing true-positive samples significantly increases the accuracy and stability performance of classification.

Effects of maternal protein or energy restriction during late gestation on immune status and responses to lipopolysaccharide challenge in postnatal young goats.

Science.gov (United States)

He, Z X; Sun, Z H; Yang, W Z; Beauchemin, K A; Tang, S X; Zhou, C S; Han, X F; Wang, M; Kang, J H; Tan, Z L

2014-11-01

Knowledge of maternal malnutrition of ruminants and effects on development of the immune system of their offspring is lacking. A study was conducted to investigate the effects of maternal protein or energy restriction during late gestation on immune status of their offspring at different ages. Sixty-three pregnant goats (local breed, Liuyang black goat, 22.2 ± 1.5 kg at d 90 of gestation) were fed control (CON, ME = 9.34 MJ/kg and CP = 12.5%, DM basis), 40% protein restricted (PR), or 40% energy restricted (ER) diets from d 91 of gestation to parturition, after which all animals received an adequate diet for nutritional recovery. Plasma concentrations of complement components (C3, C4), C-reactive protein (CRP) and immunoglobulins (IgG and IgM), jejunum cytokines (IL-2, IL-6, and IL-10) expression levels and morphology in the offspring were measured. Additionally, plasma concentration of complement and IL-6, and cytokines expression levels in gastrointestinal tract obtained at 6 wk from young goats were assessed under saline or lipopolysaccharide (LPS) challenging conditions. Maternal PR or ER decreased (P 0.05) plasma CRP concentration. The IL-10 mRNA expression of jejunum from PR kids was also less (P 0.05) in any plasma or tissue immune parameters among the 3 treatments. However, when given a LPS challenge, ER and PR kids had greater (P = 0.02) IL-6 concentration compared with CON kids. Our results suggest that both PR and ER during late gestation induced short-term as well as long-lasting alterations on immune responses in their offspring, which may make the animals more susceptible to a bacterial pathogen challenge. The present findings expand the existing knowledge in immunological mechanisms responsible for the development of disease in later life.

Different expression patterns of renal Na+/K+-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

Science.gov (United States)

Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

2016-12-01

Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na + /K + -ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences. Copyright © 2016 Elsevier Inc. All rights reserved.

Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

Science.gov (United States)

de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

2015-04-01

Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

Directory of Open Access Journals (Sweden)

Marina De Filette

Full Text Available West Nile virus (WNV is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime--boost regime with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical. In parallel a heterologous boost with purified recombinant WNV envelope (E protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8(+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.

Proteomic investigation of Vibrio alginolyticus challenged Caenorhabditis elegans revealed regulation of cellular homeostasis proteins and their role in supporting innate immune system.

Science.gov (United States)

Durai, Sellegounder; Singh, Nirpendra; Kundu, Suman; Balamurugan, Krishnaswamy

2014-08-01

Caenorhabditis elegans has been the preferred model system for many investigators to study pathogenesis. In the present investigation, regulation of C. elegans proteome was explored against V. alginolyticus infection using quantitative proteomics approach. Proteins were separated using 2D-DIGE and the differentially regulated proteins were identified using PMF and MALDI TOF/TOF analysis. The results thus obtained were validated using Western blotting for candidate proteins. The corresponding transcriptional regulation was quantified subsequently using real-time PCR. Interaction network for candidate proteins was predicted using search tool for the retrieval of interacting genes/proteins (STRING) and functional validation was performed using respective mutant strains. Out of the 25 proteins identified, 21 proteins appeared to be upregulated while four were downregulated. Upregulated proteins included those involved in stress-response (PDI-2, HSP-6), immune-response (protein kinase -18, GST-8) and energy-production (ATP-2) while proteins involved in structural maintenance (IFB-2) and lipid metabolism (SODH-1) were downregulated. The roles of these players in the host system during Vibrio infection was analyzed in vivo using wild type and mutant C. elegans. Survival assays using mutants lacking pdi-2, ire-1, and xbp-1 displayed enhanced susceptibility to V. alginolyticus. Cellular stress generated by V. alginolyticus was determined using ROS assay. This is the first report of proteome changes in C. elegans against V. alginolyticus challenge and highlights the significance of unfolded protein response (UPR) pathway during bacterial infection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

D3R Grand Challenge 2: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies

Science.gov (United States)

Gaieb, Zied; Liu, Shuai; Gathiaka, Symon; Chiu, Michael; Yang, Huanwang; Shao, Chenghua; Feher, Victoria A.; Walters, W. Patrick; Kuhn, Bernd; Rudolph, Markus G.; Burley, Stephen K.; Gilson, Michael K.; Amaro, Rommie E.

2018-01-01

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank (http://www.pdb.org), and in affinity ranking and scoring of bound ligands.

Inferring domain-domain interactions from protein-protein interactions with formal concept analysis.

Directory of Open Access Journals (Sweden)

Susan Khor

Full Text Available Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains.

Inferring Domain-Domain Interactions from Protein-Protein Interactions with Formal Concept Analysis

Science.gov (United States)

Khor, Susan

2014-01-01

Identifying reliable domain-domain interactions will increase our ability to predict novel protein-protein interactions, to unravel interactions in protein complexes, and thus gain more information about the function and behavior of genes. One of the challenges of identifying reliable domain-domain interactions is domain promiscuity. Promiscuous domains are domains that can occur in many domain architectures and are therefore found in many proteins. This becomes a problem for a method where the score of a domain-pair is the ratio between observed and expected frequencies because the protein-protein interaction network is sparse. As such, many protein-pairs will be non-interacting and domain-pairs with promiscuous domains will be penalized. This domain promiscuity challenge to the problem of inferring reliable domain-domain interactions from protein-protein interactions has been recognized, and a number of work-arounds have been proposed. This paper reports on an application of Formal Concept Analysis to this problem. It is found that the relationship between formal concepts provides a natural way for rare domains to elevate the rank of promiscuous domain-pairs and enrich highly ranked domain-pairs with reliable domain-domain interactions. This piggybacking of promiscuous domain-pairs onto less promiscuous domain-pairs is possible only with concept lattices whose attribute-labels are not reduced and is enhanced by the presence of proteins that comprise both promiscuous and rare domains. PMID:24586450

Challenges in biotechnology at LLNL: from genes to proteins; TOPICAL

International Nuclear Information System (INIS)

Albala, J S

1999-01-01

This effort has undertaken the task of developing a link between the genomics, DNA repair and structural biology efforts within the Biology and Biotechnology Research Program at LLNL. Through the advent of the I.M.A.G.E. (Integrated Molecular Analysis of Genomes and their Expression) Consortium, a world-wide effort to catalog the largest public collection of genes, accepted and maintained within BBRP, it is now possible to systematically express the protein complement of these to further elucidate novel gene function and structure. The work has ensued in four phases, outlined as follows: (1) Gene and System selection; (2) Protein expression and purification; (3) Structural analysis; and (4) biological integration. Proteins to be expressed have been those of high programmatic interest. This includes, in particular, proteins involved in the maintenance of genome integrity, particularly those involved in the repair of DNA damage, including ERCC1, ERCC4, XRCC2, XRCC3, XRCC9, HEX1, APN1, p53, RAD51B, RAD51C, and RAD51. Full-length cDNA cognates of selected genes were isolated, and cloned into baculovirus-based expression vectors. The baculoviral expression system for protein over-expression is now well-established in the Albala laboratory. Procedures have been successfully optimized for full-length cDNA clining into expression vectors for protein expression from recombinant constructs. This includes the reagents, cell lines, techniques necessary for expression of recombinant baculoviral constructs in Spodoptera frugiperda (Sf9) cells. The laboratory has also generated a high-throughput baculoviral expression paradigm for large scale expression and purification of human recombinant proteins amenable to automation

Protein docking prediction using predicted protein-protein interface

Directory of Open Access Journals (Sweden)

Li Bin

2012-01-01

Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Protein docking prediction using predicted protein-protein interface.

Science.gov (United States)

Li, Bin; Kihara, Daisuke

2012-01-10

Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

Recombinant M. bovis BCG expressing the PLD protein promotes survival in mice challenged with a C. pseudotuberculosis virulent strain.

Science.gov (United States)

Leal, Karen Silva; de Oliveira Silva, Mara Thais; de Fátima Silva Rezende, Andréa; Bezerra, Francisco Silvestre Brilhante; Begnini, Karine; Seixas, Fabiana; Collares, Tiago; Dellagostin, Odir; Portela, Ricardo Wagner; de Carvalho Azevedo, Vasco Ariston; Borsuk, Sibele

2018-06-14

The aim of this study was to evaluate the survival of mice inoculated with M. bovis BCG Pasteur recombinant expressing the PLD protein and challenged with a C. pseudotuberculosis virulent strain. Four groups were immunized with a sterile 0.9% saline solution (G1), 10 6  CFU of M. bovis BCG Pasteur (G2), 10 6  CFU of M. bovis BCG/pld (G3) or 10 6  CFU of M. bovis BCG/pld with a booster with rPLD (G4) and challenged with 10 4 CFU of C. pseudotuberculosis MIC-6 strain. The highest survival rate of 88% was observed in G4, followed by 77% in G3 and 66% in G2. A significant statistical difference was observed in the levels of cytokines IFN-γ and IL-10 in vaccinated groups (G3 and G4) when compared with the control group (G1) (p < 0.05). The results seem promising as the recombinant vaccine elicited a cellular immune response and provided significant survival after a high virulent challenge. Copyright © 2018 Elsevier Ltd. All rights reserved.

Challenges in the local delivery of peptides and proteins for oral mucositis management.

Science.gov (United States)

Campos, João C; Cunha, João D; Ferreira, Domingos C; Reis, Salette; Costa, Paulo J

2018-04-24

Oral mucositis, a common inflammatory side effect of oncological treatments, is a disorder of the oral mucosa that can cause painful ulcerations, local motor disabilities, and an increased risk of infections. Due to the discomfort it produces and the associated health risks, it can lead to cancer treatment restrains, such as the need for dose reduction, cycle delays or abandonment. Current mucositis management has low efficiency in prevention and treatment. A topical drug application for a local action can be a more effective approach than systemic routes when addressing oral cavity pathologies. Local delivery of growth factors, antibodies, and anti-inflammatory cytokines have shown promising results. However, due to the peptide and protein nature of these novel agents, and the several anatomic, physiological and environmental challenges of the oral cavity, their local action might be limited when using traditional delivering systems. This review is an awareness of the issues and strategies in the local delivery of macromolecules for the management of oral mucositis. Copyright © 2018. Published by Elsevier B.V.

An Efficient Two-Objective Hybrid Local Search Algorithm for Solving the Fuel Consumption Vehicle Routing Problem

OpenAIRE

Rao, Weizhen; Liu, Feng; Wang, Shengbin

2016-01-01

The classical model of vehicle routing problem (VRP) generally minimizes either the total vehicle travelling distance or the total number of dispatched vehicles. Due to the increased importance of environmental sustainability, one variant of VRPs that minimizes the total vehicle fuel consumption has gained much attention. The resulting fuel consumption VRP (FCVRP) becomes increasingly important yet difficult. We present a mixed integer programming model for the FCVRP, and fuel consumption is ...

An Adjuvanted A(H5N1) Subvirion Vaccine Elicits Virus-Specific Antibody Response and Improves Protection Against Lethal Influenza Viral Challenge in Mouse Model of Protein Energy Malnutrition.

Science.gov (United States)

Jones, Enitra N; Amoah, Samuel; Cao, Weiping; Sambhara, Suryaprakash; Gangappa, Shivaprakash

2017-09-15

Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including influenza infection, but no studies have addressed the potential influences of PEM on the immunogenicity and protective efficacy of avian influenza A(H5N1) vaccine. We investigated the role of PEM on vaccine-mediated protection after a lethal challenge with recombinant A(H5N1) virus using isocaloric diets providing either adequate protein (AP; 18% protein) or very low protein (VLP; 2% protein) in an established murine model of influenza vaccination. We demonstrated that mice maintained on a VLP diet succumb to lethal challenge at greater rates than mice maintained on an AP diet, despite comparable immunization regimens. Importantly, there was no virus-induced mortality in both VLP and AP groups of mice when either group was immunized with adjuvanted low-dose A(H5N1) subvirion vaccine. Our results suggest that adjuvanted vaccination in populations where PEM is endemic may be one strategy to boost vaccination-promoted immunity and improve outcomes associated with highly pathogenic A(H5N1). Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Preparing and evaluating delivery systems for proteins

DEFF Research Database (Denmark)

Jorgensen, L; Moeller, E H; van de Weert, M

2006-01-01

From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...... and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge...... for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions....

CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction

KAUST Repository

Cui, Xuefeng

2016-06-15

Motivation: Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. Method: We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence–structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. Results: We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM–HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods.

Vehicle Routing Problem for Fashion Supply Chains with Cross-Docking

Directory of Open Access Journals (Sweden)

Zhi-Hua Hu

2013-01-01

Full Text Available Cross-docking, as a strategy to reduce lead time and enhance the efficiency of the fashion supply chain, has attracted substantial attention from both the academy and the industry. Cross-docking is a critical part of many fashion and textiles supply chains in practice because it can help to achieve many supply chain strategies such as postponement. We consider a model where there are multiple suppliers and customers in a single cross-docking center. With such a model setting, the issue concerning the coordinated routing between the inbound and outbound routes is much more complex than many traditional vehicle routing problems (VRPs. We formulate the optimal route selection problems from the suppliers to the cross-docking center and from the cross-docking center to the customers as the respective VRPs. Based on the relationships between the suppliers and the customers, we integrate the two VRP models to optimize the overall traveling time, distance, and waiting time at the cross-docking center. In addition, we propose a novel mixed 0/1 integer linear programming model by which the complexity of the problem can be reduced significantly. As demonstrated by the simulation analysis, our proposed model can be solved very efficiently by a commonly used optimization software package.

Detection of protein complex from protein-protein interaction network using Markov clustering

International Nuclear Information System (INIS)

Ochieng, P J; Kusuma, W A; Haryanto, T

2017-01-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks. (paper)

Cytokine profile after oral food challenge in infants with food protein-induced enterocolitis syndrome.

Science.gov (United States)

Kimura, Mitsuaki; Ito, Yasunori; Shimomura, Masaki; Morishita, Hideaki; Meguro, Takaaki; Adachi, Yuichi; Seto, Shiro

2017-07-01

Although food protein-induced enterocolitis syndrome (FPIES) is supposed to be caused by inflammation, the role of cytokines has not yet been clarified. To elucidate the role of cytokines in the development of symptoms and abnormal laboratory findings at an oral food challenge (OFC), changes in serum cytokine levels were analyzed for 6 OFCs in 4 patients with FPIES. The result of OFC was judged positive if any gastrointestinal (GI) symptoms (vomiting, diarrhea, or bloody stool) were induced. Among 11 cytokines profiled, serum levels of interleukin (IL)-2, IL-5, and IL-8 were clearly increased in all 4 positive OFCs in which elevations of the serum level of C-reactive protein (CRP) and peripheral blood neutrophilia were also seen. The level of serum IL-10 also rose in 2 positive OFCs. Remarkable increases in the serum level of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-6, and IL-12 were observed in a positive OFC where the serum level of CRP rose markedly (6.75 mg/dL). The serum levels of IL-5 were also elevated in 2 negative OFCs. No apparent specific correlations were found between cytokines and GI symptoms. These results suggest that IL-2 and IL-8 are involved in the antigen-specific immune responses in most patients with FPIES. Further studies are needed to elucidate the significance of these cytokine in the pathogenesis of FPIES. Copyright © 2016 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

Large, dynamic, multi-protein complexes: a challenge for structural biology

Czech Academy of Sciences Publication Activity Database

Rozycki, B.; Bouřa, Evžen

2014-01-01

Roč. 26, č. 46 (2014), 463103/1-463103/11 ISSN 0953-8984 R&D Projects: GA MŠk LO1302 EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : protein structure * multi-protein complexes * hybrid methods of structural biology Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.346, year: 2014

Chimeric vaccine composed of viral peptide and mammalian heat-shock protein 60 peptide protects against West Nile virus challenge.

Science.gov (United States)

Gershoni-Yahalom, Orly; Landes, Shimon; Kleiman-Shoval, Smadar; Ben-Nathan, David; Kam, Michal; Lachmi, Bat-El; Khinich, Yevgeny; Simanov, Michael; Samina, Itzhak; Eitan, Anat; Cohen, Irun R; Rager-Zisman, Bracha; Porgador, Angel

2010-08-01

The protective efficacy and immunogenicity of a chimeric peptide against West Nile virus (WNV) was evaluated. This virus is the aetiological agent of West Nile fever, which has recently emerged in the western hemisphere. The rapid spread of WNV throughout North America, as well as the constantly changing epidemiology and transmission of the virus by blood transfusion and transplantation, have raised major public-health concerns. Currently, there are no effective treatments for WNV or vaccine for human use. We previously identified a novel, continuous B-cell epitope from domain III of the WNV envelope protein, termed Ep15. To test whether this epitope can protect against WNV infection, we synthesized a linear chimeric peptide composed of Ep15 and the heat-shock protein 60 peptide, p458. The p458 peptide is an effective carrier peptide for subunit vaccines against other infectious agents. We now report that mice immunized with the chimeric peptide, p458-Ep15, were resistant to lethal challenges with three different WNV strains. Moreover, their brains were free of viral genome and infectious virus. Mice immunized with Ep15 alone or with p431-Ep15, a control conjugate, were not protected. The chimeric p458-Ep15 peptide induced WNV-specific immunoglobulin G antibodies that neutralized the virus and induced the secretion of interferon-gammain vitro. Challenge of chimeric peptide-immunized mice considerably enhanced WNV-specific neutralizing antibodies. We conclude that this chimeric peptide can be used for formulation of a human vaccine against WNV.

Challenges in pKa Predictions for Proteins: The case of Asp213 in Human Proteinase 3

Science.gov (United States)

Hajjar, Eric; Dejaegere, Annick; Reuter, Nathalie

2009-09-01

Knowledge of the protonation states of the ionizable residues in an enzyme is a prerequisite to an accurate description of its structure and mechanism. In practice, the use of the inappropriate protonation state for an amino acid in a molecular modeling computation (e.g., molecular dynamics simulation) is likely to lead to unrealistic results. Although methods using solvers of the linearized Poisson-Boltzmann equation have proven to yield accurate pKa predictions, they bear a number of limitations. They are quite demanding in terms of computational power and are sensitive to representation of the charges and their position (force field and protein conformation). Moreover they depend on the choice of a dielectric constant for the protein interior. In this manuscript, we describe the difficulties met when trying to predict the protonation state of a buried amino acid, located in a protein for which very little biochemical data is available. Such a case is highly representative of the challenges faced in theoretical biology studies. Proteinase 3 (PR3) is an enzyme involved in proteolytic events associated with inflammation. It is a potential target in the development of new anti-inflammatory therapeutic strategies. We report the results of pKa predictions of the aspartic acid 213 of PR3 with a FDPB solver. We probed the influence of the choice of the dielectric constant for the protein interior ɛp and the benefits of conformational sampling by molecular dynamics (MD) on the pKa prediction of this carboxylate group. Using only the FDPB calculations, we could not conclude on the protonation state of Asp213. MD simulations confronted to knowledge of the ligand-binding and reaction mechanism led us to decide on a protonated form of this aspartic acid. We also demonstrate that the use of the wrong protonation state leads to an unreliable structural model for PR3. pKa prediction with a fast empirical method yielded a pKa of 8.4 for Asp213, which is in agreement with our

ProteinTracker: an application for managing protein production and purification.

Science.gov (United States)

Ponko, Stefan C; Bienvenue, David

2012-05-10

Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org.

Kinetics and Thermodynamics of Membrane Protein Folding

Directory of Open Access Journals (Sweden)

Ernesto A. Roman

2014-03-01

Full Text Available Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane β-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

Cellular strategies to cope with protein aggregation

NARCIS (Netherlands)

Scior, Annika; Juenemann, Katrin; Kirstein, Janine

2016-01-01

Nature has evolved several mechanisms to detoxify intracellular protein aggregates that arise upon proteotoxic challenges. These include the controlled deposition of misfolded proteins at distinct cellular sites, the protein disaggregation and refolding by molecular chaperones and/or degradation of

Sampling and energy evaluation challenges in ligand binding protein design.

Science.gov (United States)

Dou, Jiayi; Doyle, Lindsey; Jr Greisen, Per; Schena, Alberto; Park, Hahnbeom; Johnsson, Kai; Stoddard, Barry L; Baker, David

2017-12-01

The steroid hormone 17α-hydroxylprogesterone (17-OHP) is a biomarker for congenital adrenal hyperplasia and hence there is considerable interest in development of sensors for this compound. We used computational protein design to generate protein models with binding sites for 17-OHP containing an extended, nonpolar, shape-complementary binding pocket for the four-ring core of the compound, and hydrogen bonding residues at the base of the pocket to interact with carbonyl and hydroxyl groups at the more polar end of the ligand. Eight of 16 designed proteins experimentally tested bind 17-OHP with micromolar affinity. A co-crystal structure of one of the designs revealed that 17-OHP is rotated 180° around a pseudo-two-fold axis in the compound and displays multiple binding modes within the pocket, while still interacting with all of the designed residues in the engineered site. Subsequent rounds of mutagenesis and binding selection improved the ligand affinity to nanomolar range, while appearing to constrain the ligand to a single bound conformation that maintains the same "flipped" orientation relative to the original design. We trace the discrepancy in the design calculations to two sources: first, a failure to model subtle backbone changes which alter the distribution of sidechain rotameric states and second, an underestimation of the energetic cost of desolvating the carbonyl and hydroxyl groups of the ligand. The difference between design model and crystal structure thus arises from both sampling limitations and energy function inaccuracies that are exacerbated by the near two-fold symmetry of the molecule. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

Energy Technology Data Exchange (ETDEWEB)

Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

2015-06-15

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

International Nuclear Information System (INIS)

Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

2015-01-01

Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

Science.gov (United States)

Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction between plate make and protein in protein crystallisation screening.

Directory of Open Access Journals (Sweden)

Gordon J King

Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

ProteinTracker: an application for managing protein production and purification

Directory of Open Access Journals (Sweden)

Ponko Stefan C

2012-05-01

Full Text Available Abstract Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks, or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS. Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes, cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org.

ProteinTracker: an application for managing protein production and purification

Science.gov (United States)

2012-01-01

Background Laboratories that produce protein reagents for research and development face the challenge of deciding whether to track batch-related data using simple file based storage mechanisms (e.g. spreadsheets and notebooks), or commit the time and effort to install, configure and maintain a more complex laboratory information management system (LIMS). Managing reagent data stored in files is challenging because files are often copied, moved, and reformatted. Furthermore, there is no simple way to query the data if/when questions arise. Commercial LIMS often include additional modules that may be paid for but not actually used, and often require software expertise to truly customize them for a given environment. Findings This web-application allows small to medium-sized protein production groups to track data related to plasmid DNA, conditioned media samples (supes), cell lines used for expression, and purified protein information, including method of purification and quality control results. In addition, a request system was added that includes a means of prioritizing requests to help manage the high demand of protein production resources at most organizations. ProteinTracker makes extensive use of existing open-source libraries and is designed to track essential data related to the production and purification of proteins. Conclusions ProteinTracker is an open-source web-based application that provides organizations with the ability to track key data involved in the production and purification of proteins and may be modified to meet the specific needs of an organization. The source code and database setup script can be downloaded from http://sourceforge.net/projects/proteintracker. This site also contains installation instructions and a user guide. A demonstration version of the application can be viewed at http://www.proteintracker.org. PMID:22574679

[The protective properties of the endotoxin protein].

Science.gov (United States)

Levenson, V I; Belkin, Z P; Egorova, T P

1991-08-01

The isolation and properties of endotoxin protein, or lipid A-associated protein (LAP), from Shigella sonnei were described earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 11-17, and No. 7). In this report the data on its protective activity are presented. In experiments on mice one nanogram of LAP injected i. v. protected 50% of the animals against i. p. challenge with 40 LD50 of virulent S. sonnei. Guinea pigs injected s. c. with 10 micrograms of LAP were protected against local (keratoconjunctival) challenge with S. sonnei, the efficiency of immunization being 58%. LAP preparations containing no detectable amounts of O-antigen (less than 0.003%) were found to have a protective effect. Hyperimmune anti-LAP rabbit serum prevented local infection when incubated with S. sonnei challenge inoculum before injection into guinea pigs. Both active and passive protection induced by LAP was specific since no effect was observed in animals challenged with Shigella flexneri. In the homologous system the protective effect of anti-LAP serum was abolished by the addition of protein-free LPS. These results are compatible with the hypothesis that the protective activity of LAP depends on the presence of minute amounts of O-antigen whose immunogenic effect is greatly amplified by the protein component of the natural endotoxin complex.

Can infrared spectroscopy provide information on protein-protein interactions?

Science.gov (United States)

Haris, Parvez I

2010-08-01

For most biophysical techniques, characterization of protein-protein interactions is challenging; this is especially true with methods that rely on a physical phenomenon that is common to both of the interacting proteins. Thus, for example, in IR spectroscopy, the carbonyl vibration (1600-1700 cm(-1)) associated with the amide bonds from both of the interacting proteins will overlap extensively, making the interpretation of spectral changes very complicated. Isotope-edited infrared spectroscopy, where one of the interacting proteins is uniformly labelled with (13)C or (13)C,(15)N has been introduced as a solution to this problem, enabling the study of protein-protein interactions using IR spectroscopy. The large shift of the amide I band (approx. 45 cm(-1) towards lower frequency) upon (13)C labelling of one of the proteins reveals the amide I band of the unlabelled protein, enabling it to be used as a probe for monitoring conformational changes. With site-specific isotopic labelling, structural resolution at the level of individual amino acid residues can be achieved. Furthermore, the ability to record IR spectra of proteins in diverse environments means that isotope-edited IR spectroscopy can be used to structurally characterize difficult systems such as protein-protein complexes bound to membranes or large insoluble peptide/protein aggregates. In the present article, examples of application of isotope-edited IR spectroscopy for studying protein-protein interactions are provided.

Hot-spot analysis for drug discovery targeting protein-protein interactions.

Science.gov (United States)

Rosell, Mireia; Fernández-Recio, Juan

2018-04-01

Protein-protein interactions are important for biological processes and pathological situations, and are attractive targets for drug discovery. However, rational drug design targeting protein-protein interactions is still highly challenging. Hot-spot residues are seen as the best option to target such interactions, but their identification requires detailed structural and energetic characterization, which is only available for a tiny fraction of protein interactions. Areas covered: In this review, the authors cover a variety of computational methods that have been reported for the energetic analysis of protein-protein interfaces in search of hot-spots, and the structural modeling of protein-protein complexes by docking. This can help to rationalize the discovery of small-molecule inhibitors of protein-protein interfaces of therapeutic interest. Computational analysis and docking can help to locate the interface, molecular dynamics can be used to find suitable cavities, and hot-spot predictions can focus the search for inhibitors of protein-protein interactions. Expert opinion: A major difficulty for applying rational drug design methods to protein-protein interactions is that in the majority of cases the complex structure is not available. Fortunately, computational docking can complement experimental data. An interesting aspect to explore in the future is the integration of these strategies for targeting PPIs with large-scale mutational analysis.

RAIN: RNA-protein Association and Interaction Networks

DEFF Research Database (Denmark)

Junge, Alexander; Refsgaard, Jan Christian; Garde, Christian

2017-01-01

is challenging due to data heterogeneity. Here, we present a database of ncRNA-RNA and ncRNA-protein interactions and its integration with the STRING database of protein-protein interactions. These ncRNA associations cover four organisms and have been established from curated examples, experimental data...

HitPredict version 4: comprehensive reliability scoring of physical protein?protein interactions from more than 100 species

OpenAIRE

L?pez, Yosvany; Nakai, Kenta; Patil, Ashwini

2015-01-01

HitPredict is a consolidated resource of experimentally identified, physical protein?protein interactions with confidence scores to indicate their reliability. The study of genes and their inter-relationships using methods such as network and pathway analysis requires high quality protein?protein interaction information. Extracting reliable interactions from most of the existing databases is challenging because they either contain only a subset of the available interactions, or a mixture of p...

From the Protein's Perspective: The Benefits and Challenges of Protein Structure-Based Pharmacophore Modeling

NARCIS (Netherlands)

Sanders, M.P.A.; McGuire, R; Roumen, L.; de Esch, I.J.P.; de Vlieg, J; Klomp, J.P.G; de Graaf, C.

2011-01-01

A pharmacophore describes the arrangement of molecular features a ligand must contain to efficaciously bind a receptor. Pharmacophore models are developed to improve molecular understanding of ligand-protein interactions, and can be used as a tool to identify novel compounds that fulfil the

Presidential Green Chemistry Challenge: 2001 Small Business Award

Science.gov (United States)

Presidential Green Chemistry Challenge 2001 award winner, EDEN Bioscience, discovered and commercialized harpins: nontoxic, naturally occurring, biodegradable proteins that activate a plant's defense and growth mechanisms.

SDSL-ESR-based protein structure characterization

NARCIS (Netherlands)

Strancar, J.; Kavalenka, A.A.; Urbancic, I.; Ljubetic, A.; Hemminga, M.A.

2010-01-01

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be

Protein 3D structure computed from evolutionary sequence variation.

Directory of Open Access Journals (Sweden)

Debora S Marks

Full Text Available The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing.In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy.We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues, including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7-4.8 Å C(α-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org. This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of

Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges

DEFF Research Database (Denmark)

Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

2010-01-01

-synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live...

Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

Science.gov (United States)

Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

2017-01-01

The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses.

Science.gov (United States)

Agnihothram, Sudhakar; Gopal, Robin; Yount, Boyd L; Donaldson, Eric F; Menachery, Vineet D; Graham, Rachel L; Scobey, Trevor D; Gralinski, Lisa E; Denison, Mark R; Zambon, Maria; Baric, Ralph S

2014-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs.  Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs.  Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43.  Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

HYDROGEL-BASED NANOCOMPOSITES OF THERAPEUTIC PROTEINS FOR TISSUE REPAIR.

Science.gov (United States)

Zhu, Suwei; Segura, Tatiana

2014-05-01

The ability to design artificial extracellular matrices as cell instructive scaffolds has opened the door to technologies capable of studying cell fates in vitro and to guide tissue repair in vivo . One main component of the design of artificial extracellular matrices is the incorporation of protein-based biochemical cues to guide cell phenotypes and multicellular organizations. However, promoting the long-term bioactivity, controlling the bioavailability and understanding how the physical presentations of these proteins impacts cellular fates are among the challenges of the field. Nanotechnolgy has advanced to meet the challenges of protein therapeutics. For example, the approaches to incorporating proteins into tissue repairing scaffolds have ranged from bulk encapsulations to smart nanodepots that protect proteins from degradations and allow opportunities for controlled release.

Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

Science.gov (United States)

Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

2013-01-01

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

LemA and Erp Y-like recombinant proteins from Leptospira interrogans protect hamsters from challenge using AddaVax™ as adjuvant.

Science.gov (United States)

Oliveira, Thaís Larré; Schuch, Rodrigo Andrade; Inda, Guilherme Roig; Roloff, Bárbara Couto; Neto, Amilton Clair Pinto Seixas; Amaral, Marta; Dellagostin, Odir Antonio; Hartwig, Daiane Drawanz

2018-05-03

Recombinant subunit vaccines have been extensively evaluated as promising alternatives against leptospirosis. Here, we evaluated two proteins in formulations containing the adjuvant AddaVax™ as vaccine candidates for prevention and control of leptospirosis. Recombinant proteins rErp Y-like and rLemA were characterized by ELISA to assess their ability to bind extracellular matrix (ECM) components and fibrinogen. Groups of eight hamsters were immunized intramuscularly with rErp Y-like or rLemA mixed with a squalene-based adjuvant (AddaVax), and then vaccine efficacy was determined in terms of protection against a lethal challenge. The humoral immune response was determined by ELISA, and the evidence of sub-lethal infection was evaluated by histopathology and kidney culture. rLemA protein binds laminin, fibrinogen, and collagen type IV, while rErp Y-like interacts with fibrinogen. Significant protection was achieved for rLemA and rErp Y-like vaccines, which showed 87.5% and 62.5% survivals, respectively. On day 28, the humoral immune response was significantly greater in the vaccine groups as compared to that in the control group, and the response was predominantly based on IgG2/3. The surviving animals showed negative results in culture isolation but presented with tissue lesions in the lungs and kidneys. Cumulatively, our findings suggest that LemA and Erp Y-like proteins act as adhesins and are able to protect against mortality, but not against tissue lesions. Moreover, AddaVax is a novel adjuvant with potential for improving the immunogenicity of leptospiral vaccines. Copyright © 2018 Elsevier Ltd. All rights reserved.

Protein Analysis in Human Cerebrospinal Fluid: Physiological Aspects, Current Progress and Future Challenges

Directory of Open Access Journals (Sweden)

Andreas F. Hühmer

2006-01-01

Full Text Available The introduction of lumbar puncture into clinical medicine over 100 years ago marks the beginning of the study of central nervous system diseases using the human cerebrospinal fluid (CSF. Ever since, CSF has been analyzed extensively to elucidate the physiological and biochemical bases of neurological disease. The proximity of CSF to the brain makes it a good target for studying the pathophysiology of brain functions, but the barrier function of the CSF also impedes its diagnostic value. Today, measurements to determine alterations in the composition of CSF are central in the differential diagnosis of specific diseases of the central nervous system (CNS. In particular, the analysis of the CSF protein composition provides crucial information in the diagnosis of CNS diseases. This enables the assessment of the physiology of the blood-CSF barrier and of the immunology of intrathecial responses. Besides those routine measurements, protein compositional studies of CSF have been extended recently to many other proteins in the expectation that comprehensive analysis of lower abundance CSF proteins will lead to the discovery of new disease markers. Disease marker discovery by molecular profiling of the CSF tissue has the enormous potential of providing many new disease relevant molecules. New developments in protein profiling techniques hold promise for the discovery and validation of relevant disease markers. In this review, we summarize the current efforts and progress in CSF protein profiling measurements using conventional and current protein analysis tools. We also discuss necessary development in methodology in order to have the highest impact on the study of the molecular composition of CSF proteins.

Arraying proteins by cell-free synthesis.

Science.gov (United States)

He, Mingyue; Wang, Ming-Wei

2007-10-01

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

The challenging measurement of protein in complex biomass-derived samples

DEFF Research Database (Denmark)

Haven, M.O.; Jørgensen, H.

2014-01-01

and fast protein measurement on this type of samples was the ninhydrin assay. This method has also been used widely for this purpose, but with two different methods for protein hydrolysis prior to the assay - alkaline or acidic hydrolysis. In samples containing glucose or ethanol, there was significant...... that the presence of cellulose, lignin and glucose (above 50 g/kg) could significantly affect the results of the assay. Comparison of analyses performed with the ninhydrin assay and with a CN analyser revealed that there was good agreement between these two analytical methods, but care has to be taken when applying...... the ninhydrin assay. If used correctly, the ninhydrin assay can be used as a fast method to evaluate the adsorption of cellulases to lignin....

A credit-card library approach for disrupting protein-protein interactions.

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Xu, Yang; Shi, Jin; Yamamoto, Noboru; Moss, Jason A; Vogt, Peter K; Janda, Kim D

2006-04-15

Protein-protein interfaces are prominent in many therapeutically important targets. Using small organic molecules to disrupt protein-protein interactions is a current challenge in chemical biology. An important example of protein-protein interactions is provided by the Myc protein, which is frequently deregulated in human cancers. Myc belongs to the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors. It is biologically active only as heterodimer with the bHLH-ZIP protein Max. Herein, we report a new strategy for the disruption of protein-protein interactions that has been corroborated through the design and synthesis of a small parallel library composed of 'credit-card' compounds. These compounds are derived from a planar, aromatic scaffold and functionalized with four points of diversity. From a 285 membered library, several hits were obtained that disrupted the c-Myc-Max interaction and cellular functions of c-Myc. The IC50 values determined for this small focused library for the disruption of Myc-Max dimerization are quite potent, especially since small molecule antagonists of protein-protein interactions are notoriously difficult to find. Furthermore, several of the compounds were active at the cellular level as shown by their biological effects on Myc action in chicken embryo fibroblast assays. In light of our findings, this approach is considered a valuable addition to the armamentarium of new molecules being developed to interact with protein-protein interfaces. Finally, this strategy for disrupting protein-protein interactions should prove applicable to other families of proteins.

Degradation of Akt using protein-catalyzed capture agents.

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Henning, Ryan K; Varghese, Joseph O; Das, Samir; Nag, Arundhati; Tang, Grace; Tang, Kevin; Sutherland, Alexander M; Heath, James R

2016-04-01

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Sensitizing properties of proteins

DEFF Research Database (Denmark)

Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott

2014-01-01

The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

Current strategies for protein production and purification enabling membrane protein structural biology.

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Pandey, Aditya; Shin, Kyungsoo; Patterson, Robin E; Liu, Xiang-Qin; Rainey, Jan K

2016-12-01

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

Rational design of new materials using recombinant structural proteins: Current state and future challenges.

Science.gov (United States)

Sutherland, Tara D; Huson, Mickey G; Rapson, Trevor D

2018-01-01

Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

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Chánique, Andrea M; Parra, Loreto P

2018-01-01

Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa . Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

Classification of protein-protein interaction full-text documents using text and citation network features.

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Kolchinsky, Artemy; Abi-Haidar, Alaa; Kaur, Jasleen; Hamed, Ahmed Abdeen; Rocha, Luis M

2010-01-01

We participated (as Team 9) in the Article Classification Task of the Biocreative II.5 Challenge: binary classification of full-text documents relevant for protein-protein interaction. We used two distinct classifiers for the online and offline challenges: 1) the lightweight Variable Trigonometric Threshold (VTT) linear classifier we successfully introduced in BioCreative 2 for binary classification of abstracts and 2) a novel Naive Bayes classifier using features from the citation network of the relevant literature. We supplemented the supplied training data with full-text documents from the MIPS database. The lightweight VTT classifier was very competitive in this new full-text scenario: it was a top-performing submission in this task, taking into account the rank product of the Area Under the interpolated precision and recall Curve, Accuracy, Balanced F-Score, and Matthew's Correlation Coefficient performance measures. The novel citation network classifier for the biomedical text mining domain, while not a top performing classifier in the challenge, performed above the central tendency of all submissions, and therefore indicates a promising new avenue to investigate further in bibliome informatics.

Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene

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Acutis Pier Luigi

2012-02-01

Full Text Available Abstract Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP. Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002. Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.

Presidential Green Chemistry Challenge: 2009 Greener Reaction Conditions Award

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Presidential Green Chemistry Challenge 2009 award winner, CEM Corporation, developed a fast, automated analytical process using less toxic reagents and less energy to distinguish protein from the food adulterant, melamine.

Exploring the context of the lung proteome within the airway mucosa following allergen challenge.

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Fehniger, Thomas E; Sato-Folatre, José-Gabriel; Malmström, Johan; Berglund, Magnus; Lindberg, Claes; Brange, Charlotte; Lindberg, Henrik; Marko-Varga, György

2004-01-01

The lung proteome is a dynamic collection of specialized proteins related to pulmonary function. Many cells of different derivations, activation states, and levels of maturity contribute to the changing environment, which produces the lung proteome. Inflammatory cells reacting to environmental challenge, for example from allergens, produce and secrete proteins which have profound effects on both resident and nonresident cells located in airways, alveoli, and the vascular tree which provides blood cells to the parenchyma alveolar bed for gas exchange. In an experimental model of allergic airway inflammation, we have compared control and allergen challenged lung compartments to determine global protein expression patterns using 2D-gel electrophoresis and subsequent spot identification by MS/MS mass spectrometry. We have then specifically isolated the epithelial mucosal layer, which lines conducting airways, from control and allergen challenged lungs, using laser capture technology and performed proteome identification on these selected cell samples. A central component of our investigations has been to contextually relate the histological features of the dynamic pulmonary environment to the changes in protein expression observed following challenge. Our results provide new information of the complexity of the submucosa/epithelium interface and the mechanisms behind the transformation of airway epithelium from normal steady states to functionally activated states.

Mild hypothermic culture conditions affect residual host cell protein composition post-Protein A chromatography.

Science.gov (United States)

Goey, Cher Hui; Bell, David; Kontoravdi, Cleo

2018-04-01

Host cell proteins (HCPs) are endogenous impurities, and their proteolytic and binding properties can compromise the integrity, and, hence, the stability and efficacy of recombinant therapeutic proteins such as monoclonal antibodies (mAbs). Nonetheless, purification of mAbs currently presents a challenge because they often co-elute with certain HCP species during the capture step of protein A affinity chromatography. A Quality-by-Design (QbD) strategy to overcome this challenge involves identifying residual HCPs and tracing their source to the harvested cell culture fluid (HCCF) and the corresponding cell culture operating parameters. Then, problematic HCPs in HCCF may be reduced by cell engineering or culture process optimization. Here, we present experimental results linking cell culture temperature and post-protein A residual HCP profile. We had previously reported that Chinese hamster ovary cell cultures conducted at standard physiological temperature and with a shift to mild hypothermia on day 5 produced HCCF of comparable product titer and HCP concentration, but with considerably different HCP composition. In this study, we show that differences in HCP variety at harvest cascaded to downstream purification where different residual HCPs were present in the two sets of samples post-protein A purification. To detect low-abundant residual HCPs, we designed a looping liquid chromatography-mass spectrometry method with continuous expansion of a preferred, exclude, and targeted peptide list. Mild hypothermic cultures produced 20% more residual HCP species, especially cell membrane proteins, distinct from the control. Critically, we identified that half of the potentially immunogenic residual HCP species were different between the two sets of samples.

BLAST-based structural annotation of protein residues using Protein Data Bank.

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Singh, Harinder; Raghava, Gajendra P S

2016-01-25

In the era of next-generation sequencing where thousands of genomes have been already sequenced; size of protein databases is growing with exponential rate. Structural annotation of these proteins is one of the biggest challenges for the computational biologist. Although, it is easy to perform BLAST search against Protein Data Bank (PDB) but it is difficult for a biologist to annotate protein residues from BLAST search. A web-server StarPDB has been developed for structural annotation of a protein based on its similarity with known protein structures. It uses standard BLAST software for performing similarity search of a query protein against protein structures in PDB. This server integrates wide range modules for assigning different types of annotation that includes, Secondary-structure, Accessible surface area, Tight-turns, DNA-RNA and Ligand modules. Secondary structure module allows users to predict regular secondary structure states to each residue in a protein. Accessible surface area predict the exposed or buried residues in a protein. Tight-turns module is designed to predict tight turns like beta-turns in a protein. DNA-RNA module developed for predicting DNA and RNA interacting residues in a protein. Similarly, Ligand module of server allows one to predicted ligands, metal and nucleotides ligand interacting residues in a protein. In summary, this manuscript presents a web server for comprehensive annotation of a protein based on similarity search. It integrates number of visualization tools that facilitate users to understand structure and function of protein residues. This web server is available freely for scientific community from URL http://crdd.osdd.net/raghava/starpdb .

Protein Dynamics in the Plant Extracellular Space

Directory of Open Access Journals (Sweden)

Leonor Guerra-Guimarães

2016-07-01

Full Text Available The extracellular space (ECS or apoplast is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF. The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization

DEFF Research Database (Denmark)

Andersen, Tonni Grube; Nintemann, Sebastian; Marek, Magdalena

2016-01-01

When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true-from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased se...

Improved protein surface comparison and application to low-resolution protein structure data

Directory of Open Access Journals (Sweden)

Kihara Daisuke

2010-12-01

Full Text Available Abstract Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM, which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs. The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

Improved protein surface comparison and application to low-resolution protein structure data.

Science.gov (United States)

Sael, Lee; Kihara, Daisuke

2010-12-14

Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

Nanostructures for protein drug delivery.

Science.gov (United States)

Pachioni-Vasconcelos, Juliana de Almeida; Lopes, André Moreni; Apolinário, Alexsandra Conceição; Valenzuela-Oses, Johanna Karina; Costa, Juliana Souza Ribeiro; Nascimento, Laura de Oliveira; Pessoa, Adalberto; Barbosa, Leandro Ramos Souza; Rangel-Yagui, Carlota de Oliveira

2016-02-01

Use of nanoscale devices as carriers for drugs and imaging agents has been extensively investigated and successful examples can already be found in therapy. In parallel, recombinant DNA technology together with molecular biology has opened up numerous possibilities for the large-scale production of many proteins of pharmaceutical interest, reflecting in the exponentially growing number of drugs of biotechnological origin. When we consider protein drugs, however, there are specific criteria to take into account to select adequate nanostructured systems as drug carriers. In this review, we highlight the main features, advantages, drawbacks and recent developments of nanostructures for protein encapsulation, such as nanoemulsions, liposomes, polymersomes, single-protein nanocapsules and hydrogel nanoparticles. We also discuss the importance of nanoparticle stabilization, as well as future opportunities and challenges in nanostructures for protein drug delivery.

Prospective estimation of IgG, IgG subclass and IgE antibodies to dietary proteins in infants with cow milk allergy. Levels of antibodies to whole milk protein, BLG and ovalbumin in relation to repeated milk challenge and clinical course of cow milk allergy

DEFF Research Database (Denmark)

Høst, A; Husby, S; Gjesing, B

1992-01-01

Prospectively, serum levels of IgE, specific IgE antibodies (AB) to whole cow milk protein (CMP), bovine se-albumin, bovine immunoglobulin, bovine lactoferrin, bovine lactalbumin and beta-lactoglobulin (BLG), IgG and IgG subclass antibodies to ovalbumin (OA) and BLG, and IgG4 RAST to CMP (bovine...... whey) were measured in 39 infants with cow milk protein allergy (CMPA) at birth (cord blood), at time of diagnosis and before and after milk challenge at the age of 12 months. Immunological measurements were also undertaken in 33 control infants without CMPA at birth, at 6 months and at 18 months...... of the type of CMPA (IgE-mediated (CMA) or non-IgE-mediated (CMI)), and irrespective of whether remission had occurred. In cord blood 25/33 (76%) of the infants with CMPA had specific IgE-AB to one or more of the bovine milk proteins indicating a prenatal intrauterine sensitization to cow milk protein. At 6...

Protein biomarker enrichment by biomarker antibody complex elution for immunoassay biosensing

NARCIS (Netherlands)

Sabatté, G.S.; Feitsma, H.; Evers, T.H.; Prins, M.W.J.

2011-01-01

It is very challenging to perform sample enrichment for protein biomarkers because proteins can easily change conformation and denature. In this paper we demonstrate protein enrichment suited for high-sensitivity integrated immuno-biosensing. The method enhances the concentration of the biomarkers

Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

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Sun, Bingyun; Hood, Leroy

2014-06-06

The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

AnchorDock for Blind Flexible Docking of Peptides to Proteins.

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Slutzki, Michal; Ben-Shimon, Avraham; Niv, Masha Y

2017-01-01

Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The "blind" docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.This protocol narrows the docking search to the most relevant parts of the conformational space. This is achieved by pre-folding the free peptide and by computationally detecting anchoring spots on the surface of the unbound protein. Multiple flexible simulated annealing molecular dynamics (SAMD) simulations are subsequently carried out, starting from pre-folded peptide conformations, constrained to the various precomputed anchoring spots.Here, AnchorDock is demonstrated using two known protein-peptide complexes. A PDZ-peptide complex provides a relatively easy case due to the relatively small size of the protein, and a typical peptide conformation and binding region; a more challenging example is a complex between USP7 N-term and a p53-derived peptide, where the protein is larger, and the peptide conformation and a binding site are generally assumed to be unknown. AnchorDock returned native-like solutions ranked first and third for the PDZ and USP7 complexes, respectively. We describe the procedure step by step and discuss possible modifications where applicable.

Protein-lipid nanohybrids as emerging platforms for drug and gene delivery: Challenges and outcomes.

Science.gov (United States)

Gaber, Mohamed; Medhat, Waseem; Hany, Mark; Saher, Nourhan; Fang, Jia-You; Elzoghby, Ahmed

2017-05-28

Nanoparticulate drug delivery systems have been long used to deliver a vast range of drugs and bioactives owing to their ability to demonstrate novel physical, chemical, and/or biological properties. An exponential growth has spurred in research and development of these nanocarriers which led to the evolution of a great number of diverse nanosystems including liposomes, nanoemulsions, solid lipid nanoparticles (SLNs), micelles, dendrimers, polymeric nanoparticles (NPs), metallic NPs, and carbon nanotubes. Among them, lipid-based nanocarriers have made the largest progress whether commercially or under development. Despite this progress, these lipid-based nanocarriers suffer from several limitations that led to the development of many protein-coated lipid nanocarriers. To less extent, protein-based nanocarriers suffer from limitations that led to the fabrication of some lipid bilayer enveloping protein nanocarriers. This review discusses in-depth some limitations associated with the lipid-based or protein-based nanocarriers and the fruitful outcomes brought by protein-lipid hybridization. Also discussed are the various hybridization techniques utilized to formulate these protein-lipid nanohybrids and the mechanisms involved in the drug loading process. Copyright © 2017 Elsevier B.V. All rights reserved.

Mass spectrometry compatible surfactant for optimized in-gel protein digestion.

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Saveliev, Sergei V; Woodroofe, Carolyn C; Sabat, Grzegorz; Adams, Christopher M; Klaubert, Dieter; Wood, Keith; Urh, Marjeta

2013-01-15

Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.

Physiological Importance and Mechanisms of Protein Hydrolysate Absorption

Science.gov (United States)

Zhanghi, Brian M.; Matthews, James C.

Understanding opportunities to maximize the efficient digestion and assimilation by production animals of plant- and animal-derived protein products is critical for farmers, nutritionists, and feed manufacturers to sustain and expand the affordable production of high quality animal products for human consumption. The challenge to nutritionists is to match gastrointestinal tract load to existing or ­inducible digestive and absorptive capacities. The challenge to feed manufacturers is to develop products that are efficient substrates for digestion, absorption, and/or both events. Ultimately, the efficient absorption of digesta proteins depends on the mediated passage (transport) of protein hydrosylate products as dipeptides and unbound amino acids across the lumen- and blood-facing membranes of intestinal absorptive cells. Data testing the relative efficiency of supplying protein as hydrolysates or specific dipeptides versus as free amino acids, and the response of animals in several physiological states to feeding of protein hydrolysates, are presented and reviewed in this chapter. Next, data describing the transport mechanisms responsible for absorbing protein hydrolysate digestion products, and the known and putative regulation of these mechanisms by their substrates (small peptides) and hormones are presented and reviewed. Several conclusions are drawn regarding the efficient use of protein hydrolysate-based diets for particular physiological states, the economically-practical application of which likely will depend on technological advances in the manufacture of protein hydrolysate products.

Experimental parameterization of an energy function for the simulation of unfolded proteins

DEFF Research Database (Denmark)

Norgaard, A.B.; Ferkinghoff-Borg, Jesper; Lindorff-Larsen, K.

2008-01-01

The determination of conformational preferences in unfolded and disordered proteins is an important challenge in structural biology. We here describe an algorithm to optimize energy functions for the simulation of unfolded proteins. The procedure is based on the maximum likelihood principle and e...... and can be applied to a range of experimental data and energy functions including the force fields used in molecular dynamics simulations.......The determination of conformational preferences in unfolded and disordered proteins is an important challenge in structural biology. We here describe an algorithm to optimize energy functions for the simulation of unfolded proteins. The procedure is based on the maximum likelihood principle...

A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

Directory of Open Access Journals (Sweden)

Li Min

2012-03-01

Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

Vikash Verma

Full Text Available 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.

Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

Science.gov (United States)

Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

2015-01-01

Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

Paediatric oral peanut challenges: a comparison of practice in London and western Switzerland.

Science.gov (United States)

Ludman, S; Wassenberg, J; Du Toit, G; Fox, A T; Lack, G; Eigenmann, P A

2013-04-01

There are guidelines on how to develop a food challenge protocol, but at present there is no gold standard guidance on method, and separate units produce differing protocols. We performed a retrospective analysis of 200 patients' data from the paediatric allergy units in Lausanne and Geneva, Western Switzerland, and St Thomas' Hospital (STH), UK. St Thomas' Hospital has a younger cohort with a lower overall mean spIgE (2.36 kU/l vs. 8.00 kU/l, P = 0.004). The target peanut protein volumes differed: Switzerland 4.4 g vs. STH 8.4 g. Despite this, the dose actually achieved in positive challenges was not significantly different (2.33 g vs. 1.49 g, P = 0.16). 26% of challenges reacted at 4 g or more of peanut protein. The differences in results highlight how the variation in reasoning behind food challenge alters the outcome. Standardization of food challenges would allow easy comparison between hospitals and geographical areas for research purposes. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

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Eric A Yen

2014-05-01

Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

Creating a specialist protein resource network: a meeting report for the protein bioinformatics and community resources retreat

DEFF Research Database (Denmark)

Babbitt, Patricia C.; Bagos, Pantelis G.; Bairoch, Amos

2015-01-01

During 11–12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from...... protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication...

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge.

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Subaschandrabose Rajesh Kumar

Full Text Available Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9 was protected against both H7N9 (A/Sh2/2013 and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211 in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HAm and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HAm constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA

K-nearest uphill clustering in the protein structure space

KAUST Repository

Cui, Xuefeng

2016-08-26

The protein structure classification problem, which is to assign a protein structure to a cluster of similar proteins, is one of the most fundamental problems in the construction and application of the protein structure space. Early manually curated protein structure classifications (e.g., SCOP and CATH) are very successful, but recently suffer the slow updating problem because of the increased throughput of newly solved protein structures. Thus, fully automatic methods to cluster proteins in the protein structure space have been designed and developed. In this study, we observed that the SCOP superfamilies are highly consistent with clustering trees representing hierarchical clustering procedures, but the tree cutting is very challenging and becomes the bottleneck of clustering accuracy. To overcome this challenge, we proposed a novel density-based K-nearest uphill clustering method that effectively eliminates noisy pairwise protein structure similarities and identifies density peaks as cluster centers. Specifically, the density peaks are identified based on K-nearest uphills (i.e., proteins with higher densities) and K-nearest neighbors. To our knowledge, this is the first attempt to apply and develop density-based clustering methods in the protein structure space. Our results show that our density-based clustering method outperforms the state-of-the-art clustering methods previously applied to the problem. Moreover, we observed that computational methods and human experts could produce highly similar clusters at high precision values, while computational methods also suggest to split some large superfamilies into smaller clusters. © 2016 Elsevier B.V.

Technologies and challenges in large-scale phosphoproteomics

DEFF Research Database (Denmark)

Engholm-Keller, Kasper; Larsen, Martin Røssel

2013-01-01

become the main technique for discovery and characterization of phosphoproteins in a nonhypothesis driven fashion. In this review, we describe methods for state-of-the-art MS-based analysis of protein phosphorylation as well as the strategies employed in large-scale phosphoproteomic experiments...... with focus on the various challenges and limitations this field currently faces....

Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms.

Science.gov (United States)

Mattick, John S

2003-10-01

The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoietic development and phenotypic variation. Copyright 2003 Wiley Periodicals, Inc.

A large-scale evaluation of computational protein function prediction

NARCIS (Netherlands)

Radivojac, P.; Clark, W.T.; Oron, T.R.; Schnoes, A.M.; Wittkop, T.; Kourmpetis, Y.A.I.; Dijk, van A.D.J.; Friedberg, I.

2013-01-01

Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be

Canola/rapeseed protein-functionality and nutrition

Directory of Open Access Journals (Sweden)

Wanasundara Janitha P.D.

2016-07-01

Full Text Available Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S and napin (2S dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed.

SDSL-ESR-based protein structure characterization.

Science.gov (United States)

Strancar, Janez; Kavalenka, Aleh; Urbancic, Iztok; Ljubetic, Ajasja; Hemminga, Marcus A

2010-03-01

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

Improving recombinant protein purification yield

Science.gov (United States)

Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

Progressing neurobiological strategies against proteostasis failure: Challenges in neurodegeneration.

Science.gov (United States)

Amanullah, Ayeman; Upadhyay, Arun; Joshi, Vibhuti; Mishra, Ribhav; Jana, Nihar Ranjan; Mishra, Amit

2017-12-01

Proteins are ordered useful cellular entities, required for normal health and organism's survival. The proteome is the absolute set of cellular expressed proteins, which regulates a wide range of physiological functions linked with all domains of life. In aging cells or under unfavorable cellular conditions, misfolding of proteins generates common pathological events linked with neurodegenerative diseases and aging. Current advances of proteome studies systematically generates some progress in our knowledge that how misfolding of proteins or their accumulation can contribute to the impairment or depletion of proteome functions. Still, the underlying causes of this unrecoverable loss are not clear that how such unsolved transitions give rise to multifactorial challengeable degenerative pathological conditions in neurodegeneration. In this review, we specifically focus and systematically summarize various molecular mechanisms of proteostasis maintenance, as well as discuss progressing neurobiological strategies, promising natural and pharmacological candidates, which can be useful to counteract the problem of proteopathies. Our article emphasizes an urgent need that now it is important for us to recognize the fundamentals of proteostasis to design a new molecular framework and fruitful strategies to uncover how the proteome defects are associated with aging and neurodegenerative diseases. A enhance understanding of progress link with proteome and neurobiological challenges may provide new basic concepts in the near future, based on pharmacological agents, linked with impaired proteostasis and neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Optimization of protein samples for NMR using thermal shift assays

International Nuclear Information System (INIS)

Kozak, Sandra; Lercher, Lukas; Karanth, Megha N.; Meijers, Rob; Carlomagno, Teresa; Boivin, Stephane

2016-01-01

Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor"® provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

Optimization of protein samples for NMR using thermal shift assays

Energy Technology Data Exchange (ETDEWEB)

Kozak, Sandra [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Lercher, Lukas; Karanth, Megha N. [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Meijers, Rob [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany); Carlomagno, Teresa, E-mail: teresa.carlomagno@oci.uni-hannover.de [European Molecular Biology Laboratory (EMBL), SCB Unit (Germany); Boivin, Stephane, E-mail: sboivin77@hotmail.com, E-mail: s.boivin@embl-hamburg.de [European Molecular Biology Laboratory (EMBL), Hamburg Outstation, SPC Facility (Germany)

2016-04-15

Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor{sup ®} provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies.

Lessons learned in induced fit docking and metadynamics in the Drug Design Data Resource Grand Challenge 2

Science.gov (United States)

Baumgartner, Matthew P.; Evans, David A.

2018-01-01

Two of the major ongoing challenges in computational drug discovery are predicting the binding pose and affinity of a compound to a protein. The Drug Design Data Resource Grand Challenge 2 was developed to address these problems and to drive development of new methods. The challenge provided the 2D structures of compounds for which the organizers help blinded data in the form of 35 X-ray crystal structures and 102 binding affinity measurements and challenged participants to predict the binding pose and affinity of the compounds. We tested a number of pose prediction methods as part of the challenge; we found that docking methods that incorporate protein flexibility (Induced Fit Docking) outperformed methods that treated the protein as rigid. We also found that using binding pose metadynamics, a molecular dynamics based method, to score docked poses provided the best predictions of our methods with an average RMSD of 2.01 Å. We tested both structure-based (e.g. docking) and ligand-based methods (e.g. QSAR) in the affinity prediction portion of the competition. We found that our structure-based methods based on docking with Smina (Spearman ρ = 0.614), performed slightly better than our ligand-based methods (ρ = 0.543), and had equivalent performance with the other top methods in the competition. Despite the overall good performance of our methods in comparison to other participants in the challenge, there exists significant room for improvement especially in cases such as these where protein flexibility plays such a large role.

Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests.

Science.gov (United States)

Javaid, Shaista; Naz, Sehrish; Amin, Imran; Jander, Georg; Ul-Haq, Zaheer; Mansoor, Shahid

2018-03-19

Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis (mealybug), a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.

Computational prediction of protein hot spot residues.

Science.gov (United States)

Morrow, John Kenneth; Zhang, Shuxing

2012-01-01

Most biological processes involve multiple proteins interacting with each other. It has been recently discovered that certain residues in these protein-protein interactions, which are called hot spots, contribute more significantly to binding affinity than others. Hot spot residues have unique and diverse energetic properties that make them challenging yet important targets in the modulation of protein-protein complexes. Design of therapeutic agents that interact with hot spot residues has proven to be a valid methodology in disrupting unwanted protein-protein interactions. Using biological methods to determine which residues are hot spots can be costly and time consuming. Recent advances in computational approaches to predict hot spots have incorporated a myriad of features, and have shown increasing predictive successes. Here we review the state of knowledge around protein-protein interactions, hot spots, and give an overview of multiple in silico prediction techniques of hot spot residues.

Upgrading protein products using bioprocessing on agricultural crops

DEFF Research Database (Denmark)

Sulewska, Anna Maria; Sørensen, Jens Christian; Markedal, Keld Ejdrup

to sustainability leads to a demand for plant protein products made from locally grown crops. Novel bioprocessing methods have been developed to generate protein products which are nutritious, readily available and do not generate hazardous waste. The processing focus has therefore been on developing protein......Due to increasing world population, higher average income, and changes in food preferences, there is a growing demand for proteins, especially novel plant-based protein sources, that can substitute animal proteins and supplement currently used soya proteins. Increased customer awareness......-enriched products with minimized content of antinutritional compounds. For every crop it is a challenge to obtain protein fractions with sufficient added value to make processing economically feasible. In this work we present the characterization of protein products developed in pilot scale using the novel...

Protein stability in pulmonary drug delivery via nebulization.

Science.gov (United States)

Hertel, Sebastian P; Winter, Gerhard; Friess, Wolfgang

2015-10-01

Protein inhalation is a delivery route which offers high potential for direct local lung application of proteins. Liquid formulations are usually available in early stages of biopharmaceutical development and nebulizers are the device of choice for atomization avoiding additional process steps like drying and enabling fast progression to clinical trials. While some proteins were proven to remain stable throughout aerosolization e.g. DNase, many biopharmaceuticals are more susceptible towards the stresses encountered during nebulization. The main reason for protein instability is unfolding and aggregation at the air-liquid interface, a problem which is of particular challenge in the case of ultrasound and jet nebulizers due to recirculation of much of the generated droplets. Surfactants are an important formulation component to protect the sensitive biomolecules. A second important challenge is warming of ultrasound and vibrating mesh devices, which can be overcome by overfilling, precooled solutions or cooling of the reservoir. Ultimately, formulation development has to go hand in hand with device evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.

Handling Gene and Protein Names in the Age of Bioinformatics: The Special Challenge of Secreted Multimodular Bacterial Enzymes such as the cbhA/cbh9A Gene of Clostridium thermocellum

Energy Technology Data Exchange (ETDEWEB)

Brunecky, Roman [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Schwarz, Wolfgang H. [Technical University of Munich; Broeker, Jannis [Technical University of Munich; Liebl, Wolfgang [Technical University of Munich; Zverlov, Vladimir V. [Technical University of Munich; Russian Academy of Science

2018-02-26

An increasing number of researchers working in biology, biochemistry, biotechnology, bioengineering, bioinformatics and other related fields of science are using biological molecules. As the scientific background of the members of different scientific communities is more diverse than ever before, the number of scientists not familiar with the rules for non-ambiguous designation of genetic elements is increasing. However, with biological molecules gaining importance through biotechnology, their functional and unambiguous designation is vital. Unfortunately, naming genes and proteins is not an easy task. In addition, the traditional concepts of bioinformatics are challenged with the appearance of proteins comprising different modules with a respective function in each module. This article highlights basic rules and novel solutions in designation recently used within the community of bacterial geneticists, and we discuss the present-day handling of gene and protein designations. As an example we will utilize a recent mischaracterization of gene nomenclature. We make suggestions for better handling of names in future literature as well as in databases and annotation projects. Our methodology emphasizes the hydrolytic function of multi-modular genes and extracellular proteins from bacteria.

Challenges and opportunities in synthetic biology for chemical engineers.

Science.gov (United States)

Luo, Yunzi; Lee, Jung-Kul; Zhao, Huimin

2013-11-15

Synthetic biology provides numerous great opportunities for chemical engineers in the development of new processes for large-scale production of biofuels, value-added chemicals, and protein therapeutics. However, challenges across all scales abound. In particular, the modularization and standardization of the components in a biological system, so-called biological parts, remain the biggest obstacle in synthetic biology. In this perspective, we will discuss the main challenges and opportunities in the rapidly growing synthetic biology field and the important roles that chemical engineers can play in its advancement.

[Immunogenicity and protective efficacy of pertactin recombinants against Bordetella bronchiseptica challenge].

Science.gov (United States)

Zhao, Zhanqin; Wang, Chen; Xue, Yun; Ding, Ke; Zhang, Chunjie; Cheng, Xiangchao; Li, Yinju; Liu, Yichen; Wu, Tingcai

2010-09-01

In this study we showed the immunogenicity and protective efficacy of five pertactin recombinants against Bordetella bronchiseptica (Bb) challenge. The complete coding sequence (2040 bp) of the prn gene (PRN) and its fragments,5'-terminal 1173 bp fragment (PN),3'-terminal 867 bp fragment (PC), two copies of region I (654 bp; PR I) in PN, and 2 copies of region II (678 bp; PR II) in PC, were separately cloned into the prokaryotic expression vector pGEX-KG, and expressed in the Eschierichia coli BL21 (DE3) using induction by isopropyl-beta-D-thiogalactopyranoside. The recombinant proteins were named GST-PRN, GST-PN, GST-PC, GST-2PR I and GST-2PR II. All five recombinant proteins showed immunological reactivity in the Western-blot analysis. Mice, immunized subcutaneously with two doses of the purified proteins mixed with an equal volume of Freund's adjuvant,produced robust PRN-specific IgG antibody levels. When challenged, 6 of 9 mice in GST-2PR I group and all 9 mice in the other groups survived intranasal challenge with three times the 50% lethal dose (LD50) of virulent Bb HH0809. After challenge with 10 LD50 7/9,3/9,6/9,1/10 and 6/10 of the mice survived. Furthermore, complete protection against intraperitoneal (i.p.) challenge with 10 LD50 of HH0809 was observed in mice that were injected i.p. with 0.5 ml rabbit anti-GST-PRN, GST-PN,GST-PC or GST-2PR II serum. Only 1 of 10 mice survived in the group of mice that received anti-GST-2PR I, and no survivors were noted in the group of mice that received PRN-absorbed rabbit antiserum (0/5). In this study,we showed that all of five pertactin recombinants had differential immunogenicity and protective efficacy against Bb challenge. Mice immunized with GST-PC had better survival against fatal Bb challenge than did those immunized with GST-PN. In addition, GST-2PR II and GST-2PR I provided the similar results These data may have implications for the development of safe and efficacious subunit vaccines for the prevention of

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

Science.gov (United States)

A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or an...

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

Science.gov (United States)

Yim, W J; Kim, K Y; Lee, Y W; Sundaram, S P; Lee, Y; Sa, T M

2014-07-15

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato. Copyright © 2014 Elsevier GmbH. All rights reserved.

Key challenges for the creation and maintenance of specialist protein resources

NARCIS (Netherlands)

Holliday, G.L.; Bairoch, A.; Bagos, P.G.; Chatonnet, A.; Craik, D.J.; Finn, R.D.; Henrissat, B.; Landsman, D.; Manning, G.; Nagano, N.; O'Donovan, C.; Pruitt, K.D.; Rawlings, N.D.; Saier, M.; Sowdhamini, R.; Spedding, M.; Srinivasan, N.; Vriend, G.; Babbitt, P.C.; Bateman, A.

2015-01-01

As the volume of data relating to proteins increases, researchers rely more and more on the analysis of published data, thus increasing the importance of good access to these data that vary from the supplemental material of individual articles, all the way to major reference databases with

EHD proteins: Key conductors of endocytic transport

Science.gov (United States)

Naslavsky, Naava; Caplan, Steve

2010-01-01

Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field. PMID:21067929

Modeling complexes of modeled proteins.

Science.gov (United States)

Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

2017-03-01

Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Small Proteins Can No Longer Be Ignored

Science.gov (United States)

Storz, Gisela; Wolf, Yuri I.; Ramamurthi, Kumaran S.

2014-01-01

Small proteins, here defined as proteins of 50 amino acids or less in the absence of processing, have traditionally been overlooked due to challenges in their annotation and biochemical detection. In the past several years however, increasing numbers of small proteins have been identified either through the realization that mutations in “intergenic” regions actually are within unannotated small protein genes, or through the discovery that some small, regulatory RNAs (sRNAs) encode small proteins. These insights together with comparative sequence analysis indicate that tens if not hundreds of small proteins are synthesized in a given organism. This review will summarize what has been learned about the functions of several of these bacterial small proteins, most of which act at the membrane, illustrating the astonishing range of processes in which the small proteins act and pointing to several general conclusions. Important questions for future studies of these overlooked proteins also will be discussed. PMID:24606146

From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

Directory of Open Access Journals (Sweden)

Mu Gao

2009-03-01

Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

Science.gov (United States)

Van Molle, Inge; Thomann, Andreas; Buckley, Dennis L; So, Ernest C; Lang, Steffen; Crews, Craig M; Ciulli, Alessio

2012-10-26

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

The language of the protein universe.

Science.gov (United States)

Scaiewicz, Andrea; Levitt, Michael

2015-12-01

Proteins, the main cell machinery which play a major role in nearly every cellular process, have always been a central focus in biology. We live in the post-genomic era, and inferring information from massive data sets is a steadily growing universal challenge. The increasing availability of fully sequenced genomes can be regarded as the 'Rosetta Stone' of the protein universe, allowing the understanding of genomes and their evolution, just as the original Rosetta Stone allowed Champollion to decipher the ancient Egyptian hieroglyphics. In this review, we consider aspects of the protein domain architectures repertoire that are closely related to those of human languages and aim to provide some insights about the language of proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

An overview of site-specific delivery of orally administered proteins ...

African Journals Online (AJOL)

Oral delivery of proteins and peptides poses one of the greatest challenges in controlled drug delivery due to degradation by proteolytic enzymes, poor membrane permeability and large molecular size. Therapeutic proteins/peptides are useful in correcting metabolic disorders (e.g., insulin in diabetes mellitus), ...

Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

International Nuclear Information System (INIS)

Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike; Zhang Liqun; Yang Lijuan; Ward, Jerrold M.; Dorward, David W.; Pickles, Raymond J.; Murphy, Brian R.; Feldmann, Heinz; Collins, Peter L.

2009-01-01

We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

Network based approaches reveal clustering in protein point patterns

Science.gov (United States)

Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

2014-03-01

Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

Challenges and opportunities in synthetic biology for chemical engineers

Science.gov (United States)

Luo, Yunzi; Lee, Jung-Kul; Zhao, Huimin

2012-01-01

Synthetic biology provides numerous great opportunities for chemical engineers in the development of new processes for large-scale production of biofuels, value-added chemicals, and protein therapeutics. However, challenges across all scales abound. In particular, the modularization and standardization of the components in a biological system, so-called biological parts, remain the biggest obstacle in synthetic biology. In this perspective, we will discuss the main challenges and opportunities in the rapidly growing synthetic biology field and the important roles that chemical engineers can play in its advancement. PMID:24222925

Protein instability and immunogenicity: roadblocks to clinical application of injectable protein delivery systems for sustained release.

Science.gov (United States)

Jiskoot, Wim; Randolph, Theodore W; Volkin, David B; Middaugh, C Russell; Schöneich, Christian; Winter, Gerhard; Friess, Wolfgang; Crommelin, Daan J A; Carpenter, John F

2012-03-01

Protein instability and immunogenicity are two main roadblocks to the clinical success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address immunogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both conventional and novel therapeutic protein dosage forms. Next, we provide a summary of the stresses on proteins arising during preparation of drug delivery systems and subsequent in vivo release. We note the challenges and difficulties in achieving the absolute requirement of quantitatively assessing the degradation of protein molecules in a drug delivery system. We describe the potential roles for academic research in further improving protein stability and developing new analytical technologies to detect protein degradation byproducts in novel drug delivery systems. Finally, we provide recommendations for the appropriate approaches to formulation design and assay development to ensure that stable, minimally immunogenic formulations of therapeutic proteins are created. These approaches should help to increase the probability that novel drug delivery systems for sustained protein release will become more readily available as effective therapeutic agents to treat and benefit patients. Copyright © 2011 Wiley Periodicals, Inc.

Protein supplementation with sports protein bars in renal patients.

Science.gov (United States)

Meade, Anthony

2007-05-01

Malnutrition prevalence in patients on dialysis is well established. The protein requirements for both hemodialysis and peritoneal dialysis have been documented elsewhere, including the Kidney Disease Outcomes Quality Initiative Clinical Practice Guidelines for Nutrition in Chronic Renal Failure. The clinical challenge is to assist patients in meeting these targets, especially in those with anorexia. Traditional supplements have included fluid, which is an issue for patients who are fluid restricted. The study objectives were to (1) investigate the range of sports protein supplements that may be suitable for patients on hemodialysis to use and (2) trial nonfluid protein supplements in patients on hemodialysis. Known manufacturers of sports protein bars and other sports supplements available in Australia were contacted for the nutrient breakdown of high-protein products, specifically potassium, protein, and phosphorus contents. As a result, selected high-protein sports bars (Protein FX, Aussie Bodies, Port Melbourne, Victoria, Australia) were used as an alternative to the more commonly used renal-specific fluid supplements (Nepro, Abbott Laboratories, Abbott Park, IL; Novasource Renal, Novartis Nutrition Corporation, Fremont, MI; and Renilon, Nutricia, Wiltshire, UK) in patients with poor nutritional status requiring supplementation. Patient satisfaction and clinical nutrition markers were investigated. The study took place at inpatient, in-center, and satellite hemodialysis settings in Adelaide, South Australia. A total of 32 patients (16 females and 16 males) with an average age of 62.9 years (range 32-86 years) undergoing hemodialysis (acute and maintenance) were included. Subjects were selected by the author as part of routine clinical nutrition care. Patients trialed sports protein bars as a protein supplement alone or in conjunction with other supplementary products. All patients were in favor of the trial, with 22 of 32 patients continuing with the protein

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions

KAUST Repository

Najibi, Seyed Morteza; Maadooliat, Mehdi; Zhou, Lan; Huang, Jianhua Z.; Gao, Xin

2017-01-01

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions

KAUST Repository

Najibi, Seyed Morteza

2017-02-08

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

Harnessing Aptamers to Overcome Challenges in Gluten Detection

Directory of Open Access Journals (Sweden)

Rebeca Miranda-Castro

2016-04-01

Full Text Available Celiac disease is a lifelong autoimmune disorder triggered by foods containing gluten, the storage protein in wheat, rye, and barley. The rapidly escalating number of patients diagnosed with this disease poses a great challenge to both food industry and authorities to guarantee food safety for all. Therefore, intensive efforts are being made to establish minimal disease-eliciting doses of gluten and consequently to improve gluten-free labeling. These efforts depend to a high degree on the availability of methods capable of detecting the protein in food samples at levels as low as possible. Current analytical approaches rely on the use of antibodies as selective recognition elements. With limited sensitivity, these methods exhibit some deficiencies that compromise the accuracy of the obtained results. Aptamers provide an ideal alternative for designing biosensors for fast and selective measurement of gluten in foods. This article highlights the challenges in gluten detection, the current status of the use of aptamers for solving this problem, and what remains to be done to move these systems into commercial applications.

Creating a specialist protein resource network: a meeting report for the protein bioinformatics and community resources retreat.

Science.gov (United States)

Babbitt, Patricia C; Bagos, Pantelis G; Bairoch, Amos; Bateman, Alex; Chatonnet, Arnaud; Chen, Mark Jinan; Craik, David J; Finn, Robert D; Gloriam, David; Haft, Daniel H; Henrissat, Bernard; Holliday, Gemma L; Isberg, Vignir; Kaas, Quentin; Landsman, David; Lenfant, Nicolas; Manning, Gerard; Nagano, Nozomi; Srinivasan, Narayanaswamy; O'Donovan, Claire; Pruitt, Kim D; Sowdhamini, Ramanathan; Rawlings, Neil D; Saier, Milton H; Sharman, Joanna L; Spedding, Michael; Tsirigos, Konstantinos D; Vastermark, Ake; Vriend, Gerrit

2015-01-01

During 11-12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue.

Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases

Science.gov (United States)

Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah; Abdelhameed, Ali Saber; Khan, Rizwan Hasan

2016-11-01

In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially misfolded intermediates aggregate via the interaction with the complementary intermediates and consequently enhance oligomers formation that grows into fibrils and proto-fibrils. The amyloid fibrils for example, accumulate in the brain and central nervous system (CNS) as amyloid deposits in the Parkinson's disease (PD), Alzheimer's disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other neurodegenerative diseases. Treatment of such misfolding maladies is considered as one of the most important challenges of the 21st century. Currently, several treatments strategies have been and are being discovered. These therapeutic interventions partly reversed or prevented the pathological state. More recently, a new approach was discovered, which employs nanobodies that targets multisteps in fibril formation pathway that may possibly completely cure these misfolding diseases. Keeping the above views in mind in the current review, we have comprehensively discussed the different mechanisms underlying protein misfolding thereby leading to diseases conditions and their therapeutic interventions.

Rogue athletes and recombinant DNA technology: challenges for doping control.

Science.gov (United States)

Azzazy, Hassan M E; Mansour, Mai M H

2007-10-01

The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

Immunogenicity of NYVAC Prime-Protein Boost Human Immunodeficiency Virus Type 1 Envelope Vaccination and Simian-Human Immunodeficiency Virus Challenge of Nonhuman Primates.

Science.gov (United States)

Saunders, Kevin O; Santra, Sampa; Parks, Robert; Yates, Nicole L; Sutherland, Laura L; Scearce, Richard M; Balachandran, Harikrishnan; Bradley, Todd; Goodman, Derrick; Eaton, Amanda; Stanfield-Oakley, Sherry A; Tartaglia, James; Phogat, Sanjay; Pantaleo, Giuseppe; Esteban, Mariano; Gomez, Carmen E; Perdiguero, Beatriz; Jacobs, Bertram; Kibler, Karen; Korber, Bette; Montefiori, David C; Ferrari, Guido; Vandergrift, Nathan; Liao, Hua-Xin; Tomaras, Georgia D; Haynes, Barton F

2018-04-15

A preventive human immunodeficiency virus type 1 (HIV-1) vaccine is an essential part of the strategy to eradicate AIDS. A critical question is whether antibodies that do not neutralize primary isolate (tier 2) HIV-1 strains can protect from infection. In this study, we investigated the ability of an attenuated poxvirus vector (NYVAC) prime-envelope gp120 boost to elicit potentially protective antibody responses in a rhesus macaque model of mucosal simian-human immunodeficiency virus (SHIV) infection. NYVAC vector delivery of a group M consensus envelope, trivalent mosaic envelopes, or a natural clade B isolate B.1059 envelope elicited antibodies that mediated neutralization of tier 1 viruses, cellular cytotoxicity, and phagocytosis. None of the macaques made neutralizing antibodies against the tier 2 SHIV SF162P3 used for mucosal challenge. Significant protection from infection was not observed for the three groups of vaccinated macaques compared to unvaccinated macaques, although binding antibody to HIV-1 Env correlated with decreased viremia after challenge. Thus, NYVAC Env prime-gp120 boost vaccination elicited polyfunctional, nonneutralizing antibody responses with minimal protective activity against tier 2 SHIV mucosal challenge. IMPORTANCE The antibody responses that confer protection against HIV-1 infection remain unknown. Polyfunctional antibody responses correlated with time to infection in previous macaque studies. Determining the ability of vaccines to induce these types of responses is critical for understanding how to improve upon the one efficacious human HIV-1 vaccine trial completed thus far. We characterized the antibody responses induced by a NYVAC-protein vaccine and determined the protective capacity of polyfunctional antibody responses in an R5, tier 2 mucosal SHIV infection model. Copyright © 2018 American Society for Microbiology.

Designing coarse grained-and atom based-potentials for protein-protein docking

Directory of Open Access Journals (Sweden)

Tobi Dror

2010-11-01

Full Text Available Abstract Background Protein-protein docking is a challenging computational problem in functional genomics, particularly when one or both proteins undergo conformational change(s upon binding. The major challenge is to define a scoring function soft enough to tolerate these changes and specific enough to distinguish between near-native and "misdocked" conformations. Results Using a linear programming (LP technique, we developed two types of potentials: (i Side chain-based and (ii Heavy atom-based. To achieve this we considered a set of 161 transient complexes and generated a large set of putative docked structures (decoys, based on a shape complementarity criterion, for each complex. The demand on the potentials was to yield, for the native (correctly docked structure, a potential energy lower than those of any of the non-native (misdocked structures. We show that the heavy atom-based potentials were able to comply with this requirement but not the side chain-based one. Thus, despite the smaller number of parameters, the capability of heavy atom-based potentials to discriminate between native and "misdocked" conformations is improved relative to those of the side chain-based potentials. The performance of the atom-based potentials was evaluated by a jackknife test on a set of 50 complexes taken from the Zdock2.3 decoys set. Conclusions Our results show that, using the LP approach, we were able to train our potentials using a dataset of transient complexes only the newly developed potentials outperform three other known potentials in this test.

Hydration shells exchange charge with their protein

DEFF Research Database (Denmark)

Abitan, Haim; Lindgård, Per-Anker; Nielsen, Bjørn Gilbert

2010-01-01

. In our experiments, the amplitude of an ultrasonic pressure wave is gradually increased (0–20 atm) while we simultaneously measure the Raman spectra from the hydrated protein (β-lactoglobulin and lysozyme). We detected two types of spectral changes: first, up to 70% increase in the intensity......Investigation of the interaction between a protein and its hydration shells is an experimental and theoretical challenge. Here, we used ultrasonic pressure waves in aqueous solutions of a protein to explore the conformational states of the protein and its interaction with its hydration shells...... the presence of an ultrasonic pressure, a protein and its hydration shells are in thermodynamic and charge equilibrium, i.e. a protein and its hydration shells exchange charges. The ultrasonic wave disrupts these equilibria which are regained within 30–45 min after the ultrasonic pressure is shut off....

Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

Science.gov (United States)

Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

2015-07-21

Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

Biomolecular simulations on petascale: promises and challenges

International Nuclear Information System (INIS)

Agarwal, Pratul K; Alam, Sadaf R

2006-01-01

Proteins work as highly efficient machines at the molecular level and are responsible for a variety of processes in all living cells. There is wide interest in understanding these machines for implications in biochemical/biotechnology industries as well as in health related fields. Over the last century, investigations of proteins based on a variety of experimental techniques have provided a wealth of information. More recently, theoretical and computational modeling using large scale simulations is providing novel insights into the functioning of these machines. The next generation supercomputers with petascale computing power, hold great promises as well as challenges for the biomolecular simulation scientists. We briefly discuss the progress being made in this area

Light assisted drying (LAD) for protein stabilization: optical characterization of samples

Science.gov (United States)

Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

2018-02-01

Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

Molecular recognition in protein modification with rhodium metallopeptides

Science.gov (United States)

Ball, Zachary T.

2015-01-01

Chemical manipulation of natural, unengineered proteins is a daunting challenge which tests the limits of reaction design. By combining transition-metal or other catalysts with molecular recognition ideas, it is possible to achieve site-selective protein reactivity without the need for engineered recognition sequences or reactive sites. Some recent examples in this area have used ruthenium photocatalysis, pyridine organocatalysis, and rhodium(II) metallocarbene catalysis, indicating that the fundamental ideas provide opportunities for using diverse reactivity on complex protein substrates and in complex cell-like environments. PMID:25588960

The Protein Model Portal.

Science.gov (United States)

Arnold, Konstantin; Kiefer, Florian; Kopp, Jürgen; Battey, James N D; Podvinec, Michael; Westbrook, John D; Berman, Helen M; Bordoli, Lorenza; Schwede, Torsten

2009-03-01

Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. One of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. Often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. This has been done by all of the PSI centers, and by various independent modeling groups. The goal of the Protein Model Portal (PMP) is to provide a single portal which gives access to the various models that can be leveraged from PSI targets and other experimental protein structures. A single interface allows all existing pre-computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target-template alignment, model building, and quality assessment. The current release of the portal consists of 7.6 million model structures provided by different partner resources (CSMP, JCSG, MCSG, NESG, NYSGXRC, JCMM, ModBase, SWISS-MODEL Repository). The PMP is available at http://www.proteinmodelportal.org and from the PSI Structural Genomics Knowledgebase.

Cellular Handling of Protein Aggregates by Disaggregation Machines.

Science.gov (United States)

Mogk, Axel; Bukau, Bernd; Kampinga, Harm H

2018-01-18

Both acute proteotoxic stresses that unfold proteins and expression of disease-causing mutant proteins that expose aggregation-prone regions can promote protein aggregation. Protein aggregates can interfere with cellular processes and deplete factors crucial for protein homeostasis. To cope with these challenges, cells are equipped with diverse folding and degradation activities to rescue or eliminate aggregated proteins. Here, we review the different chaperone disaggregation machines and their mechanisms of action. In all these machines, the coating of protein aggregates by Hsp70 chaperones represents the conserved, initializing step. In bacteria, fungi, and plants, Hsp70 recruits and activates Hsp100 disaggregases to extract aggregated proteins. In the cytosol of metazoa, Hsp70 is empowered by a specific cast of J-protein and Hsp110 co-chaperones allowing for standalone disaggregation activity. Both types of disaggregation machines are supported by small Hsps that sequester misfolded proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Quantification and Classification of E. coli Proteome Utilization and Unused Protein Costs across Environments

DEFF Research Database (Denmark)

O'Brien, Edward J.; Utrilla, Jose; Palsson, Bernhard

2016-01-01

The costs and benefits of protein expression are balanced through evolution. Expression of un-utilized protein (that have no benefits in the current environment) incurs a quantifiable fitness costs on cellular growth rates; however, the magnitude and variability of un-utilized protein expression...... in varying environments. Thus, unused protein expression is the source of large and pervasive fitness costs that may provide the benefit of hedging against environmental change....... in natural settings is unknown, largely due to the challenge in determining environment-specific proteome utilization. We address this challenge using absolute and global proteomics data combined with a recently developed genome-scale model of Escherichia coli that computes the environment-specific cost...

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

Science.gov (United States)

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Computational design of proteins with novel structure and functions

International Nuclear Information System (INIS)

Yang Wei; Lai Lu-Hua

2016-01-01

Computational design of proteins is a relatively new field, where scientists search the enormous sequence space for sequences that can fold into desired structure and perform desired functions. With the computational approach, proteins can be designed, for example, as regulators of biological processes, novel enzymes, or as biotherapeutics. These approaches not only provide valuable information for understanding of sequence–structure–function relations in proteins, but also hold promise for applications to protein engineering and biomedical research. In this review, we briefly introduce the rationale for computational protein design, then summarize the recent progress in this field, including de novo protein design, enzyme design, and design of protein–protein interactions. Challenges and future prospects of this field are also discussed. (topical review)

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

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Krawczenko, Agnieszka; Bielawska-Pohl, Aleksandra; Wojtowicz, Karolina; Jura, Roksana; Paprocka, Maria; Wojdat, Elżbieta; Kozłowska, Urszula; Klimczak, Aleksandra; Grillon, Catherine; Kieda, Claudine; Duś, Danuta

2017-01-01

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Fluorescent Protein Approaches in Alpha Herpesvirus Research

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Ian B. Hogue

2015-11-01

Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

Preventive Effects of Resveratrol on Endocannabinoid System and Synaptic Protein Modifications in Rat Cerebral Cortex Challenged by Bilateral Common Carotid Artery Occlusion and Reperfusion

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Gianfranca Carta

2018-01-01

Full Text Available This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R and to investigate RVT’s ability to preserve the neuronal structural integrity. Frontal and temporal-occipital cortices were examined in two groups of adult Wistar rats, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half the rats were gavage-fed with a single dose of RVT (40 mg/per rat in 300 µL of sunflower oil as the vehicle, while the second half received the vehicle alone. In the frontal cortex, RVT pre-treatment prevented the BCCAO/R-induced increase of lipoperoxides, augmented concentrations of palmitoylethanolamide and docosahexaenoic acid, increased relative levels of the cannabinoid receptors type 1 (CB1 and 2 (CB2, and peroxisome-proliferator-activated-receptor (PPAR-α proteins. Increased expression of CB1/CB2 receptors mirrored that of synaptophysin and post-synaptic density-95 protein. No BCCAO/R-induced changes occurred in the temporal-occipital cortex. Collectively, our results demonstrate that, in the frontal cortex, RVT pre-treatment prevents the BCCAO/R-induced oxidative stress and modulates the endocannabinoid and PPAR-α systems. The increased expression of synaptic structural proteins further suggests the possible efficacy of RVT as a dietary supplement to preserve the nervous tissue metabolism and control the physiological response to the hypoperfusion/reperfusion challenge.

Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

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Cuncong Zhong

2016-07-01

Full Text Available Analyses of metagenome data (MG and metatranscriptome data (MT are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE genes. Finally, HMM

Studying Membrane Protein Structure and Function Using Nanodiscs

DEFF Research Database (Denmark)

Huda, Pie

The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

Computational protein design: a review

International Nuclear Information System (INIS)

Coluzza, Ivan

2017-01-01

Proteins are one of the most versatile modular assembling systems in nature. Experimentally, more than 110 000 protein structures have been identified and more are deposited every day in the Protein Data Bank. Such an enormous structural variety is to a first approximation controlled by the sequence of amino acids along the peptide chain of each protein. Understanding how the structural and functional properties of the target can be encoded in this sequence is the main objective of protein design. Unfortunately, rational protein design remains one of the major challenges across the disciplines of biology, physics and chemistry. The implications of solving this problem are enormous and branch into materials science, drug design, evolution and even cryptography. For instance, in the field of drug design an effective computational method to design protein-based ligands for biological targets such as viruses, bacteria or tumour cells, could give a significant boost to the development of new therapies with reduced side effects. In materials science, self-assembly is a highly desired property and soon artificial proteins could represent a new class of designable self-assembling materials. The scope of this review is to describe the state of the art in computational protein design methods and give the reader an outline of what developments could be expected in the near future. (topical review)

Seeing the trees through the forest : sequence-based homo- and heteromeric protein-protein interaction sites prediction using random forest

NARCIS (Netherlands)

Hou, Qingzhen; De Geest, Paul F.G.; Vranken, Wim F.; Heringa, Jaap; Feenstra, K. Anton

2017-01-01

Motivation: Genome sequencing is producing an ever-increasing amount of associated protein sequences. Few of these sequences have experimentally validated annotations, however, and computational predictions are becoming increasingly successful in producing such annotations. One key challenge remains

Efficient extraction of protein-protein interactions from full-text articles.

Science.gov (United States)

Hakenberg, Jörg; Leaman, Robert; Vo, Nguyen Ha; Jonnalagadda, Siddhartha; Sullivan, Ryan; Miller, Christopher; Tari, Luis; Baral, Chitta; Gonzalez, Graciela

2010-01-01

Proteins and their interactions govern virtually all cellular processes, such as regulation, signaling, metabolism, and structure. Most experimental findings pertaining to such interactions are discussed in research papers, which, in turn, get curated by protein interaction databases. Authors, editors, and publishers benefit from efforts to alleviate the tasks of searching for relevant papers, evidence for physical interactions, and proper identifiers for each protein involved. The BioCreative II.5 community challenge addressed these tasks in a competition-style assessment to evaluate and compare different methodologies, to make aware of the increasing accuracy of automated methods, and to guide future implementations. In this paper, we present our approaches for protein-named entity recognition, including normalization, and for extraction of protein-protein interactions from full text. Our overall goal is to identify efficient individual components, and we compare various compositions to handle a single full-text article in between 10 seconds and 2 minutes. We propose strategies to transfer document-level annotations to the sentence-level, which allows for the creation of a more fine-grained training corpus; we use this corpus to automatically derive around 5,000 patterns. We rank sentences by relevance to the task of finding novel interactions with physical evidence, using a sentence classifier built from this training corpus. Heuristics for paraphrasing sentences help to further remove unnecessary information that might interfere with patterns, such as additional adjectives, clauses, or bracketed expressions. In BioCreative II.5, we achieved an f-score of 22 percent for finding protein interactions, and 43 percent for mapping proteins to UniProt IDs; disregarding species, f-scores are 30 percent and 55 percent, respectively. On average, our best-performing setup required around 2 minutes per full text. All data and pattern sets as well as Java classes that

Long-term delivery of protein therapeutics.

Science.gov (United States)

Vaishya, Ravi; Khurana, Varun; Patel, Sulabh; Mitra, Ashim K

2015-03-01

Proteins are effective biotherapeutics with applications in diverse ailments. Despite being specific and potent, their full clinical potential has not yet been realized. This can be attributed to short half-lives, complex structures, poor in vivo stability, low permeability, frequent parenteral administrations and poor adherence to treatment in chronic diseases. A sustained release system, providing controlled release of proteins, may overcome many of these limitations. This review focuses on recent development in approaches, especially polymer-based formulations, which can provide therapeutic levels of proteins over extended periods. Advances in particulate, gel-based formulations and novel approaches for extended protein delivery are discussed. Emphasis is placed on dosage form, method of preparation, mechanism of release and stability of biotherapeutics. Substantial advancements have been made in the field of extended protein delivery via various polymer-based formulations over last decade despite the unique delivery-related challenges posed by protein biologics. A number of injectable sustained-release formulations have reached market. However, therapeutic application of proteins is still hampered by delivery-related issues. A large number of protein molecules are under clinical trials, and hence, there is an urgent need to develop new methods to deliver these highly potent biologics.

Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

Science.gov (United States)

Golshani, Maryam; Rafati, Sima; Nejati-Moheimani, Mehdi; Ghasemian, Melina; Bouzari, Saeid

2016-12-25

In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection. Copyright © 2016 Elsevier B.V. All rights reserved.

SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

Energy Technology Data Exchange (ETDEWEB)

JOHN C WALKER

2011-11-01

Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

Controlled release and intracellular protein delivery from mesoporous silica nanoparticles.

Science.gov (United States)

Deodhar, Gauri V; Adams, Marisa L; Trewyn, Brian G

2017-01-01

Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized. This serves to control the movement of molecules in and out of the pores, thus entrapping guest molecules until a specific stimulus triggers release. In this review, we will cover the benefits of using MSN as protein therapeutic carriers, demonstrating that there is great diversity in the ways MSN can be used to service proteins. Methods for controlling the physical dimensions of pores via synthetic conditions, applications of therapeutic protein loaded MSN materials in cancer therapies, delivering protein loaded MSN materials to plant cells using biolistic methods, and common stimuli-responsive functionalities will be discussed. New and exciting strategies for controlled release and manipulation of proteins are also covered in this review. While research in this area has advanced substantially, we conclude this review with future challenges to be tackled by the scientific community. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ProNormz--an integrated approach for human proteins and protein kinases normalization.

Science.gov (United States)

Subramani, Suresh; Raja, Kalpana; Natarajan, Jeyakumar

2014-02-01

The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein-protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz. Copyright © 2013 Elsevier Inc. All rights reserved.

Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

Directory of Open Access Journals (Sweden)

Gorin Andrey A

2008-05-01

Full Text Available Abstract Background Protein-protein interactions are ubiquitous and essential for all cellular processes. High-resolution X-ray crystallographic structures of protein complexes can reveal the details of their function and provide a basis for many computational and experimental approaches. Differentiation between biological and non-biological contacts and reconstruction of the intact complex is a challenging computational problem. A successful solution can provide additional insights into the fundamental principles of biological recognition and reduce errors in many algorithms and databases utilizing interaction information extracted from the Protein Data Bank (PDB. Results We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1 comprehensively collecting all protein-protein interfaces; (2 clustering similar protein-protein interfaces together; (3 estimating the probability that each cluster is relevant based on a diverse set of properties; and (4 combining these scores for each PDB entry in order to predict the complex structure. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions. These interfaces, as well as the predicted protein complexes, are available from the Protein Interface Server (PInS website (see Availability and requirements section. Conclusion Our method demonstrates an almost two-fold reduction of the annotation error rate as evaluated on a large benchmark set of complexes validated from the literature. We also estimate relative contributions of each interface property to the accurate discrimination of biologically relevant interfaces and discuss possible directions for further improving the prediction method.

False positive reduction in protein-protein interaction predictions using gene ontology annotations

Directory of Open Access Journals (Sweden)

Lin Yen-Han

2007-07-01

Full Text Available Abstract Background Many crucial cellular operations such as metabolism, signalling, and regulations are based on protein-protein interactions. However, the lack of robust protein-protein interaction information is a challenge. One reason for the lack of solid protein-protein interaction information is poor agreement between experimental findings and computational sets that, in turn, comes from huge false positive predictions in computational approaches. Reduction of false positive predictions and enhancing true positive fraction of computationally predicted protein-protein interaction datasets based on highly confident experimental results has not been adequately investigated. Results Gene Ontology (GO annotations were used to reduce false positive protein-protein interactions (PPI pairs resulting from computational predictions. Using experimentally obtained PPI pairs as a training dataset, eight top-ranking keywords were extracted from GO molecular function annotations. The sensitivity of these keywords is 64.21% in the yeast experimental dataset and 80.83% in the worm experimental dataset. The specificities, a measure of recovery power, of these keywords applied to four predicted PPI datasets for each studied organisms, are 48.32% and 46.49% (by average of four datasets in yeast and worm, respectively. Based on eight top-ranking keywords and co-localization of interacting proteins a set of two knowledge rules were deduced and applied to remove false positive protein pairs. The 'strength', a measure of improvement provided by the rules was defined based on the signal-to-noise ratio and implemented to measure the applicability of knowledge rules applying to the predicted PPI datasets. Depending on the employed PPI-predicting methods, the strength varies between two and ten-fold of randomly removing protein pairs from the datasets. Conclusion Gene Ontology annotations along with the deduced knowledge rules could be implemented to partially

Expression and Purification of Soluble STAT5b/STAT3 Proteins for SH2 Domain Binding Assay.

Science.gov (United States)

Asai, Akira; Takakuma, Kazuyuki

2017-01-01

When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.

Molecularly imprinted polymers for the recognition of proteins: the state of the art.

Science.gov (United States)

Bossi, A; Bonini, F; Turner, A P F; Piletsky, S A

2007-01-15

Molecular imprinting has proved to be an effective technique for the creation of recognition sites on a polymer scaffold. Protein imprinting has been a focus for many chemists working in the area of molecular recognition, since the creation of synthetic polymers that can specifically recognise proteins is a very challenging but potentially extremely rewarding objective. It is expected that molecularly imprinted polymers (MIPs) with specificity for proteins will find application in medicine, diagnostics, proteomics, environmental analysis, sensors and drug delivery. In this review, the authors provide an overview of the progress achieved in the decade between 1994 and 2005, with respect to the challenging area of MIPs for protein recognition. The discussion furnishes a comparative analysis of different approaches developed, underlining their relative advantages and disadvantages and highlighting trends and possible future directions.

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

Science.gov (United States)

Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

2015-11-12

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

Peritrophin-like protein from Litopenaeus vannamei (LvPT) involved in white spot syndrome virus (WSSV) infection in digestive tract challenged with reverse gavage

Science.gov (United States)

Xie, Shijun; Li, Fuhua; Zhang, Xiaojun; Zhang, Jiquan; Xiang, Jianhai

2017-11-01

The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus (WSSV) in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of Litopenaeus vannamei (LvPT) was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of LvPT was predicted with the GenScript OptimumAntigen™ design tool. An anti-LvPT polyclonal antibody was produced and shown to specifically bind a band at 27 kDa, identified as LvPT. The LvPT protein was expressed and its concentration determined. LvPT dsRNA (4 μg per shrimp) was used to inhibit LvPT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection (hpi). Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after LvPT dsRNA interference than in those of the controls at 48 and 72 hpi. Our results imply that LvPT plays an important role during WSSV infection of the digestive tract.

Detection of Locally Over-Represented GO Terms in Protein-Protein Interaction Networks

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LAVALLÉE-ADAM, MATHIEU; COULOMBE, BENOIT; BLANCHETTE, MATHIEU

2015-01-01

High-throughput methods for identifying protein-protein interactions produce increasingly complex and intricate interaction networks. These networks are extremely rich in information, but extracting biologically meaningful hypotheses from them and representing them in a human-readable manner is challenging. We propose a method to identify Gene Ontology terms that are locally over-represented in a subnetwork of a given biological network. Specifically, we propose several methods to evaluate the degree of clustering of proteins associated to a particular GO term in both weighted and unweighted PPI networks, and describe efficient methods to estimate the statistical significance of the observed clustering. We show, using Monte Carlo simulations, that our best approximation methods accurately estimate the true p-value, for random scale-free graphs as well as for actual yeast and human networks. When applied to these two biological networks, our approach recovers many known complexes and pathways, but also suggests potential functions for many subnetworks. Online Supplementary Material is available at www.liebertonline.com. PMID:20377456

Sensitivity of the appetite control system in obese subjects to nutritional and serotoninergic challenges.

Science.gov (United States)

Hill, A J; Blundell, J E

1990-03-01

The sensitivity of the appetite system of a group of obese individuals was assessed in response to two challenges known to reduce hunger and enhance satiety in lean people. The challenges were the presentation of a caloric (high protein) load and the activation of serotonin systems. Eight obese female adults (BMI = 38) received 2 X 15 mg d-fenfluramine or placebo daily for 3 days, the study conforming to a 2 X 2 factor (drug X lunch type), double blind, repeated measures design. Three hours after dosing on day 3 they ate either a high carbohydrate (63 percent of total energy) or high protein (54 percent) lunchtime meal (the caloric load). These fixed meal challenges were equal in energy (475 kcal), weight and fat content. Ratings of hunger motivation and food preferences were tracked over the course of lunch and for a further 3 hours, at which point subjects returned for a self-selection test meal. Intakes from this second open meal revealed significant main effects of both caloric load and drug on energy intake, with the high protein d-fenfluramine combination being the most potent anorectic pairing. These findings were supported by the profiles of hunger motivation. This study has confirmed that the appetite system of these subjects was responsive to these biologically relevant challenges. The results suggest that the combination of an appetite modulating drug with specific dietary intervention may represent an effective strategy for the management of hunger arising from caloric restriction.

MEGADOCK 4.0: an ultra-high-performance protein-protein docking software for heterogeneous supercomputers.

Science.gov (United States)

Ohue, Masahito; Shimoda, Takehiro; Suzuki, Shuji; Matsuzaki, Yuri; Ishida, Takashi; Akiyama, Yutaka

2014-11-15

The application of protein-protein docking in large-scale interactome analysis is a major challenge in structural bioinformatics and requires huge computing resources. In this work, we present MEGADOCK 4.0, an FFT-based docking software that makes extensive use of recent heterogeneous supercomputers and shows powerful, scalable performance of >97% strong scaling. MEGADOCK 4.0 is written in C++ with OpenMPI and NVIDIA CUDA 5.0 (or later) and is freely available to all academic and non-profit users at: http://www.bi.cs.titech.ac.jp/megadock. akiyama@cs.titech.ac.jp Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

Science.gov (United States)

Tackett, Alan J; DeGrasse, Jeffrey A; Sekedat, Matthew D; Oeffinger, Marlene; Rout, Michael P; Chait, Brian T

2005-01-01

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

The potential of protein-nanomaterial interaction for advanced drug delivery

DEFF Research Database (Denmark)

Peng, Qiang; Mu, Huiling

2016-01-01

Nanomaterials, like nanoparticles, micelles, nano-sheets, nanotubes and quantum dots, have great potentials in biomedical fields. However, their delivery is highly limited by the formation of protein corona upon interaction with endogenous proteins. This new identity, instead of nanomaterial itself...... of such interaction for advanced drug delivery are presented........ Therefore, protein-nanomaterial interaction is a great challenge for nanomaterial systems and should be inhibited. However, this interaction can also be used to functionalize nanomaterials by forming a selected protein corona. Unlike other decoration using exogenous molecules, nanomaterials functionalized...

Nasopharyngeal Protein Biomarkers of Acute Respiratory Virus Infection

Directory of Open Access Journals (Sweden)

Thomas W. Burke

2017-03-01

Full Text Available Infection of respiratory mucosa with viral pathogens triggers complex immunologic events in the affected host. We sought to characterize this response through proteomic analysis of nasopharyngeal lavage in human subjects experimentally challenged with influenza A/H3N2 or human rhinovirus, and to develop targeted assays measuring peptides involved in this host response allowing classification of acute respiratory virus infection. Unbiased proteomic discovery analysis identified 3285 peptides corresponding to 438 unique proteins, and revealed that infection with H3N2 induces significant alterations in protein expression. These include proteins involved in acute inflammatory response, innate immune response, and the complement cascade. These data provide insights into the nature of the biological response to viral infection of the upper respiratory tract, and the proteins that are dysregulated by viral infection form the basis of signature that accurately classifies the infected state. Verification of this signature using targeted mass spectrometry in independent cohorts of subjects challenged with influenza or rhinovirus demonstrates that it performs with high accuracy (0.8623 AUROC, 75% TPR, 97.46% TNR. With further development as a clinical diagnostic, this signature may have utility in rapid screening for emerging infections, avoidance of inappropriate antibacterial therapy, and more rapid implementation of appropriate therapeutic and public health strategies.

Immunogenic and protective efficacy of recombinant protein GtxA-N against Gallibacterium anatis challenge in chickens.

Science.gov (United States)

Pedersen, Ida J; Pors, Susanne E; Bager Skjerning, Ragnhild J; Nielsen, Søren S; Bojesen, Anders M

2015-10-01

Gallibacterium anatis is a major cause of reproductive tract infections in chickens. Here, we aimed to evaluate the efficacy of the recombinant protein GtxA-N at protecting hens, by addressing three objectives; (i) evaluating the antibody response following immunization (ii) scoring and comparing lesions, following challenge with G. anatis, in immunized and non-immunized hens and (iii) investigating if the anti-GtxA-N antibody titre in individual hens correlated with the observed lesions. Two consecutive experiments were performed in hens. In the first experiment hens were immunized with GtxA-N on day 0 and day 14, infected with G. anatis on day 28 and euthanized on day 56. The GtxA-N antibody response was assessed in pooled serum samples throughout the experiment, using an indirect enzyme-linked immunosorbent assay (ELISA). In the second experiment the GtxA-N antibody titres were assessed in individual hens before and after immunization. Subsequently, the hens were inoculated with G. anatis and finally all hens where euthanized and submitted for post mortem examination 48 h after inoculation. Immunization elicited strong antibody responses that lasted at least 8 weeks (P scores following G. anatis infection were significantly lower in immunized hens compared to non-immunized hens (P = .004). Within the immunized group, no correlation was found between the individual antibody titres and the lesion scores. This study clearly demonstrated GtxA-N as a vaccine antigen able of inducing protective immunity against G. anatis.

Regulation of protein homeostasis in neurodegenerative diseases : the role of coding and non-coding genes

NARCIS (Netherlands)

Alvarenga Fernandes Sin, Olga; Nollen, Ellen A. A.

Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell's response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding,

Food-cooking processes modulate allergenic properties of hen's egg white proteins.

Science.gov (United States)

Liu, Xiaoyu; Feng, Bai-Sui; Kong, Xiaoli; Xu, Hong; Li, Xiumin; Yang, Ping-Chang; Liu, Zhigang

2013-01-01

Reducing the allergenicity of food allergens can suppress the clinical symptoms of food allergy. The objective of the present study was to investigate the effects of processing on the allergenic properties of hen's egg white proteins. Eggs were processed by traditional Chinese cooking, including steaming, water boiling, frying, spicing and tea boiling. The contents of processed egg protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; the allergenicity was evaluated by Western blotting, enzyme-linked immunosorbent assay and enzyme allergosorbent test inhibition. Circular dichroism spectrum analysis of four major egg allergens from various egg products was performed as well. A mouse model of food allergy was developed to test the allergenicity of processed egg protein in vivo. Protein degradation was significant following tea boiling and spiced-tea boiling. The total allergenic potential of water-boiled egg and fried egg was relatively higher than that of steamed egg, spiced egg and tea-boiled egg. Challenge with proteins from raw egg, water-boiled egg and fried egg induced skewed T-helper 2 pattern responses (Th2 responses) in the intestine of mice sensitized to egg proteins; however, when the mice sensitized to egg proteins were challenged with proteins from steamed egg, spiced egg and tea-boiled egg, respectively, only weak Th2 responses were induced in their intestine. Processing by steaming, spicing, or tea boiling can weaken the allergenicity of egg proteins. Copyright © 2012 S. Karger AG, Basel.

Proteomic analysis of an environmental isolate of Rhodotorula mucilaginosa after arsenic and cadmium challenge: Identification of a protein expression signature for heavy metal exposure.

Science.gov (United States)

Ilyas, Sidra; Rehman, Abdul; Coelho, Ana Varela; Sheehan, David

2016-06-01

A metal-resistant Rhodotorula mucilaginosa strain was isolated from an industrial wastewater. Effects on reduced/oxidized glutathione (GSSG/GSH), antioxidant enzymes and proteome were assessed on metal challenge (100mg/L). Increased GSH (mM/g) was found with CdCl2 (18.43±3.34), NaAsO2 (14.76±2.14), CuSO4 (14.73±2.49), and Pb(NO3)2 (15.74±5.3) versus control (7.67±0.95). GSH:GSSG ratio decreased with CdCl2, NaAsO2, and Pb(NO3)2 but not with CuSO4 and cysteine-containing protein levels increased with CdCl2 and NaAsO2. NaAsO2 exposure enhanced glutathione transferase activity but this decreased with CdCl2. Both metals significantly increased glutathione reductase and catalase activities. Metabolism-dependent uptake of Cd and As (12-day exposure) of approximately 65mg/g was observed in live cells with greater cell surface interaction for As compared to Cd. A particular role for arsenic oxidase in As resistance was identified. One dimensional electrophoresis revealed higher oxidation of protein thiols in response to NaAsO2 than to CdCl2. Two dimensional electrophoresis showed altered abundance of some proteins on metal treatment. Selected spots were excised for mass spectrometry and seven proteins identified. Under oxidative stress conditions, xylose reductase, putative chitin deacetylase, 20S proteasome subunit, eukaryotic translation elongation factor 2, valine-tRNA ligase and a metabolic enzyme F0F1 ATP synthase alpha subunit were all expressed as well as a unique hypothetical protein. These may comprise a protein expression signature for metal-induced oxidation in this yeast. Fungi are of widespread importance in agriculture, biodegradation and often show extensive tolerance to heavy metals. This makes them of interest from the perspective of bioremediation. In this study an environmental isolate of R. mucilaginosa showing extensive tolerance of a panel of heavy metals, in particular cadmium and arsenic, was studied. Several biochemical parameters such as

DIRProt: a computational approach for discriminating insecticide resistant proteins from non-resistant proteins.

Science.gov (United States)

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Banchariya, Anjali; Rao, Atmakuri Ramakrishna

2017-03-24

Insecticide resistance is a major challenge for the control program of insect pests in the fields of crop protection, human and animal health etc. Resistance to different insecticides is conferred by the proteins encoded from certain class of genes of the insects. To distinguish the insecticide resistant proteins from non-resistant proteins, no computational tool is available till date. Thus, development of such a computational tool will be helpful in predicting the insecticide resistant proteins, which can be targeted for developing appropriate insecticides. Five different sets of feature viz., amino acid composition (AAC), di-peptide composition (DPC), pseudo amino acid composition (PAAC), composition-transition-distribution (CTD) and auto-correlation function (ACF) were used to map the protein sequences into numeric feature vectors. The encoded numeric vectors were then used as input in support vector machine (SVM) for classification of insecticide resistant and non-resistant proteins. Higher accuracies were obtained under RBF kernel than that of other kernels. Further, accuracies were observed to be higher for DPC feature set as compared to others. The proposed approach achieved an overall accuracy of >90% in discriminating resistant from non-resistant proteins. Further, the two classes of resistant proteins i.e., detoxification-based and target-based were discriminated from non-resistant proteins with >95% accuracy. Besides, >95% accuracy was also observed for discrimination of proteins involved in detoxification- and target-based resistance mechanisms. The proposed approach not only outperformed Blastp, PSI-Blast and Delta-Blast algorithms, but also achieved >92% accuracy while assessed using an independent dataset of 75 insecticide resistant proteins. This paper presents the first computational approach for discriminating the insecticide resistant proteins from non-resistant proteins. Based on the proposed approach, an online prediction server DIRProt has

A Machine Learning Approach for Hot-Spot Detection at Protein-Protein Interfaces

Directory of Open Access Journals (Sweden)

Rita Melo

2016-07-01

Full Text Available Understanding protein-protein interactions is a key challenge in biochemistry. In this work, we describe a more accurate methodology to predict Hot-Spots (HS in protein-protein interfaces from their native complex structure compared to previous published Machine Learning (ML techniques. Our model is trained on a large number of complexes and on a significantly larger number of different structural- and evolutionary sequence-based features. In particular, we added interface size, type of interaction between residues at the interface of the complex, number of different types of residues at the interface and the Position-Specific Scoring Matrix (PSSM, for a total of 79 features. We used twenty-seven algorithms from a simple linear-based function to support-vector machine models with different cost functions. The best model was achieved by the use of the conditional inference random forest (c-forest algorithm with a dataset pre-processed by the normalization of features and with up-sampling of the minor class. The method has an overall accuracy of 0.80, an F1-score of 0.73, a sensitivity of 0.76 and a specificity of 0.82 for the independent test set.

The first decade of MALDI protein profiling

DEFF Research Database (Denmark)

Albrethsen, Jakob

2011-01-01

MALDI protein profiling has identified several important challenges in omics-based biomarker research. First, research into the analytical performance of a novel omics-platform of potential diagnostic impact must be carried out in a critical manner and according to common guidelines. Evaluation s...

Predicting protein structures with a multiplayer online game

OpenAIRE

Cooper, Seth; Khatib, Firas; Treuille, Adrien; Barbero, Janos; Lee, Jeehyung; Beenen, Michael; Leaver-Fay, Andrew; Baker, David; Popović, Zoran

2010-01-01

People exert significant amounts of problem solving effort playing computer games. Simple image- and text-recognition tasks have been successfully crowd-sourced through gamesi, ii, iii, but it is not clear if more complex scientific problems can be similarly solved with human-directed computing. Protein structure prediction is one such problem: locating the biologically relevant native conformation of a protein is a formidable computational challenge given the very large size of the search sp...

Single-molecule protein sequencing through fingerprinting: computational assessment

Science.gov (United States)

Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

2015-10-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

Single-molecule protein sequencing through fingerprinting: computational assessment

International Nuclear Information System (INIS)

Yao, Yao; Docter, Margreet; Van Ginkel, Jetty; Joo, Chirlmin; De Ridder, Dick

2015-01-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences. (paper)

Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

DEFF Research Database (Denmark)

Chi, Celestine Ngang; Bach, Anders; Engström, Åke

2011-01-01

The E6 protein of human papillomavirus exhibits complex interaction patterns with several host proteins and their roles in HPV mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor......, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues...

Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus

2013-01-01

Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange...... in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor...

Recombinant erythropoietin and analogues: a challenge for doping control.

Science.gov (United States)

Pascual, J A; Belalcazar, V; de Bolos, C; Gutiérrez, R; Llop, E; Segura, J

2004-04-01

Erythropoietin (EPO) increases the number of circulating erythrocytes and thus muscle oxygenation. The availability of the recombinant protein (rEPO) has increased the risk of its illegal use in sports, its detection being a difficult challenge. Five different hematopoietic parameters were initially chosen as indirect markers of rEPO abuse: concentration of serum EPO, concentration of serum-soluble transferrin receptors (sTFr), hematocrit, percentage of reticulocytes, and percentage of macrocytes. New models considering only hemoglobin, serum EPO concentration, and percentage of reticulocytes are simpler and seem to be more sensitive when low doses of rEPO are used. A more direct method of urine analysis (isoelectrofocusing, double blotting, and chemiluminescent detection) based on the charge differences between rEPO and endogenous EPO, related to their carbohydrate composition, provides proof of rEPO use. Furthermore, this approach permits the detection of darbepoetin, a direct analogue of EPO also known as NESP ("new erythropoiesis stimulating protein"). Recently a protein conjugate, "synthetic erythropoiesis protein" (SEP), containing precision-length, monodisperse, negatively charged polymers instead of oligosaccharides has been synthesized. Finally, EPO-mimetics are molecules capable of acting as EPO in dimerizing the EPO receptor. Two kinds of EPO-mimetics have been described: peptides and nonpeptides. The enhancement of oxygen availability to muscles by rEPO, analogues, and mimetics constitutes one of the main challenges to doping control. Major steps have already been developed for detection ofrEPO and some analogues. In the near future, the transfection to an athlete's body of genes that code for erythropoietin might be an emerging doping issue, and sports authorities have incorporated "gene doping" among the prohibited practices.

Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

Directory of Open Access Journals (Sweden)

Roberts Paul C

2009-04-01

Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

Ranking beta sheet topologies of proteins

DEFF Research Database (Denmark)

Fonseca, Rasmus; Helles, Glennie; Winter, Pawel

2010-01-01

One of the challenges of protein structure prediction is to identify long-range interactions between amino acids. To reliably predict such interactions, we enumerate, score and rank all beta-topologies (partitions of beta-strands into sheets, orderings of strands within sheets and orientations...... of paired strands) of a given protein. We show that the beta-topology corresponding to the native structure is, with high probability, among the top-ranked. Since full enumeration is very time-consuming, we also suggest a method to deal with proteins with many beta-strands. The results reported...... in this paper are highly relevant for ab initio protein structure prediction methods based on decoy generation. The top-ranked beta-topologies can be used to find initial conformations from which conformational searches can be started. They can also be used to filter decoys by removing those with poorly...

The protein side of the central dogma: permanence and change.

Science.gov (United States)

Morange, Michel

2006-01-01

There are two facets to the central dogma proposed by Francis Crick in 1957. One concerns the relation between the sequence of nucleotides and the sequence of amino acids, the second is devoted to the relation between the sequence of amino acids and the native three-dimensional structure of proteins. 'Folding is simply a function of the order of the amino acids,' i.e. no information is required for the proper folding of a protein other than the information contained in its sequence. This protein side of the central dogma was elaborated in a scientific context in which the characteristics and functions of proteins, and the mechanisms of protein folding, were seen very differently. This context, which made the folding problem a simple one, supported the bold proposition of Francis Crick. The protein side of the central dogma was not challenged by the discovery of prions if one adopts the definition of information given by Francis Crick. It might have been challenged by the discovery that regulatory enzymes exist in different conformations, and the evidence for the existence of chaperones assisting protein folding. But it was not, and folding remains what it was for Francis Crick, 'simply a function of the order of amino acids'. But the meaning of 'function' has dramatically changed. It is no longer the result of simple physicochemical laws, but that of a long evolutionary process which has optimized protein folding. Molecular mechanistic explanations have to be allied with evolutionary explanations, in a way characteristic of present biology.

"Meatless days" or "less but better"? Exploring strategies to adapt Western meat consumption to health and sustainability challenges

NARCIS (Netherlands)

de Boer, J.; Schösler, H.; Aiking, H.

2014-01-01

Adapting Western meat consumption to health and sustainability challenges requires an overall reduction of industrially produced animal proteins plus a partial replacement by plant proteins. Combining insights on food, environment, and consumers, this paper aims to explore change strategies that may

Combination of pneumococcal surface protein A (PspA with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice.

Directory of Open Access Journals (Sweden)

Maria Leonor S Oliveira

Full Text Available Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP that is currently part of the DTP (diphtheria-tetanus-pertussis vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low protected mice against challenge with two

[Study on the anti-NTHi infection of Hap recombinant protein in vivo].

Science.gov (United States)

Li, Wan-yi; Wang, Bao-ning; Zuo, Feng-qiong; Zeng, Wei; Feng, Feng; Kuang, Yu; Jiang, Zhong-hua; Li, Ming-yuan

2010-07-01

To observe the immune effect of Hap recombinant protein on murine model of bronchopneumonia infected with NTHi, and explore the mechanism about the anti-NTHi infection. The C57BL/6 mice intranasally immunized with purified Hap recombinant protein and CT-B were challenged by NTHi encased in agar beads. The immunifaction of anti-infection was observed through encocyte counting of BALF, bacteria detection of lung and the pathologyical change of lung tissue. In the challenge with NTHi experiment, the inflammatory exudation of the infected murine and pathological change of lung tissue was relieved by combined immunization of Hap recombinant protein and CT-B, and quantity of NTHi in lung of the infected murine was reduced obviously. The Hap recombinant protein also had good ability of anti-NTHi infection in the murine model of NTHi bronchopneumonia. This study could offer the oretical and experimental basis for development of new vaccine against NTHi.

CombiMotif: A new algorithm for network motifs discovery in protein-protein interaction networks

Science.gov (United States)

Luo, Jiawei; Li, Guanghui; Song, Dan; Liang, Cheng

2014-12-01

Discovering motifs in protein-protein interaction networks is becoming a current major challenge in computational biology, since the distribution of the number of network motifs can reveal significant systemic differences among species. However, this task can be computationally expensive because of the involvement of graph isomorphic detection. In this paper, we present a new algorithm (CombiMotif) that incorporates combinatorial techniques to count non-induced occurrences of subgraph topologies in the form of trees. The efficiency of our algorithm is demonstrated by comparing the obtained results with the current state-of-the art subgraph counting algorithms. We also show major differences between unicellular and multicellular organisms. The datasets and source code of CombiMotif are freely available upon request.

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

Science.gov (United States)

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.

In Vitro Analysis of Metabolite Transport Proteins.

Science.gov (United States)

Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P M

2017-01-01

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

Building blocks for protein interaction devices

Science.gov (United States)

Grünberg, Raik; Ferrar, Tony S.; van der Sloot, Almer M.; Constante, Marco; Serrano, Luis

2010-01-01

Here, we propose a framework for the design of synthetic protein networks from modular protein–protein or protein–peptide interactions and provide a starter toolkit of protein building blocks. Our proof of concept experiments outline a general work flow for part–based protein systems engineering. We streamlined the iterative BioBrick cloning protocol and assembled 25 synthetic multidomain proteins each from seven standardized DNA fragments. A systematic screen revealed two main factors controlling protein expression in Escherichia coli: obstruction of translation initiation by mRNA secondary structure or toxicity of individual domains. Eventually, 13 proteins were purified for further characterization. Starting from well-established biotechnological tools, two general–purpose interaction input and two readout devices were built and characterized in vitro. Constitutive interaction input was achieved with a pair of synthetic leucine zippers. The second interaction was drug-controlled utilizing the rapamycin-induced binding of FRB(T2098L) to FKBP12. The interaction kinetics of both devices were analyzed by surface plasmon resonance. Readout was based on Förster resonance energy transfer between fluorescent proteins and was quantified for various combinations of input and output devices. Our results demonstrate the feasibility of parts-based protein synthetic biology. Additionally, we identify future challenges and limitations of modular design along with approaches to address them. PMID:20215443

Current Peptide and Protein Candidates Challenging HIV Therapy beyond the Vaccine Era

Directory of Open Access Journals (Sweden)

Koollawat Chupradit

2017-09-01

Full Text Available Human immunodeficiency virus (HIV is a causative agent of acquired immune deficiency syndrome (AIDS. Highly active antiretroviral therapy (HAART can slow down the replication of HIV-1, leading to an improvement in the survival of HIV-1-infected patients. However, drug toxicities and poor drug administration has led to the emergence of a drug-resistant strain. HIV-1 immunotherapy has been continuously developed, but antibody therapy and HIV vaccines take time to improve its efficiency and have limitations. HIV-1-specific chimeric antigen receptor (CAR-based immunotherapy founded on neutralizing antibodies is now being developed. In HIV-1 therapy, anti-HIV chimeric antigen receptors showed promising data in the suppression of HIV-1 replication; however, autologous transfusion is still a problem. This has led to the development of effective peptides and proteins for an alternative HIV-1 treatment. In this paper, we provide a comprehensive review of potent anti-HIV-1 peptides and proteins that reveal promising therapeutic activities. The inhibitory mechanisms of each therapeutic molecule in the different stages of the HIV-1 life cycle will be discussed herein.

Chaperone-protease networks in mitochondrial protein homeostasis.

Science.gov (United States)

Voos, Wolfgang

2013-02-01

As essential organelles, mitochondria are intimately integrated into the metabolism of a eukaryotic cell. The maintenance of the functional integrity of the mitochondrial proteome, also termed protein homeostasis, is facing many challenges both under normal and pathological conditions. First, since mitochondria are derived from bacterial ancestor cells, the proteins in this endosymbiotic organelle have a mixed origin. Only a few proteins are encoded on the mitochondrial genome, most genes for mitochondrial proteins reside in the nuclear genome of the host cell. This distribution requires a complex biogenesis of mitochondrial proteins, which are mostly synthesized in the cytosol and need to be imported into the organelle. Mitochondrial protein biogenesis usually therefore comprises complex folding and assembly processes to reach an enzymatically active state. In addition, specific protein quality control (PQC) processes avoid an accumulation of damaged or surplus polypeptides. Mitochondrial protein homeostasis is based on endogenous enzymatic components comprising a diverse set of chaperones and proteases that form an interconnected functional network. This review describes the different types of mitochondrial proteins with chaperone functions and covers the current knowledge of their roles in protein biogenesis, folding, proteolytic removal and prevention of aggregation, the principal reactions of protein homeostasis. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

Flow-induced structuring of dense protein dispersions

NARCIS (Netherlands)

Manski, J.M.

2007-01-01

Both health and sustainability are drivers for the increased interest in the creation of novel foods comprising a high protein content. The key challenge is the formation of an attractive, stable and palatable food texture, which is mainly determined by the food structure. In this research, new

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions.

Science.gov (United States)

Delaforge, Elise; Milles, Sigrid; Huang, Jie-Rong; Bouvier, Denis; Jensen, Malene Ringkjøbing; Sattler, Michael; Hart, Darren J; Blackledge, Martin

2016-01-01

Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Obtaining classical swine fever virus E2 recombinant protein and DNA-vaccine on the basis of one subunit

International Nuclear Information System (INIS)

Deryabin, O.; Deryabina, O.; Verbitskiy, P.; Kordyum, V.

2005-01-01

Three forms of E2 recombinant protein were expressed in E. coli. Swine sera obtained against different forms of the recombinant protein were cross-studied with indirect ELISA. Using individual proteins as an antigen, only 15% of sera against other forms of protein reacted positively, while 100% of heterologous sera showed positive reaction with fused protein. Challenge experiments showed the existence of protective action only from the individual protein. Specificity and activity of sera obtained from the animals after control challenge was confirmed in a blocking variant of ELISA. Genetic construction used a eukaryotic vector that contained the E2 protein gene. Immunization of mice with the resulting DNA induced synthesis of specific antibodies, the titre of which increased considerably after additional single immunization with the E2 recombinant protein, expressed in E. coli. This demonstrated the effectiveness of animal priming by DNA vaccine, and the possibility of using the E2 recombinant protein in E. coli for booster vaccination. (author)

Computational fragment-based screening using RosettaLigand: the SAMPL3 challenge

Science.gov (United States)

Kumar, Ashutosh; Zhang, Kam Y. J.

2012-05-01

SAMPL3 fragment based virtual screening challenge provides a valuable opportunity for researchers to test their programs, methods and screening protocols in a blind testing environment. We participated in SAMPL3 challenge and evaluated our virtual fragment screening protocol, which involves RosettaLigand as the core component by screening a 500 fragments Maybridge library against bovine pancreatic trypsin. Our study reaffirmed that the real test for any virtual screening approach would be in a blind testing environment. The analyses presented in this paper also showed that virtual screening performance can be improved, if a set of known active compounds is available and parameters and methods that yield better enrichment are selected. Our study also highlighted that to achieve accurate orientation and conformation of ligands within a binding site, selecting an appropriate method to calculate partial charges is important. Another finding is that using multiple receptor ensembles in docking does not always yield better enrichment than individual receptors. On the basis of our results and retrospective analyses from SAMPL3 fragment screening challenge we anticipate that chances of success in a fragment screening process could be increased significantly with careful selection of receptor structures, protein flexibility, sufficient conformational sampling within binding pocket and accurate assignment of ligand and protein partial charges.

Rationally designed synthetic protein hydrogels with predictable mechanical properties.

Science.gov (United States)

Wu, Junhua; Li, Pengfei; Dong, Chenling; Jiang, Heting; Bin Xue; Gao, Xiang; Qin, Meng; Wang, Wei; Bin Chen; Cao, Yi

2018-02-12

Designing synthetic protein hydrogels with tailored mechanical properties similar to naturally occurring tissues is an eternal pursuit in tissue engineering and stem cell and cancer research. However, it remains challenging to correlate the mechanical properties of protein hydrogels with the nanomechanics of individual building blocks. Here we use single-molecule force spectroscopy, protein engineering and theoretical modeling to prove that the mechanical properties of protein hydrogels are predictable based on the mechanical hierarchy of the cross-linkers and the load-bearing modules at the molecular level. These findings provide a framework for rationally designing protein hydrogels with independently tunable elasticity, extensibility, toughness and self-healing. Using this principle, we demonstrate the engineering of self-healable muscle-mimicking hydrogels that can significantly dissipate energy through protein unfolding. We expect that this principle can be generalized for the construction of protein hydrogels with customized mechanical properties for biomedical applications.

Thermodynamics of protein destabilization in live cells.

Science.gov (United States)

Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael

2015-10-06

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

[Progress in the spectral library based protein identification strategy].

Science.gov (United States)

Yu, Derui; Ma, Jie; Xie, Zengyan; Bai, Mingze; Zhu, Yunping; Shu, Kunxian

2018-04-25

Exponential growth of the mass spectrometry (MS) data is exhibited when the mass spectrometry-based proteomics has been developing rapidly. It is a great challenge to develop some quick, accurate and repeatable methods to identify peptides and proteins. Nowadays, the spectral library searching has become a mature strategy for tandem mass spectra based proteins identification in proteomics, which searches the experiment spectra against a collection of confidently identified MS/MS spectra that have been observed previously, and fully utilizes the abundance in the spectrum, peaks from non-canonical fragment ions, and other features. This review provides an overview of the implement of spectral library search strategy, and two key steps, spectral library construction and spectral library searching comprehensively, and discusses the progress and challenge of the library search strategy.

Development of a stealth carrier system for structural studies of membrane proteins in solution

DEFF Research Database (Denmark)

Maric, Selma

Structural studies of membrane proteins remain a great experimental challenge. Functional reconstitution into artificial carriers that mimic the native bilayer environment allows for the handling of membrane proteins in solution and enables the use of small-angle scattering techniques for fast...... and reliable structural analysis. The difficulty with this approach is that the carrier discs contribute to the measured scattering intensity in a highly non-trivial fashion, making subsequent data analysis challenging. This thesis presents the development of a specifically deuterated, stealth nanodisc system...

Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

Science.gov (United States)

Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

2016-11-01

Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features. Copyright © 2016. Published by Elsevier Inc.

A glimpse of the future in animal nutrition science. 1. Past and future challenges

OpenAIRE

Tedeschi, Luis Orlindo; Almeida, Amélia Katiane de; Atzori, Alberto Stanislao; Muir, James Pierre; Fonseca, Mozart Alves; Cannas, Antonello

2017-01-01

ABSTRACT If the world population continues to increase exponentially, wealth and education inequalities might become more pronounced in the developing world. Thus, offering affordable, high-quality protein food to people will become more important and daunting than ever. Past and future challenges will increasingly demand quicker and more innovative and efficient solutions. Animal scientists around the globe currently face many challenging issues: from ensuring food security to prevent excess...

Advancements of two dimensional correlation spectroscopy in protein researches

Science.gov (United States)

Tao, Yanchun; Wu, Yuqing; Zhang, Liping

2018-05-01

The developments of two-dimensional correlation spectroscopy (2DCOS) applications in protein studies are discussed, especially for the past two decades. The powerful utilities of 2DCOS combined with various analytical techniques in protein studies are summarized. The emphasis is on the vibration spectroscopic techniques including IR, NIR, Raman and optical activity (ROA), as well as vibration circular dichroism (VCD) and fluorescence spectroscopy. In addition, some new developments, such as hetero-spectral 2DCOS, moving-window correlation, and model based correlation, are also reviewed for their utility in the investigation of the secondary structure, denaturation, folding and unfolding changes of protein. Finally, the new possibility and challenges of 2DCOS in protein research are highlighted as well.

Protein-Protein Interaction Article Classification Using a Convolutional Recurrent Neural Network with Pre-trained Word Embeddings.

Science.gov (United States)

Matos, Sérgio; Antunes, Rui

2017-12-13

Curation of protein interactions from scientific articles is an important task, since interaction networks are essential for the understanding of biological processes associated with disease or pharmacological action for example. However, the increase in the number of publications that potentially contain relevant information turns this into a very challenging and expensive task. In this work we used a convolutional recurrent neural network for identifying relevant articles for extracting information regarding protein interactions. Using the BioCreative III Article Classification Task dataset, we achieved an area under the precision-recall curve of 0.715 and a Matthew's correlation coefficient of 0.600, which represents an improvement over previous works.

Structural interface parameters are discriminatory in recognising near-native poses of protein-protein interactions.

Directory of Open Access Journals (Sweden)

Sony Malhotra

Full Text Available Interactions at the molecular level in the cellular environment play a very crucial role in maintaining the physiological functioning of the cell. These molecular interactions exist at varied levels viz. protein-protein interactions, protein-nucleic acid interactions or protein-small molecules interactions. Presently in the field, these interactions and their mechanisms mark intensively studied areas. Molecular interactions can also be studied computationally using the approach named as Molecular Docking. Molecular docking employs search algorithms to predict the possible conformations for interacting partners and then calculates interaction energies. However, docking proposes number of solutions as different docked poses and hence offers a serious challenge to identify the native (or near native structures from the pool of these docked poses. Here, we propose a rigorous scoring scheme called DockScore which can be used to rank the docked poses and identify the best docked pose out of many as proposed by docking algorithm employed. The scoring identifies the optimal interactions between the two protein partners utilising various features of the putative interface like area, short contacts, conservation, spatial clustering and the presence of positively charged and hydrophobic residues. DockScore was first trained on a set of 30 protein-protein complexes to determine the weights for different parameters. Subsequently, we tested the scoring scheme on 30 different protein-protein complexes and native or near-native structure were assigned the top rank from a pool of docked poses in 26 of the tested cases. We tested the ability of DockScore to discriminate likely dimer interactions that differ substantially within a homologous family and also demonstrate that DOCKSCORE can distinguish correct pose for all 10 recent CAPRI targets.

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy

International Nuclear Information System (INIS)

Zhou, Donghua H.; Nieuwkoop, Andrew J.; Berthold, Deborah A.; Comellas, Gemma; Sperling, Lindsay J.; Tang, Ming; Shah, Gautam J.; Brea, Elliott J.; Lemkau, Luisel R.; Rienstra, Chad M.

2012-01-01

Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

Challenges in Commercializing Biomimetic Membranes.

Science.gov (United States)

Perry, Mark; Madsen, Steen Ulrik; Jørgensen, Tine; Braekevelt, Sylvie; Lauritzen, Karsten; Hélix-Nielsen, Claus

2015-11-05

The discovery of selective water channel proteins-aquaporins-has prompted growing interest in using these proteins, as the building blocks for designing new types of membranes. However, as with any other new and potentially disruptive technology, barriers for successful market entry exist. One category includes customer-related barriers, which can be influenced to some extent. Another category includes market-technical-related barriers, which can be very difficult to overcome by an organization/company aiming at successfully introducing their innovation on the market-in particular if both the organization and the technology are at early stages. Often, one faces barriers from both these categories at the same time, which makes it necessary to gain insight of the particular market when introducing a new innovative product. In this review we present the basic concepts and discuss some of these barriers and challenges associated with introducing biomimetic aquaporin membranes. These include technical issues in membrane production and product testing. Then we discuss possible business models for introducing new technologies in general, followed by a presentation of beach-head market segments relevant for biomimetic aquaporin membranes.

A novel Multi-Agent Ada-Boost algorithm for predicting protein structural class with the information of protein secondary structure.

Science.gov (United States)

Fan, Ming; Zheng, Bin; Li, Lihua

2015-10-01

Knowledge of the structural class of a given protein is important for understanding its folding patterns. Although a lot of efforts have been made, it still remains a challenging problem for prediction of protein structural class solely from protein sequences. The feature extraction and classification of proteins are the main problems in prediction. In this research, we extended our earlier work regarding these two aspects. In protein feature extraction, we proposed a scheme by calculating the word frequency and word position from sequences of amino acid, reduced amino acid, and secondary structure. For an accurate classification of the structural class of protein, we developed a novel Multi-Agent Ada-Boost (MA-Ada) method by integrating the features of Multi-Agent system into Ada-Boost algorithm. Extensive experiments were taken to test and compare the proposed method using four benchmark datasets in low homology. The results showed classification accuracies of 88.5%, 96.0%, 88.4%, and 85.5%, respectively, which are much better compared with the existing methods. The source code and dataset are available on request.

Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria

Directory of Open Access Journals (Sweden)

Sachith D. Gunasinghe

2017-05-01

Full Text Available Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.

The synthesis of recombinant membrane proteins in yeast for structural studies.

Science.gov (United States)

Routledge, Sarah J; Mikaliunaite, Lina; Patel, Anjana; Clare, Michelle; Cartwright, Stephanie P; Bawa, Zharain; Wilks, Martin D B; Low, Floren; Hardy, David; Rothnie, Alice J; Bill, Roslyn M

2016-02-15

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Patterning protein complexes on DNA nanostructures using a GFP nanobody.

Science.gov (United States)

Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

2016-11-01

DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

NARCIS (Netherlands)

Sluyter, Van S.C.; Warnock, N.I.; Schmidt, S.; Anderson, P.; Kan, van J.A.L.; Bacic, A.; Waters, E.J.

2013-01-01

White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the

The Protein Identifier Cross-Referencing (PICR service: reconciling protein identifiers across multiple source databases

Directory of Open Access Journals (Sweden)

Leinonen Rasko

2007-10-01

Full Text Available Abstract Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR service, a web application that provides interactive and programmatic (SOAP and REST access to a mapping algorithm that uses the UniProt Archive (UniParc as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV or Microsoft Excel (XLS files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

Science.gov (United States)

Yamniuk, Aaron P; Newitt, John A; Doyle, Michael L; Arisaka, Fumio; Giannetti, Anthony M; Hensley, Preston; Myszka, David G; Schwarz, Fred P; Thomson, James A; Eisenstein, Edward

2015-12-01

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors.

Science.gov (United States)

Sable, Rushikesh; Jois, Seetharama

2015-06-23

Blocking protein-protein interactions (PPI) using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

A Hands-On Approach to Teaching Protein Translation & Translocation into the ER

Science.gov (United States)

LaBonte, Michelle L.

2013-01-01

The process of protein translation and translocation into the endoplasmic reticulum (ER) can often be challenging for introductory college biology students to visualize. To help them understand how proteins become oriented in the ER membrane, I developed a hands-on activity in which students use Play-Doh to simulate the process of protein…

Quantification and Classification of E. coli Proteome Utilization and Unused Protein Costs across Environments.

Directory of Open Access Journals (Sweden)

Edward J O'Brien

2016-06-01

Full Text Available The costs and benefits of protein expression are balanced through evolution. Expression of un-utilized protein (that have no benefits in the current environment incurs a quantifiable fitness costs on cellular growth rates; however, the magnitude and variability of un-utilized protein expression in natural settings is unknown, largely due to the challenge in determining environment-specific proteome utilization. We address this challenge using absolute and global proteomics data combined with a recently developed genome-scale model of Escherichia coli that computes the environment-specific cost and utility of the proteome on a per gene basis. We show that nearly half of the proteome mass is unused in certain environments and accounting for the cost of this unused protein expression explains >95% of the variance in growth rates of Escherichia coli across 16 distinct environments. Furthermore, reduction in unused protein expression is shown to be a common mechanism to increase cellular growth rates in adaptive evolution experiments. Classification of the unused protein reveals that the unused protein encodes several nutrient- and stress- preparedness functions, which may convey fitness benefits in varying environments. Thus, unused protein expression is the source of large and pervasive fitness costs that may provide the benefit of hedging against environmental change.

The Protein Data Bank in Europe (PDBe): bringing structure to biology

International Nuclear Information System (INIS)

Velankar, Sameer; Kleywegt, Gerard J.

2011-01-01

Some future challenges for the PDB and its guardians are discussed and current and future activities in structural bioinformatics at the Protein Data Bank in Europe (PDBe) are described. The Protein Data Bank in Europe (PDBe) is the European partner in the Worldwide PDB and as such handles depositions of X-ray, NMR and EM data and structure models. PDBe also provides advanced bioinformatics services based on data from the PDB and related resources. Some of the challenges facing the PDB and its guardians are discussed, as well as some of the areas on which PDBe activities will focus in the future (advanced services, ligands, integration, validation and experimental data). Finally, some recent developments at PDBe are described

The Protein Data Bank in Europe (PDBe): bringing structure to biology

Energy Technology Data Exchange (ETDEWEB)

Velankar, Sameer; Kleywegt, Gerard J., E-mail: gerard@ebi.ac.uk [Protein Data Bank in Europe (PDBe), EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom)

2011-04-01

Some future challenges for the PDB and its guardians are discussed and current and future activities in structural bioinformatics at the Protein Data Bank in Europe (PDBe) are described. The Protein Data Bank in Europe (PDBe) is the European partner in the Worldwide PDB and as such handles depositions of X-ray, NMR and EM data and structure models. PDBe also provides advanced bioinformatics services based on data from the PDB and related resources. Some of the challenges facing the PDB and its guardians are discussed, as well as some of the areas on which PDBe activities will focus in the future (advanced services, ligands, integration, validation and experimental data). Finally, some recent developments at PDBe are described.

Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions

Directory of Open Access Journals (Sweden)

Elise Delaforge

2016-09-01

Full Text Available Intrinsically disordered linkers provide multi-domain proteins with degrees of conformational freedom that are often essential for function. These highly dynamic assemblies represent a significant fraction of all proteomes, and deciphering the physical basis of their interactions represents a considerable challenge. Here we describe the difficulties associated with mapping the large-scale domain dynamics and describe two recent examples where solution state methods, in particular NMR spectroscopy, are used to investigate conformational exchange on very different timescales.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

Science.gov (United States)

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Combination of existing and alternative technologies to promote oilseeds and pulses proteins in food applications

OpenAIRE

Chéreau Denis; Videcoq Pauline; Ruffieux Cécile; Pichon Lisa; Motte Jean-Charles; Belaid Saliha; Ventureira Jorge; Lopez Michel

2016-01-01

The continuous world population growth induces a total protein demand increase based mainly on plant sources. To meet these global nutritional challenges, existing and innovative dry and wet fractionation processes will have to be combined to better valorise plant protein fraction from pulses and oilseeds. The worldwide success of soy protein isolates originate from the intrinsic qualities of soybean proteins but also from a con...

Solid-phase synthesis of protein-polymers on reversible immobilization supports.

Science.gov (United States)

Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

2018-02-27

Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

Functions of intrinsic disorder in transmembrane proteins

DEFF Research Database (Denmark)

Kjaergaard, Magnus; Kragelund, Birthe B.

2017-01-01

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane...... receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like...... mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding...

A surprising role for conformational entropy in protein function

Science.gov (United States)

Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

2014-01-01

Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

Identifying New Small Proteins in Escherichia coli.

Science.gov (United States)

VanOrsdel, Caitlin E; Kelly, John P; Burke, Brittany N; Lein, Christina D; Oufiero, Christopher E; Sanchez, Joseph F; Wimmers, Larry E; Hearn, David J; Abuikhdair, Fatimeh J; Barnhart, Kathryn R; Duley, Michelle L; Ernst, Sarah E G; Kenerson, Briana A; Serafin, Aubrey J; Hemm, Matthew R

2018-04-12

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Towson University.

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Pierced Lasso Proteins

Science.gov (United States)

Jennings, Patricia

Entanglement and knots are naturally occurring, where, in the microscopic world, knots in DNA and homopolymers are well characterized. The most complex knots are observed in proteins which are harder to investigate, as proteins are heteropolymers composed of a combination of 20 different amino acids with different individual biophysical properties. As new-knotted topologies and new proteins containing knots continue to be discovered and characterized, the investigation of knots in proteins has gained intense interest. Thus far, the principle focus has been on the evolutionary origin of tying a knot, with questions of how a protein chain `self-ties' into a knot, what the mechanism(s) are that contribute to threading, and the biological relevance and functional implication of a knotted topology in vivo gaining the most insight. Efforts to study the fully untied and unfolded chain indicate that the knot is highly stable, remaining intact in the unfolded state orders of magnitude longer than first anticipated. The persistence of ``stable'' knots in the unfolded state, together with the challenge of defining an unfolded and untied chain from an unfolded and knotted chain, complicates the study of fully untied protein in vitro. Our discovery of a new class of knotted proteins, the Pierced Lassos (PL) loop topology, simplifies the knotting approach. While PLs are not easily recognizable by the naked eye, they have now been identified in many proteins in the PDB through the use of computation tools. PL topologies are diverse proteins found in all kingdoms of life, performing a large variety of biological responses such as cell signaling, immune responses, transporters and inhibitors (http://lassoprot.cent.uw.edu.pl/). Many of these PL topologies are secreted proteins, extracellular proteins, as well as, redox sensors, enzymes and metal and co-factor binding proteins; all of which provide a favorable environment for the formation of the disulphide bridge. In the PL

Microglia Transcriptome Changes in a Model of Depressive Behavior after Immune Challenge.

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Dianelys Gonzalez-Pena

Full Text Available Depression symptoms following immune response to a challenge have been reported after the recovery from sickness. A RNA-Seq study of the dysregulation of the microglia transcriptome in a model of inflammation-associated depressive behavior was undertaken. The transcriptome of microglia from mice at day 7 after Bacille Calmette Guérin (BCG challenge was compared to that from unchallenged Control mice and to the transcriptome from peripheral macrophages from the same mice. Among the 562 and 3,851 genes differentially expressed between BCG-challenged and Control mice in microglia and macrophages respectively, 353 genes overlapped between these cells types. Among the most differentially expressed genes in the microglia, serum amyloid A3 (Saa3 and cell adhesion molecule 3 (Cadm3 were over-expressed and coiled-coil domain containing 162 (Ccdc162 and titin-cap (Tcap were under-expressed in BCG-challenged relative to Control. Many of the differentially expressed genes between BCG-challenged and Control mice were associated with neurological disorders encompassing depression symptoms. Across cell types, S100 calcium binding protein A9 (S100A9, interleukin 1 beta (Il1b and kynurenine 3-monooxygenase (Kmo were differentially expressed between challenged and control mice. Immune response, chemotaxis, and chemokine activity were among the functional categories enriched by the differentially expressed genes. Functional categories enriched among the 9,117 genes differentially expressed between cell types included leukocyte regulation and activation, chemokine and cytokine activities, MAP kinase activity, and apoptosis. More than 200 genes exhibited alternative splicing events between cell types including WNK lysine deficient protein kinase 1 (Wnk1 and microtubule-actin crosslinking factor 1(Macf1. Network visualization revealed the capability of microglia to exhibit transcriptome dysregulation in response to immune challenge still after resolution of sickness

Chinese mitten crab (Eriocheir sinensis) iron-sulphur cluster assembly protein 2 (EsIscA2) is differentially regulated after immune and oxidative stress challenges.

Science.gov (United States)

Zhang, Peng; Liu, Yu; Wang, Min; Dong, Miren; Liu, Zhaoqun; Jia, Zhihao; Wang, Weilin; Zhang, Anguo; Wang, Lingling; Song, Linsheng

2018-07-01

stimulation (p < 0.05). When the primary cultured crab hemocytes were incubated with different concentrations of H 2 O 2 for 15 min, the expression level of EsIscA2 mRNA was significantly repressed to the 0.34-0.44-fold of that in the control group. After A. hydrophila stimulation, the mRNA expression of EsGrx2 was up-regulated at 3 h (3.22-fold compared to control group, p < 0.05) and reached the peak at 12 h (4.88-fold, p < 0.05). All these results suggested that EsIscA2 had iron-binding capabilities as observed in IscA proteins from other organisms, supporting the role of EsIscA2 as a mitochondrial iron donor for ISC synthesis in Chinese mitten crab. Its differential mRNA expression after immune and oxidative stress challenges suggested the adaptations of ISC synthesis rates to these stress conditions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

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Ding Wei

2010-11-01

Full Text Available Abstract Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.

An α-Helix-Mimicking 12,13-Helix: Designed α/β/γ-Foldamers as Selective Inhibitors of Protein-Protein Interactions.

Science.gov (United States)

Grison, Claire M; Miles, Jennifer A; Robin, Sylvie; Wilson, Andrew J; Aitken, David J

2016-09-05

A major current challenge in bioorganic chemistry is the identification of effective mimics of protein secondary structures that act as inhibitors of protein-protein interactions (PPIs). In this work, trans-2-aminocyclobutanecarboxylic acid (tACBC) was used as the key β-amino acid component in the design of α/β/γ-peptides to structurally mimic a native α-helix. Suitably functionalized α/β/γ-peptides assume an α-helix-mimicking 12,13-helix conformation in solution, exhibit enhanced proteolytic stability in comparison to the wild-type α-peptide parent sequence from which they are derived, and act as selective inhibitors of the p53/hDM2 interaction. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Antivirulence Properties of an Antifreeze Protein

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Martin Heisig

2014-10-01

Full Text Available As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

Effector proteins of rust fungi.

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Petre, Benjamin; Joly, David L; Duplessis, Sébastien

2014-01-01

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.

Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study

DEFF Research Database (Denmark)

Das, Manabendra; Du, Yang; Ribeiro, Orquidea

2017-01-01

were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes......Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties....... Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n...

Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

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Zhiqiang Yan

Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

Science.gov (United States)

Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

2003-01-01

Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge.

Science.gov (United States)

Campos, Ivana B; Darrieux, Michelle; Ferreira, Daniela M; Miyaji, Eliane N; Silva, Débora A; Arêas, Ana Paula M; Aires, Karina A; Leite, Luciana C C; Ho, Paulo L; Oliveira, Maria Leonor S

2008-04-01

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

The potential biomarkers of drug addiction: proteomic and metabolomics challenges.

Science.gov (United States)

Wang, Lv; Wu, Ning; Zhao, Tai-Yun; Li, Jin

2016-07-28

Drug addiction places a significant burden on society and individuals. Proteomics and metabolomics approaches pave the road for searching potential biomarkers to assist the diagnosis and treatment. This review summarized putative drug addiction-related biomarkers in proteomics and metabolomics studies and discussed challenges and prospects in future studies. Alterations of several hundred proteins and metabolites were reported when exposure to abused drug, which enriched in energy metabolism, oxidative stress response, protein modification and degradation, synaptic function and neurotrasmission, etc. Hsp70, peroxiredoxin-6 and α- and β-synuclein, as well as n-methylserotonin and purine metabolites, were promising as potential biomarker for drug addiction.

A generative, probabilistic model of local protein structure

DEFF Research Database (Denmark)

Boomsma, Wouter; Mardia, Kanti V.; Taylor, Charles C.

2008-01-01

Despite significant progress in recent years, protein structure prediction maintains its status as one of the prime unsolved problems in computational biology. One of the key remaining challenges is an efficient probabilistic exploration of the structural space that correctly reflects the relative...... conformational stabilities. Here, we present a fully probabilistic, continuous model of local protein structure in atomic detail. The generative model makes efficient conformational sampling possible and provides a framework for the rigorous analysis of local sequence-structure correlations in the native state...

A Structural Perspective on the Modulation of Protein-Protein Interactions with Small Molecules.

Science.gov (United States)

Demirel, Habibe Cansu; Dogan, Tunca; Tuncbag, Nurcan

2018-05-31

Protein-protein interactions (PPIs) are the key components in many cellular processes including signaling pathways, enzymatic reactions and epigenetic regulation. Abnormal interactions of some proteins may be pathogenic and cause various disorders including cancer and neurodegenerative diseases. Although inhibiting PPIs with small molecules is a challenging task, it gained an increasing interest because of its strong potential for drug discovery and design. The knowledge of the interface as well as the structural and chemical characteristics of the PPIs and their roles in the cellular pathways are necessary for a rational design of small molecules to modulate PPIs. In this study, we review the recent progress in the field and detail the physicochemical properties of PPIs including binding hot spots with a focus on structural methods. Then, we review recent approaches for structural prediction of PPIs. Finally, we revisit the concept of targeting PPIs in a systems biology perspective and we refer to the non-structural approaches, usually employed when the structural information is not present. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Science.gov (United States)

Hatahet, Feras; Blazyk, Jessica L; Martineau, Eugenie; Mandela, Eric; Zhao, Yongxin; Campbell, Robert E; Beckwith, Jonathan; Boyd, Dana

2015-12-08

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Fragment-based drug discovery and its application to challenging drug targets.

Science.gov (United States)

Price, Amanda J; Howard, Steven; Cons, Benjamin D

2017-11-08

Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The continuing conundrum of the LEA proteins.

Science.gov (United States)

Tunnacliffe, Alan; Wise, Michael J

2007-10-01

Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as "space fillers" to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

Site-Specific PEGylation of Therapeutic Proteins

Directory of Open Access Journals (Sweden)

Jonathan K. Dozier

2015-10-01

Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

International Nuclear Information System (INIS)

Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

2007-01-01

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication

Selecting protein families for environmental features based on manifold regularization.

Science.gov (United States)

Jiang, Xingpeng; Xu, Weiwei; Park, E K; Li, Guangrong

2014-06-01

Recently, statistics and machine learning have been developed to identify functional or taxonomic features of environmental features or physiological status. Important proteins (or other functional and taxonomic entities) to environmental features can be potentially used as biosensors. A major challenge is how the distribution of protein and gene functions embodies the adaption of microbial communities across environments and host habitats. In this paper, we propose a novel regularization method for linear regression to adapt the challenge. The approach is inspired by local linear embedding (LLE) and we call it a manifold-constrained regularization for linear regression (McRe). The novel regularization procedure also has potential to be used in solving other linear systems. We demonstrate the efficiency and the performance of the approach in both simulation and real data.

Comparison of Immunoprotection of Leptospira Recombinant Proteins with conventional vaccine in experimental animals.

Science.gov (United States)

Parthiban, M; Kumar, S Senthil; Balachandran, C; Kumanan, K; Aarthi, K S; Nireesha, G

2015-12-01

Leptospirosis is a bacterial disease caused by bacteria of the genus Leptospira affecting humans and animals. Untreated leptospirosis may result in severe kidney damage, meningitis, liver failure, respiratory distress, and even death. Virulent leptospirosis can rapidly enter kidney fibroblasts and induce a programmed cell death. Thus, it is a challenge for immunologists to develop an effective and safe leptospirosis vaccine. Here, we compared the commercial canine leptospira vaccine and recombinant proteins (OmpL1 and LipL41) with and without adjuvant in terms of immune response and challenge studies in hamsters and immune response studies alone in experimental dogs. The outer membrane proteins viz., lipL41 and OmpL1 of leptospira interrogans serovars icterohaemorrhagiae were amplified. The primers were designed in such a way that amplified products of OmpL1 and lipL41 were ligated and cloned simultaneously into a single vector. The cloned products were expressed in E. coli BL21 cells. The immunoprotection studies were conducted for both recombinant proteins and commercial vaccine. The challenge experiment studies revealed that combination of both rLip41 and rOmpL1 and commercial vaccine gave 83% and 87% protection, respectively. Histopathological investigation revealed mild sub lethal changes were noticed in liver and kidney in commercially vaccinated group alone. The immune responses against recombinant leptospiral proteins were also demonstrated in dogs.

Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

Science.gov (United States)

Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

2014-12-01

Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

DeepQA: Improving the estimation of single protein model quality with deep belief networks

OpenAIRE

Cao, Renzhi; Bhattacharya, Debswapna; Hou, Jie; Cheng, Jianlin

2016-01-01

Background Protein quality assessment (QA) useful for ranking and selecting protein models has long been viewed as one of the major challenges for protein tertiary structure prediction. Especially, estimating the quality of a single protein model, which is important for selecting a few good models out of a large model pool consisting of mostly low-quality models, is still a largely unsolved problem. Results We introduce a novel single-model quality assessment method DeepQA based on deep belie...

Compressibility of the protein-water interface

Science.gov (United States)

Persson, Filip; Halle, Bertil

2018-06-01

The compressibility of a protein relates to its stability, flexibility, and hydrophobic interactions, but the measurement, interpretation, and computation of this important thermodynamic parameter present technical and conceptual challenges. Here, we present a theoretical analysis of protein compressibility and apply it to molecular dynamics simulations of four globular proteins. Using additively weighted Voronoi tessellation, we decompose the solution compressibility into contributions from the protein and its hydration shells. We find that positively cross-correlated protein-water volume fluctuations account for more than half of the protein compressibility that governs the protein's pressure response, while the self correlations correspond to small (˜0.7%) fluctuations of the protein volume. The self compressibility is nearly the same as for ice, whereas the total protein compressibility, including cross correlations, is ˜45% of the bulk-water value. Taking the inhomogeneous solvent density into account, we decompose the experimentally accessible protein partial compressibility into intrinsic, hydration, and molecular exchange contributions and show how they can be computed with good statistical accuracy despite the dominant bulk-water contribution. The exchange contribution describes how the protein solution responds to an applied pressure by redistributing water molecules from lower to higher density; it is negligibly small for native proteins, but potentially important for non-native states. Because the hydration shell is an open system, the conventional closed-system compressibility definitions yield a pseudo-compressibility. We define an intrinsic shell compressibility, unaffected by occupation number fluctuations, and show that it approaches the bulk-water value exponentially with a decay "length" of one shell, less than the bulk-water compressibility correlation length. In the first hydration shell, the intrinsic compressibility is 25%-30% lower than in

Compressibility of the protein-water interface.

Science.gov (United States)

Persson, Filip; Halle, Bertil

2018-06-07

The compressibility of a protein relates to its stability, flexibility, and hydrophobic interactions, but the measurement, interpretation, and computation of this important thermodynamic parameter present technical and conceptual challenges. Here, we present a theoretical analysis of protein compressibility and apply it to molecular dynamics simulations of four globular proteins. Using additively weighted Voronoi tessellation, we decompose the solution compressibility into contributions from the protein and its hydration shells. We find that positively cross-correlated protein-water volume fluctuations account for more than half of the protein compressibility that governs the protein's pressure response, while the self correlations correspond to small (∼0.7%) fluctuations of the protein volume. The self compressibility is nearly the same as for ice, whereas the total protein compressibility, including cross correlations, is ∼45% of the bulk-water value. Taking the inhomogeneous solvent density into account, we decompose the experimentally accessible protein partial compressibility into intrinsic, hydration, and molecular exchange contributions and show how they can be computed with good statistical accuracy despite the dominant bulk-water contribution. The exchange contribution describes how the protein solution responds to an applied pressure by redistributing water molecules from lower to higher density; it is negligibly small for native proteins, but potentially important for non-native states. Because the hydration shell is an open system, the conventional closed-system compressibility definitions yield a pseudo-compressibility. We define an intrinsic shell compressibility, unaffected by occupation number fluctuations, and show that it approaches the bulk-water value exponentially with a decay "length" of one shell, less than the bulk-water compressibility correlation length. In the first hydration shell, the intrinsic compressibility is 25%-30% lower than

Use of a protein engineering strategy to overcome limitations in the production of "Difficult to Express" recombinant proteins.

Science.gov (United States)

Hussain, Hirra; Fisher, David I; Abbott, W Mark; Roth, Robert G; Dickson, Alan J

2017-10-01

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant

Protein-protein interactions as a strategy towards protein-specific drug design: the example of ataxin-1.

Directory of Open Access Journals (Sweden)

Cesira de Chiara

Full Text Available A main challenge for structural biologists is to understand the mechanisms that discriminate between molecular interactions and determine function. Here, we show how partner recognition of the AXH domain of the transcriptional co-regulator ataxin-1 is fine-tuned by a subtle balance between self- and hetero-associations. Ataxin-1 is the protein responsible for the hereditary spinocerebellar ataxia type 1, a disease linked to protein aggregation and transcriptional dysregulation. Expansion of a polyglutamine tract is essential for ataxin-1 aggregation, but the sequence-wise distant AXH domain plays an important aggravating role in the process. The AXH domain is also a key element for non-aberrant function as it intervenes in interactions with multiple protein partners. Previous data have shown that AXH is dimeric in solution and forms a dimer of dimers when crystallized. By solving the structure of a complex of AXH with a peptide from the interacting transcriptional repressor CIC, we show that the dimer interface of AXH is displaced by the new interaction and that, when blocked by the CIC peptide AXH aggregation and misfolding are impaired. This is a unique example in which palindromic self- and hetero-interactions within a sequence with chameleon properties discriminate the partner. We propose a drug design strategy for the treatment of SCA1 that is based on the information gained from the AXH/CIC complex.

ORGANOFINERY: FROM GREEN CROPS TO PROTEINS, ENERGY AND FERTILISER

DEFF Research Database (Denmark)

Salces, Beatriz Molinuevo; Fernandez, Maria Santamaria; Kiel, P.

Difficulties with the supply of organic protein feed; low crop yields and low value of leguminous forage crops and a lack of organic fertilisers are nowadays some of the major challenges faced in organic farming with monogastric animals. Thus, organic farmers are forced to import feed and manure ...... from conventional farms. In order to overcome these challenges, the OrganoFinery project targets to develop a green biorefinery concept where organic crops are utilised for animal feed, fertiliser and energy production by producing biogas....

Botulinum neurotoxin: a marvel of protein design.

Science.gov (United States)

Montal, Mauricio

2010-01-01

Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

Labelling strategies for enhanced application of ICPMS in protein analysis

International Nuclear Information System (INIS)

Bettmer, J.; Kutscher, D.J.

2009-01-01

Full text: Quantitative protein analysis is one of today's challenges in analytical chemistry. Herein, mass spectrometric techniques play an important role with the use of both label-free and labelling approaches. In the field of ICPMS, the latter approach is attractive as it can provide highly sensitive detection of proteins after labelling with metal-containing compounds. Following a brief introduction to the different strategies described in the literature, this presentation will be focussed on protein labelling using a mercury compound (p-hydroxymercuribenzoic acid, pHMB). Besides fundamental studies on the derivatization process itself, a strategy will be presented in which absolute protein quantification can be achieved. Finally, the potential, but also limitations of the technique will be highlighted. (author)

Fanconi anemia proteins in telomere maintenance.

Science.gov (United States)

Sarkar, Jaya; Liu, Yie

2016-07-01

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell. Published by Elsevier B.V.

Targeted Delivery of Protein Drugs by Nanocarriers

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Antonella Battisti

2010-03-01

Full Text Available Recent advances in biotechnology demonstrate that peptides and proteins are the basis of a new generation of drugs. However, the transportation of protein drugs in the body is limited by their high molecular weight, which prevents the crossing of tissue barriers, and by their short lifetime due to immuno response and enzymatic degradation. Moreover, the ability to selectively deliver drugs to target organs, tissues or cells is a major challenge in the treatment of several human diseases, including cancer. Indeed, targeted delivery can be much more efficient than systemic application, while improving bioavailability and limiting undesirable side effects. This review describes how the use of targeted nanocarriers such as nanoparticles and liposomes can improve the pharmacokinetic properties of protein drugs, thus increasing their safety and maximizing the therapeutic effect.

Unveiling network-based functional features through integration of gene expression into protein networks.

Science.gov (United States)

Jalili, Mahdi; Gebhardt, Tom; Wolkenhauer, Olaf; Salehzadeh-Yazdi, Ali

2018-06-01

Decoding health and disease phenotypes is one of the fundamental objectives in biomedicine. Whereas high-throughput omics approaches are available, it is evident that any single omics approach might not be adequate to capture the complexity of phenotypes. Therefore, integrated multi-omics approaches have been used to unravel genotype-phenotype relationships such as global regulatory mechanisms and complex metabolic networks in different eukaryotic organisms. Some of the progress and challenges associated with integrated omics studies have been reviewed previously in comprehensive studies. In this work, we highlight and review the progress, challenges and advantages associated with emerging approaches, integrating gene expression and protein-protein interaction networks to unravel network-based functional features. This includes identifying disease related genes, gene prioritization, clustering protein interactions, developing the modules, extract active subnetworks and static protein complexes or dynamic/temporal protein complexes. We also discuss how these approaches contribute to our understanding of the biology of complex traits and diseases. This article is part of a Special Issue entitled: Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers. Copyright © 2018 Elsevier B.V. All rights reserved.

Predicting protein structures with a multiplayer online game.

Science.gov (United States)

Cooper, Seth; Khatib, Firas; Treuille, Adrien; Barbero, Janos; Lee, Jeehyung; Beenen, Michael; Leaver-Fay, Andrew; Baker, David; Popović, Zoran; Players, Foldit

2010-08-05

People exert large amounts of problem-solving effort playing computer games. Simple image- and text-recognition tasks have been successfully 'crowd-sourced' through games, but it is not clear if more complex scientific problems can be solved with human-directed computing. Protein structure prediction is one such problem: locating the biologically relevant native conformation of a protein is a formidable computational challenge given the very large size of the search space. Here we describe Foldit, a multiplayer online game that engages non-scientists in solving hard prediction problems. Foldit players interact with protein structures using direct manipulation tools and user-friendly versions of algorithms from the Rosetta structure prediction methodology, while they compete and collaborate to optimize the computed energy. We show that top-ranked Foldit players excel at solving challenging structure refinement problems in which substantial backbone rearrangements are necessary to achieve the burial of hydrophobic residues. Players working collaboratively develop a rich assortment of new strategies and algorithms; unlike computational approaches, they explore not only the conformational space but also the space of possible search strategies. The integration of human visual problem-solving and strategy development capabilities with traditional computational algorithms through interactive multiplayer games is a powerful new approach to solving computationally-limited scientific problems.

Bio-Inspired Protein-Based Nanoformulations for Cancer Theranostics

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Yi Gou

2018-04-01

Full Text Available Over the past decade, more interests have been aroused in engineering protein-based nanoformulations for cancer treatment. This excitement originates from the success of FDA approved Abraxane (Albumin-based paclitaxel nanoparticles in 2005. The new generation of biocompatible endogenous protein-based nanoformulations is currently constructed through delivering cancer therapeutic and diagnostic agents simultaneously, as named potential theranostics. Protein nanoformulations are commonly incorporated with dyes, contrast agents, drug payloads or inorganic nanoclusters, serving as imaging-guided combinatorial cancer therapeutics. Employing the nature identity of proteins, the theranostics, escape the clearance by reticuloendothelial cells and have a long blood circulation time. The nanoscale sizet allows them to be penetrated deeply into tumor tissues. In addition, stimuli release and targeted molecules are incorporated to improve the delivery efficiency. The ongoing advancement of protein-based nanoformulations for cancer theranostics in recent 5 years is reviewed in this paper. Fine-designed nanoformulations based on albumin, ferritin, gelatin, and transferrin are highlighted from the literature. Finally, the current challenges are identified in translating protein-based nanoformulations from laboratory to clinical trials.

Dependency on de novo protein synthesis and proteomic changes during metamorphosis of the marine bryozoan Bugula neritina

KAUST Repository

Wong, Yue Him; Arellano, Shawn M; Zhang, Huoming; Ravasi, Timothy; Qian, Pei-Yuan

2010-01-01

synthesis of proteins and, instead, involves post-translational modifications of existing proteins, providing a simple mechanism to quickly initiate metamorphosis. To test our hypothesis, we challenged B. neritina larvae with transcription and translation

Signatures of pleiotropy, economy and convergent evolution in a domain-resolved map of human-virus protein-protein interaction networks.

Science.gov (United States)

Garamszegi, Sara; Franzosa, Eric A; Xia, Yu

2013-01-01

A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are

Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors

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Rushikesh Sable

2015-06-01

Full Text Available Blocking protein-protein interactions (PPI using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.

Early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization.

Science.gov (United States)

Vennema, H; de Groot, R J; Harbour, D A; Dalderup, M; Gruffydd-Jones, T; Horzinek, M C; Spaan, W J

1990-01-01

The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis was recombined into the genome of vaccinia virus. The recombinant induced spike-protein-specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with feline infectious peritonitis virus, these animals succumbed earlier than did the control group immunized with wild-type vaccinia virus (early death syndrome). Images PMID:2154621

Protein adsorption on nanoparticles: model development using computer simulation

International Nuclear Information System (INIS)

Shao, Qing; Hall, Carol K

2016-01-01

The adsorption of proteins on nanoparticles results in the formation of the protein corona, the composition of which determines how nanoparticles influence their biological surroundings. We seek to better understand corona formation by developing models that describe protein adsorption on nanoparticles using computer simulation results as data. Using a coarse-grained protein model, discontinuous molecular dynamics simulations are conducted to investigate the adsorption of two small proteins (Trp-cage and WW domain) on a model nanoparticle of diameter 10.0 nm at protein concentrations ranging from 0.5 to 5 mM. The resulting adsorption isotherms are well described by the Langmuir, Freundlich, Temkin and Kiselev models, but not by the Elovich, Fowler–Guggenheim and Hill–de Boer models. We also try to develop a generalized model that can describe protein adsorption equilibrium on nanoparticles of different diameters in terms of dimensionless size parameters. The simulation results for three proteins (Trp-cage, WW domain, and GB3) on four nanoparticles (diameter  =  5.0, 10.0, 15.0, and 20.0 nm) illustrate both the promise and the challenge associated with developing generalized models of protein adsorption on nanoparticles. (paper)

Protein dynamics in individual human cells: experiment and theory.

Directory of Open Access Journals (Sweden)

Ariel Aharon Cohen

Full Text Available A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge.

Science.gov (United States)

Demberg, Thorsten; Boyer, Jean D; Malkevich, Nina; Patterson, L Jean; Venzon, David; Summers, Ebonita L; Kalisz, Irene; Kalyanaraman, V S; Lee, Eun Mi; Weiner, David B; Robert-Guroff, Marjorie

2008-11-01

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.

Death of a dogma: eukaryotic mRNAs can code for more than one protein.

Science.gov (United States)

Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

2016-01-08

mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

BALB/c Mice Vaccinated with Leishmania major Ribosomal Proteins Extracts Combined with CpG Oligodeoxynucleotides Become Resistant to Disease Caused by a Secondary Parasite Challenge

Directory of Open Access Journals (Sweden)

Laura Ramírez

2010-01-01

Full Text Available Leishmaniasis is an increasing public health problem and effective vaccines are not currently available. We have previously demonstrated that vaccination with ribosomal proteins extracts administered in combination of CpG oligodeoxynucleotides protects susceptible BALB/c mice against primary Leishmania major infection. Here, we evaluate the long-term immunity to secondary infection conferred by this vaccine. We show that vaccinated and infected BALB/c mice were able to control a secondary Leishmania major challenge, since no inflammation and very low number of parasites were observed in the site of reinfection. In addition, although an increment in the parasite burden was observed in the draining lymph nodes of the primary site of infection we did not detected inflammatory lesions at that site. Resistance against reinfection correlated to a predominant Th1 response against parasite antigens. Thus, cell cultures established from spleens and the draining lymph node of the secondary site of infection produced high levels of parasite specific IFN-γ in the absence of IL-4 and IL-10 cytokine production. In addition, reinfected mice showed a high IgG2a/IgG1 ratio for anti-Leishmania antibodies. Our results suggest that ribosomal vaccine, which prevents pathology in a primary challenge, in combination with parasite persistence might be effective for long-term maintenance of immunity.

Hydrolyzed whey protein prevents the development of food allergy to β-lactoglobulin in sensitized mice.

Science.gov (United States)

Gomes-Santos, Ana Cristina; Fonseca, Roberta Cristelli; Lemos, Luisa; Reis, Daniela Silva; Moreira, Thaís Garcias; Souza, Adna Luciana; Silva, Mauro Ramalho; Silvestre, Marialice Pinto Coelho; Cara, Denise Carmona; Faria, Ana Maria Caetano

2015-01-01

Food allergy is an adverse immune response to dietary proteins. Hydrolysates are frequently used for children with milk allergy. However, hydrolysates effects afterwards are poorly studied. The aim of this study was to investigate the immunological consequences of hydrolyzed whey protein in allergic mice. For that, we developed a novel model of food allergy in BALB/c mice sensitized with alum-adsorbed β-lactoglobulin. These mice were orally challenged with either whey protein or whey hydrolysate. Whey-challenged mice had elevated levels of specific IgE and lost weight. They also presented gut inflammation, enhanced levels of SIgA and IL-5 as well as decreased production of IL-4 and IL-10 in the intestinal mucosa. Conversely, mice challenged with hydrolyzate maintained normal levels of IgE, IL-4 and IL-5 and showed no sign of gut inflammation probably due to increased IL-12 production in the gut. Thus, consumption of hydrolysate prevented the development of clinical signs of food allergy in mice. Copyright © 2015 Elsevier Inc. All rights reserved.

HitPredict version 4: comprehensive reliability scoring of physical protein-protein interactions from more than 100 species.

Science.gov (United States)

López, Yosvany; Nakai, Kenta; Patil, Ashwini

2015-01-01

HitPredict is a consolidated resource of experimentally identified, physical protein-protein interactions with confidence scores to indicate their reliability. The study of genes and their inter-relationships using methods such as network and pathway analysis requires high quality protein-protein interaction information. Extracting reliable interactions from most of the existing databases is challenging because they either contain only a subset of the available interactions, or a mixture of physical, genetic and predicted interactions. Automated integration of interactions is further complicated by varying levels of accuracy of database content and lack of adherence to standard formats. To address these issues, the latest version of HitPredict provides a manually curated dataset of 398 696 physical associations between 70 808 proteins from 105 species. Manual confirmation was used to resolve all issues encountered during data integration. For improved reliability assessment, this version combines a new score derived from the experimental information of the interactions with the original score based on the features of the interacting proteins. The combined interaction score performs better than either of the individual scores in HitPredict as well as the reliability score of another similar database. HitPredict provides a web interface to search proteins and visualize their interactions, and the data can be downloaded for offline analysis. Data usability has been enhanced by mapping protein identifiers across multiple reference databases. Thus, the latest version of HitPredict provides a significantly larger, more reliable and usable dataset of protein-protein interactions from several species for the study of gene groups. Database URL: http://hintdb.hgc.jp/htp. © The Author(s) 2015. Published by Oxford University Press.

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

Science.gov (United States)

Zhu, Zhikai; Desaire, Heather

2015-07-01

Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

Integral membrane protein structure determination using pseudocontact shifts

Energy Technology Data Exchange (ETDEWEB)

Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

2015-04-15

Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

PDBStat: a universal restraint converter and restraint analysis software package for protein NMR

International Nuclear Information System (INIS)

Tejero, Roberto; Snyder, David; Mao, Binchen; Aramini, James M.; Montelione, Gaetano T.

2013-01-01

The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data

PDBStat: a universal restraint converter and restraint analysis software package for protein NMR

Energy Technology Data Exchange (ETDEWEB)

Tejero, Roberto [Rutgers, The State University of New Jersey, Center for Advanced Biotechnology and Medicine (United States); Snyder, David [William Paterson University, Department of Chemistry (United States); Mao, Binchen; Aramini, James M.; Montelione, Gaetano T., E-mail: guy@cabm.rutgers.edu [Rutgers, The State University of New Jersey, Center for Advanced Biotechnology and Medicine (United States)

2013-08-15

The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data.

SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm

Directory of Open Access Journals (Sweden)

Lobstein Julie

2012-05-01

Full Text Available Abstract Background Production of correctly disulfide bonded proteins to high yields remains a challenge. Recombinant protein expression in Escherichia coli is the popular choice, especially within the research community. While there is an ever growing demand for new expression strains, few strains are dedicated to post-translational modifications, such as disulfide bond formation. Thus, new protein expression strains must be engineered and the parameters involved in producing disulfide bonded proteins must be understood. Results We have engineered a new E. coli protein expression strain named SHuffle, dedicated to producing correctly disulfide bonded active proteins to high yields within its cytoplasm. This strain is based on the trxB gor suppressor strain SMG96 where its cytoplasmic reductive pathways have been diminished, allowing for the formation of disulfide bonds in the cytoplasm. We have further engineered a major improvement by integrating into its chromosome a signal sequenceless disulfide bond isomerase, DsbC. We probed the redox state of DsbC in the oxidizing cytoplasm and evaluated its role in assisting the formation of correctly folded multi-disulfide bonded proteins. We optimized protein expression conditions, varying temperature, induction conditions, strain background and the co-expression of various helper proteins. We found that temperature has the biggest impact on improving yields and that the E. coli B strain background of this strain was superior to the K12 version. We also discovered that auto-expression of substrate target proteins using this strain resulted in higher yields of active pure protein. Finally, we found that co-expression of mutant thioredoxins and PDI homologs improved yields of various substrate proteins. Conclusions This work is the first extensive characterization of the trxB gor suppressor strain. The results presented should help researchers design the appropriate protein expression conditions using

Development of quantitative proteomics using iTRAQ based on the immunological response of Galleria mellonella larvae challenged with Fusarium oxysporum microconidia.

Directory of Open Access Journals (Sweden)

Amalia Muñoz-Gómez

Full Text Available Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104

Advances in membrane protein crystallography: in situ and in meso data collection

Energy Technology Data Exchange (ETDEWEB)

Weyand, Simone, E-mail: sw644@cam.ac.uk [Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA (United Kingdom); Tate, Christopher G., E-mail: sw644@cam.ac.uk [MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge CB2 0QH (United Kingdom)

2015-05-23

Membrane protein structural biology has made tremendous advances over the last decade but there are still many challenges associated with crystallization, data collection and structure determination. Two independent groups, Axford et al. [(2015), Acta Cryst. D71, 1228–1237] and Huang et al. [(2015), Acta Cryst. D71, 1238–1256], have published methods that make a major contribution to addressing these challenges.

Advances in membrane protein crystallography: in situ and in meso data collection

International Nuclear Information System (INIS)

Weyand, Simone; Tate, Christopher G.

2015-01-01

Membrane protein structural biology has made tremendous advances over the last decade but there are still many challenges associated with crystallization, data collection and structure determination. Two independent groups, Axford et al. [(2015), Acta Cryst. D71, 1228–1237] and Huang et al. [(2015), Acta Cryst. D71, 1238–1256], have published methods that make a major contribution to addressing these challenges

Acute and chronic effects of a 24-hour intravenous triglyceride emulsion challenge on plasma lecithin : cholesterol acyltransferase, phospholipid transfer protein, and cholesteryl ester transfer protein activities

NARCIS (Netherlands)

Riemens, SC; Van Tol, A; Sluiter, WJ; Dullaart, RPF

Lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and cholesteryl ester transfer protein (CETP) are key factors in remodeling of high density lipoproteins (HDL) and triglyceride-rich lipoproteins. We examined the effect of a large, 24 h intravenous fat load on plasma

Drug discovery opportunities and challenges at G protein coupled receptors for long chain free fatty acids

Directory of Open Access Journals (Sweden)

Nicholas D Holliday

2012-01-01

Full Text Available Discovery of G protein coupled receptors for long chain free fatty acids (FFAs, FFA1 (GPR40 and GPR120, has expanded our understanding of these nutrients as signalling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signalling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focussed on agonists to replicate acute benefits of FFA receptor signalling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signalling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further preclinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes.

Technological advances in site-directed spin labeling of proteins.

Science.gov (United States)

Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

2013-10-01

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Conformational Entropy as Collective Variable for Proteins.

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Palazzesi, Ferruccio; Valsson, Omar; Parrinello, Michele

2017-10-05

Many enhanced sampling methods rely on the identification of appropriate collective variables. For proteins, even small ones, finding appropriate descriptors has proven challenging. Here we suggest that the NMR S 2 order parameter can be used to this effect. We trace the validity of this statement to the suggested relation between S 2 and conformational entropy. Using the S 2 order parameter and a surrogate for the protein enthalpy in conjunction with metadynamics or variationally enhanced sampling, we are able to reversibly fold and unfold a small protein and draw its free energy at a fraction of the time that is needed in unbiased simulations. We also use S 2 in combination with the free energy flooding method to compute the unfolding rate of this peptide. We repeat this calculation at different temperatures to obtain the unfolding activation energy.

Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

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Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

2017-03-01

"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

Science.gov (United States)

Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Emerging functions of ribosomal proteins in gene-specific transcription and translation

International Nuclear Information System (INIS)

Lindstroem, Mikael S.

2009-01-01

Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-κB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

Amyloid-related biomarkers and axonal damage proteins in parkinsonian syndromes

DEFF Research Database (Denmark)

Bech, Sara; Hjermind, Lena E; Salvesen, Lisette

2012-01-01

Clinical differentiation between parkinsonian syndromes (PS) remains a challenge despite well-established clinical diagnostic criteria. Specific diagnostic biomarkers have yet to be identified, though in recent years, studies have been published on the aid of certain brain related proteins (BRP) ...

Langmuir-Blodgett nanotemplates for protein crystallography.

Science.gov (United States)

Pechkova, Eugenia; Nicolini, Claudio

2017-12-01

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

KAUST Repository

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2015-01-01

Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

KAUST Repository

Wong, Ka-Chun

2015-06-11

Transcription Factor Binding Sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, Protein Binding Microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build motif models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement using di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Functionality of alternative protein in gluten-free product development.

Science.gov (United States)

Deora, Navneet Singh; Deswal, Aastha; Mishra, Hari Niwas

2015-07-01

Celiac disease is an immune-mediated disease triggered in genetically susceptible individuals by ingested gluten from wheat, rye, barley, and other closely related cereal grains. The current treatment for celiac disease is life-long adherence to a strict gluten-exclusion diet. The replacement of gluten presents a significant technological challenge, as it is an essential structure-building protein, which is necessary for formulating high-quality baked goods. A major limitation in the production of gluten-free products is the lack of protein functionality in non-wheat cereals. Additionally, commercial gluten-free mixes usually contain only carbohydrates, which may significantly limit the amount of protein in the diet. In the recent past, various approaches are attempted to incorporate protein-based ingredients and to modify the functional properties for gluten-free product development. This review aims to the highlight functionality of the alternative protein-based ingredients, which can be utilized for gluten-free product development both functionally as well as nutritionally. © The Author(s) 2014.

A semi-nonparametric mixture model for selecting functionally consistent proteins.

Science.gov (United States)

Yu, Lianbo; Doerge, Rw

2010-09-28

High-throughput technologies have led to a new era of proteomics. Although protein microarray experiments are becoming more common place there are a variety of experimental and statistical issues that have yet to be addressed, and that will carry over to new high-throughput technologies unless they are investigated. One of the largest of these challenges is the selection of functionally consistent proteins. We present a novel semi-nonparametric mixture model for classifying proteins as consistent or inconsistent while controlling the false discovery rate and the false non-discovery rate. The performance of the proposed approach is compared to current methods via simulation under a variety of experimental conditions. We provide a statistical method for selecting functionally consistent proteins in the context of protein microarray experiments, but the proposed semi-nonparametric mixture model method can certainly be generalized to solve other mixture data problems. The main advantage of this approach is that it provides the posterior probability of consistency for each protein.

Organizing membrane-curving proteins: the emerging dynamical picture.

Science.gov (United States)

Simunovic, Mijo; Bassereau, Patricia; Voth, Gregory A

2018-03-30

Lipid membranes play key roles in cells, such as in trafficking, division, infection, remodeling of organelles, among others. The key step in all these processes is creating membrane curvature, typically under the control of many anchored, adhered or included proteins. However, it has become clear that the membrane itself can mediate the interactions among proteins to produce highly ordered assemblies. Computer simulations are ideally suited to investigate protein organization and the dynamics of membrane remodeling at near-micron scales, something that is extremely challenging to tackle experimentally. We review recent computational efforts in modeling protein-caused membrane deformation mechanisms, specifically focusing on coarse-grained simulations. We highlight work that exposed the membrane-mediated ordering of proteins into lines, meshwork, spirals and other assemblies, in what seems to be a very generic mechanism driven by a combination of short and long-ranged forces. Modulating the mechanical properties of membranes is an underexplored signaling mechanism in various processes deserving of more attention in the near future. Copyright © 2018 Elsevier Ltd. All rights reserved.

Heat Shock Proteins: Pathogenic Role in Atherosclerosis and Potential Therapeutic Implications

Directory of Open Access Journals (Sweden)

Arman Kilic

2012-01-01

Full Text Available Heat shock proteins (HSPs are a highly conserved group of proteins that are constitutively expressed and function as molecular chaperones, aiding in protein folding and preventing the accumulation of misfolded proteins. In the arterial wall, HSPs have a protective role under normal physiologic conditions. In disease states, however, HSPs expressed on the vascular endothelial cell surface can act as targets for detrimental autoimmunity due to their highly conserved sequences. Developing therapeutic strategies for atherosclerosis based on HSPs is challenged by the need to balance such physiologic and pathologic roles of these proteins. This paper summarizes the role of HSPs in normal vascular wall processes as well as in the development and progression of atherosclerosis. The potential implications of HSPs in clinical therapies for atherosclerosis are also discussed.

Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways and transcription factors

DEFF Research Database (Denmark)

Deshmukh, Atul S; Murgia, Marta; Nagaraja, Nagarjuna

2015-01-01

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging due to highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art mass...

Dissecting Protein Configurational Entropy into Conformational and Vibrational Contributions.

Science.gov (United States)

Chong, Song-Ho; Ham, Sihyun

2015-10-01

Quantifying how the rugged nature of the underlying free-energy landscape determines the entropic cost a protein must incur upon folding and ligand binding is a challenging problem. Here, we present a novel computational approach that dissects the protein configurational entropy on the basis of the classification of protein dynamics on the landscape into two separate components: short-term vibrational dynamics related to individual free-energy wells and long-term conformational dynamics associated with transitions between wells. We apply this method to separate the configurational entropy of the protein villin headpiece subdomain into its conformational and vibrational components. We find that the change in configurational entropy upon folding is dominated by the conformational entropy despite the fact that the magnitude of the vibrational entropy is the significantly larger component in each of the folded and unfolded states, which is in accord with the previous empirical estimations. The straightforward applicability of our method to unfolded proteins promises a wide range of applications, including those related to intrinsically disordered proteins.

Community challenges in biomedical text mining over 10 years: success, failure and the future.

Science.gov (United States)

Huang, Chung-Chi; Lu, Zhiyong

2016-01-01

One effective way to improve the state of the art is through competitions. Following the success of the Critical Assessment of protein Structure Prediction (CASP) in bioinformatics research, a number of challenge evaluations have been organized by the text-mining research community to assess and advance natural language processing (NLP) research for biomedicine. In this article, we review the different community challenge evaluations held from 2002 to 2014 and their respective tasks. Furthermore, we examine these challenge tasks through their targeted problems in NLP research and biomedical applications, respectively. Next, we describe the general workflow of organizing a Biomedical NLP (BioNLP) challenge and involved stakeholders (task organizers, task data producers, task participants and end users). Finally, we summarize the impact and contributions by taking into account different BioNLP challenges as a whole, followed by a discussion of their limitations and difficulties. We conclude with future trends in BioNLP challenge evaluations. Published by Oxford University Press 2015. This work is written by US Government employees and is in the public domain in the US.

Protein structure prediction using bee colony optimization metaheuristic

DEFF Research Database (Denmark)

Fonseca, Rasmus; Paluszewski, Martin; Winter, Pawel

2010-01-01

of the proteins structure, an energy potential and some optimization algorithm that ¿nds the structure with minimal energy. Bee Colony Optimization (BCO) is a relatively new approach to solving opti- mization problems based on the foraging behaviour of bees. Several variants of BCO have been suggested......Predicting the native structure of proteins is one of the most challenging problems in molecular biology. The goal is to determine the three-dimensional struc- ture from the one-dimensional amino acid sequence. De novo prediction algorithms seek to do this by developing a representation...... our BCO method to generate good solutions to the protein structure prediction problem. The results show that BCO generally ¿nds better solutions than simulated annealing which so far has been the metaheuristic of choice for this problem....

Intrinsically Disordered Proteins in a Physics-Based World

Directory of Open Access Journals (Sweden)

Jianhan Chen

2010-12-01

Full Text Available Intrinsically disordered proteins (IDPs are a newly recognized class of functional proteins that rely on a lack of stable structure for function. They are highly prevalent in biology, play fundamental roles, and are extensively involved in human diseases. For signaling and regulation, IDPs often fold into stable structures upon binding to specific targets. The mechanisms of these coupled binding and folding processes are of significant importance because they underlie the organization of regulatory networks that dictate various aspects of cellular decision-making. This review first discusses the challenge in detailed experimental characterization of these heterogeneous and dynamics proteins and the unique and exciting opportunity for physics-based modeling to make crucial contributions, and then summarizes key lessons from recent de novo simulations of the structure and interactions of several regulatory IDPs.

Signatures of pleiotropy, economy and convergent evolution in a domain-resolved map of human-virus protein-protein interaction networks.

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Sara Garamszegi

Full Text Available A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1 domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2 domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral

Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Science.gov (United States)

Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

2017-03-01

Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

Science.gov (United States)

Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

2011-01-01

Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

Cytokine Expression in CD3+ Cells in an Infant with Food Protein-Induced Enterocolitis Syndrome (FPIES: Case Report

Directory of Open Access Journals (Sweden)

F. Mori

2009-01-01

Full Text Available Food protein-induced enterocolitis syndrome (FPIES is a non-IgE-mediated food allergy characterized by severe vomiting, diarrhea, and often failure to thrive in infants. Symptoms typically resolve after the triggering food-derived protein is removed from the diet and recur within few hours after the re-exposure to the causal protein. The diagnosis is based on clinical symptoms and a positive food challenge. In this study, we report a case of FPIES to rice in an 8-month-old boy. We performed a double-blind placebo-controlled food challenge (DBPCFC to rice and we measured the intracellular T cell expression of interleukin-4 (IL-4; IL-10, and interferon (IFN- pre-and post-challenge during an acute FPIES reaction and when tolerance to rice had been achieved. For the first time we describe an increase in T cell IL-4 and decrease in IFN- expression after a positive challenge with rice (i.e. rice triggered a FPIES attack and an increase in T cell IL-10 expression after rice challenge 6 months later after a negative challenge (i.e., the child had acquired tolerance to rice in an 8 month old with documented FPIES to rice. A Th2 activation associated with high IL-4 levels may contribute to the pathophysiology of the disease. On the other hand, T cell-derived IL-10 may play a role in the acquisition of immunotolerance by regulating the Th1 and Th2 responses.

Determining effects of non-synonymous SNPs on protein-protein interactions using supervised and semi-supervised learning.

Directory of Open Access Journals (Sweden)

Nan Zhao

2014-05-01

Full Text Available Single nucleotide polymorphisms (SNPs are among the most common types of genetic variation in complex genetic disorders. A growing number of studies link the functional role of SNPs with the networks and pathways mediated by the disease-associated genes. For example, many non-synonymous missense SNPs (nsSNPs have been found near or inside the protein-protein interaction (PPI interfaces. Determining whether such nsSNP will disrupt or preserve a PPI is a challenging task to address, both experimentally and computationally. Here, we present this task as three related classification problems, and develop a new computational method, called the SNP-IN tool (non-synonymous SNP INteraction effect predictor. Our method predicts the effects of nsSNPs on PPIs, given the interaction's structure. It leverages supervised and semi-supervised feature-based classifiers, including our new Random Forest self-learning protocol. The classifiers are trained based on a dataset of comprehensive mutagenesis studies for 151 PPI complexes, with experimentally determined binding affinities of the mutant and wild-type interactions. Three classification problems were considered: (1 a 2-class problem (strengthening/weakening PPI mutations, (2 another 2-class problem (mutations that disrupt/preserve a PPI, and (3 a 3-class classification (detrimental/neutral/beneficial mutation effects. In total, 11 different supervised and semi-supervised classifiers were trained and assessed resulting in a promising performance, with the weighted f-measure ranging from 0.87 for Problem 1 to 0.70 for the most challenging Problem 3. By integrating prediction results of the 2-class classifiers into the 3-class classifier, we further improved its performance for Problem 3. To demonstrate the utility of SNP-IN tool, it was applied to study the nsSNP-induced rewiring of two disease-centered networks. The accurate and balanced performance of SNP-IN tool makes it readily available to study the

Determining Effects of Non-synonymous SNPs on Protein-Protein Interactions using Supervised and Semi-supervised Learning

Science.gov (United States)

Zhao, Nan; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2014-01-01

Single nucleotide polymorphisms (SNPs) are among the most common types of genetic variation in complex genetic disorders. A growing number of studies link the functional role of SNPs with the networks and pathways mediated by the disease-associated genes. For example, many non-synonymous missense SNPs (nsSNPs) have been found near or inside the protein-protein interaction (PPI) interfaces. Determining whether such nsSNP will disrupt or preserve a PPI is a challenging task to address, both experimentally and computationally. Here, we present this task as three related classification problems, and develop a new computational method, called the SNP-IN tool (non-synonymous SNP INteraction effect predictor). Our method predicts the effects of nsSNPs on PPIs, given the interaction's structure. It leverages supervised and semi-supervised feature-based classifiers, including our new Random Forest self-learning protocol. The classifiers are trained based on a dataset of comprehensive mutagenesis studies for 151 PPI complexes, with experimentally determined binding affinities of the mutant and wild-type interactions. Three classification problems were considered: (1) a 2-class problem (strengthening/weakening PPI mutations), (2) another 2-class problem (mutations that disrupt/preserve a PPI), and (3) a 3-class classification (detrimental/neutral/beneficial mutation effects). In total, 11 different supervised and semi-supervised classifiers were trained and assessed resulting in a promising performance, with the weighted f-measure ranging from 0.87 for Problem 1 to 0.70 for the most challenging Problem 3. By integrating prediction results of the 2-class classifiers into the 3-class classifier, we further improved its performance for Problem 3. To demonstrate the utility of SNP-IN tool, it was applied to study the nsSNP-induced rewiring of two disease-centered networks. The accurate and balanced performance of SNP-IN tool makes it readily available to study the rewiring of

Small Scaffolds, Big Potential: Developing Miniature Proteins as Therapeutic Agents.

Science.gov (United States)

Holub, Justin M

2017-09-01

Preclinical Research Miniature proteins are a class of oligopeptide characterized by their short sequence lengths and ability to adopt well-folded, three-dimensional structures. Because of their biomimetic nature and synthetic tractability, miniature proteins have been used to study a range of biochemical processes including fast protein folding, signal transduction, catalysis and molecular transport. Recently, miniature proteins have been gaining traction as potential therapeutic agents because their small size and ability to fold into defined tertiary structures facilitates their development as protein-based drugs. This research overview discusses emerging developments involving the use of miniature proteins as scaffolds to design novel therapeutics for the treatment and study of human disease. Specifically, this review will explore strategies to: (i) stabilize miniature protein tertiary structure; (ii) optimize biomolecular recognition by grafting functional epitopes onto miniature protein scaffolds; and (iii) enhance cytosolic delivery of miniature proteins through the use of cationic motifs that facilitate endosomal escape. These objectives are discussed not only to address challenges in developing effective miniature protein-based drugs, but also to highlight the tremendous potential miniature proteins hold for combating and understanding human disease. Drug Dev Res 78 : 268-282, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The potential of protein-nanomaterial interaction for advanced drug delivery.

Science.gov (United States)

Peng, Qiang; Mu, Huiling

2016-03-10

Nanomaterials, like nanoparticles, micelles, nano-sheets, nanotubes and quantum dots, have great potentials in biomedical fields. However, their delivery is highly limited by the formation of protein corona upon interaction with endogenous proteins. This new identity, instead of nanomaterial itself, would be the real substance the organs and cells firstly encounter. Consequently, the behavior of nanomaterials in vivo is uncontrollable and some undesired effects may occur, like rapid clearance from blood stream; risk of capillary blockage; loss of targeting capacity; and potential toxicity. Therefore, protein-nanomaterial interaction is a great challenge for nanomaterial systems and should be inhibited. However, this interaction can also be used to functionalize nanomaterials by forming a selected protein corona. Unlike other decoration using exogenous molecules, nanomaterials functionalized by selected protein corona using endogenous proteins would have greater promise for clinical use. In this review, we aim to provide a comprehensive understanding of protein-nanomaterial interaction. Importantly, a discussion about how to use such interaction is launched and some possible applications of such interaction for advanced drug delivery are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Technical refolding of proteins: Do we have freedom to operate?

Science.gov (United States)

Eiberle, Maria K; Jungbauer, Alois

2010-06-01

Expression as inclusion bodies in Escherichia coli is a widely used method for the large-scale production of therapeutic proteins that do not require post-translational modifications. High expression yields and simple recovery steps of inclusion bodies from the host cells are attractive features industrially. However, the value of an inclusion body-based process is dominated by the solubilization and refolding technologies. Scale-invariant technologies that are economical and applicable for a wide range of proteins are requested by industry. The main challenge is to convert the denatured protein into its native conformation at high yields. Refolding competes with misfolding and aggregation. Thus, the yield of native monomer depends strongly on the initial protein concentrations in the refolding solution. Reasonable yields are attained at low concentrations (freedom to operate.

Whey protein stories - an experiment in writing a multidisciplinary biography

DEFF Research Database (Denmark)

Jensen, Tenna; Bechschøft, Rasmus L.; Giacalone, Davide

2016-01-01

This is an experimental, dual-purpose article about whey protein and how to conduct interdisciplinary analyses and writings. On the one hand, this article is a multidisciplinary commodity biography, which consists of five descriptions of whey protein written by the five different research groups...... contributes to the field of food studies with a multidisciplinary biography of whey protein - including its sensory qualities and challenges, insights into its cultural history, its nutritional value and effects on the human body and an analysis of how it is perceived by people who consume it. The biography...... thereby expands upon existing understandings of whey protein while discussing the usefulness of employing the commodity biography format in interdisciplinary writing. Moreover, the article contributes to the field of interdisciplinary research by providing a practical example of a joint publication...

Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process.

Science.gov (United States)

Singh, Anupam; Upadhyay, Vaibhav; Upadhyay, Arun Kumar; Singh, Surinder Mohan; Panda, Amulya Kumar

2015-03-25

Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.

Monitoring Protein Fouling on Polymeric Membranes Using Ultrasonic Frequency-Domain Reflectometry

Directory of Open Access Journals (Sweden)

Robin Fong

2011-08-01

Full Text Available Novel signal-processing protocols were used to extend the in situ sensitivity of ultrasonic frequency-domain reflectometry (UFDR for real-time monitoring of microfiltration (MF membrane fouling during protein purification. Different commercial membrane materials, with a nominal pore size of 0.2 µm, were challenged using bovine serum albumin (BSA and amylase as model proteins. Fouling induced by these proteins was observed in flat-sheet membrane filtration cells operating in a laminar cross-flow regime. The detection of membrane-associated proteins using UFDR was determined by applying rigorous statistical methodology to reflection spectra of ultrasonic signals obtained during membrane fouling. Data suggest that the total power reflected from membrane surfaces changes in response to protein fouling at concentrations as low as 14 μg/cm2, and results indicate that ultrasonic spectra can be leveraged to detect and monitor protein fouling on commercial MF membranes.

Post-exposure vaccination with MP-12 lacking NSs protects mice against lethal Rift Valley fever virus challenge.

Science.gov (United States)

Gowen, Brian B; Bailey, Kevin W; Scharton, Dionna; Vest, Zachery; Westover, Jonna B; Skirpstunas, Ramona; Ikegami, Tetsuro

2013-05-01

Rift Valley fever virus (RVFV) causes severe disease in humans and livestock. There are currently no approved antivirals or vaccines for the treatment or prevention of RVF disease in humans. A major virulence factor of RVFV is the NSs protein, which inhibits host transcription including the interferon (IFN)-β gene and promotes the degradation of dsRNA-dependent protein kinase, PKR. We analyzed the efficacy of the live-attenuated MP-12 vaccine strain and MP-12 variants that lack the NSs protein as post-exposure vaccinations. Although parental MP-12 failed to elicit a protective effect in mice challenged with wild-type (wt) RVFV by the intranasal route, significant protection was demonstrated by vaccination with MP-12 strains lacking NSs when they were administered at 20-30 min post-exposure. Viremia and virus replication in liver, spleen and brain were also inhibited by post-exposure vaccination with MP-12 lacking NSs. The protective effect was mostly lost when vaccination was delayed 6 or 24 h after intranasal RVFV challenge. When mice were challenged subcutaneously, efficacy of MP-12 lacking NSs was diminished, most likely due to more rapid dissemination of wt RVFV. Our findings suggest that post-exposure vaccination with MP-12 lacking NSs may be developed as a novel post-exposure treatment to prevent RVF. Copyright © 2013 Elsevier B.V. All rights reserved.

Arrhythmogenic KCNE gene variants: current knowledge and future challenges

Directory of Open Access Journals (Sweden)

Shawn M Crump

2014-01-01

Full Text Available There are twenty-five known inherited cardiac arrhythmia susceptibility genes, all of which encode either ion channel pore-forming subunits or proteins that regulate aspects of ion channel biology such as function, trafficking and localization. The human KCNE gene family comprises five potassium channel regulatory subunits, sequence variants in each of which are associated with cardiac arrhythmias. KCNE gene products exhibit promiscuous partnering and in some cases ubiquitous expression, hampering efforts to unequivocally correlate each gene to specific native potassium currents. Likewise, deducing the molecular etiology of cardiac arrhythmias in individuals harboring rare KCNE gene variants, or more common KCNE polymorphisms, can be challenging. In this review we provide an update on putative arrhythmia-causing KCNE gene variants, and discuss current thinking and future challenges in the study of molecular mechanisms of KCNE-associated cardiac rhythm disturbances.

Constitutive, but not challenge-induced, interleukin-10 production is robust in acute pre-pubescent protein and energy deficits: new support for the tolerance hypothesis of malnutrition-associated immune depression based on cytokine production in vivo.

Science.gov (United States)

Monk, Jennifer M; Steevels, Tessa A M; Hillyer, Lyn M; Woodward, Bill

2011-01-01

The tolerance model of acute (i.e., wasting) pre-pubescent protein and energy deficits proposes that the immune depression characteristic of these pathologies reflects an intact anti-inflammatory form of immune competence that reduces the risk of autoimmune reactions to catabolically released self antigens. A cornerstone of this proposition is the finding that constitutive (first-tier) interleukin(IL)-10 production is sustained even into the advanced stages of acute malnutrition. The IL-10 response to inflammatory challenge constitutes a second tier of anti-inflammatory regulation and was the focus of this investigation. Weanling mice consumed a complete diet ad libitum, a low-protein diet ad libitum (mimicking incipient kwashiorkor), or the complete diet in restricted daily quantities (mimicking marasmus), and their second-tier IL-10 production was determined both in vitro and in vivo using lipopolysaccharide (LPS) and anti-CD3 as stimulants of innate and adaptive defences, respectively. Both early (3 days) and advanced (14 days) stages of wasting pathology were examined and three main outcomes emerged. First, classic in vitro systems are unreliable for discerning cytokine production in vivo. Secondly, in diverse forms of acute malnutrition declining challenge-induced IL-10 production may provide an early sign that anti-inflammatory control over immune competence is failing. Thirdly, and most fundamentally, the investigation provides new support for the tolerance model of malnutrition-associated inflammatory immune depression.

Constitutive, but Not Challenge-Induced, Interleukin-10 Production Is Robust in Acute Pre-Pubescent Protein and Energy Deficits: New Support for the Tolerance Hypothesis of Malnutrition-Associated Immune Depression Based on Cytokine Production in vivo

Directory of Open Access Journals (Sweden)

Bill Woodward

2011-01-01

Full Text Available The tolerance model of acute (i.e., wasting pre-pubescent protein and energy deficits proposes that the immune depression characteristic of these pathologies reflects an intact anti-inflammatory form of immune competence that reduces the risk of autoimmune reactions to catabolically released self antigens. A cornerstone of this proposition is the finding that constitutive (first-tier interleukin(IL-10 production is sustained even into the advanced stages of acute malnutrition. The IL-10 response to inflammatory challenge constitutes a second tier of anti-inflammatory regulation and was the focus of this investigation. Weanling mice consumed a complete diet ad libitum, a low-protein diet ad libitum (mimicking incipient kwashiorkor, or the complete diet in restricted daily quantities (mimicking marasmus, and their second-tier IL-10 production was determined both in vitro and in vivo using lipopolysaccharide (LPS and anti-CD3 as stimulants of innate and adaptive defences, respectively. Both early (3 days and advanced (14 days stages of wasting pathology were examined and three main outcomes emerged. First, classic in vitro systems are unreliable for discerning cytokine production in vivo. Secondly, in diverse forms of acute malnutrition declining challenge-induced IL-10 production may provide an early sign that anti-inflammatory control over immune competence is failing. Thirdly, and most fundamentally, the investigation provides new support for the tolerance model of malnutrition-associated inflammatory immune depression.

A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules.

Science.gov (United States)

Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

2014-11-15

The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

CSIR Research Space (South Africa)

Makhoba, XH

2015-09-01

Full Text Available For many years recombinant protein production has been at the center of biosciences used for structural and therapeutic purposes. The production of recombinant proteins in foreign host system such as E. coli has been a biggest challenge. This has...

Selectivity analysis of protein kinase CK2 inhibitors DMAT, TBB and resorufin in cisplatin-induced stress responses

DEFF Research Database (Denmark)

Fritz, Gerhard; Issinger, Olaf-Georg; Olsen, Birgitte Brinkmann

2009-01-01

Targeting protein kinases as a therapeutic approach to treat various diseases, especially cancer is currently a fast growing business. Although many inhibitors are available, exhibiting remarkable potency, the major challenge is their selectivity. Here we show that the protein kinase CK2 inhibito...

Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

Directory of Open Access Journals (Sweden)

Olena Zakharchenko

2011-01-01

Full Text Available Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE. This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

MU-LOC: A Machine-Learning Method for Predicting Mitochondrially Localized Proteins in Plants

DEFF Research Database (Denmark)

Zhang, Ning; Rao, R Shyama Prasad; Salvato, Fernanda

2018-01-01

-sequence or a multitude of internal signals. Compared with experimental approaches, computational predictions provide an efficient way to infer subcellular localization of a protein. However, it is still challenging to predict plant mitochondrially localized proteins accurately due to various limitations. Consequently......, the performance of current tools can be improved with new data and new machine-learning methods. We present MU-LOC, a novel computational approach for large-scale prediction of plant mitochondrial proteins. We collected a comprehensive dataset of plant subcellular localization, extracted features including amino...

Current Challenges in Cancer Treatment.

Science.gov (United States)

Zugazagoitia, Jon; Guedes, Cristiano; Ponce, Santiago; Ferrer, Irene; Molina-Pinelo, Sonia; Paz-Ares, Luis

2016-07-01

In this review, we highlight the current concepts and discuss some of the current challenges and future prospects in cancer therapy. We frequently use the example of lung cancer. We conducted a nonsystematic PubMed search, selecting the most comprehensive and relevant research articles, clinical trials, translational papers, and review articles on precision oncology and immuno-oncology. Papers were prioritized and selected based on their originality and potential clinical applicability. Two major revolutions have changed cancer treatment paradigms in the past few years: targeting actionable alterations in oncogene-driven cancers and immuno-oncology. Important challenges are still ongoing in both fields of cancer therapy. On the one hand, druggable genomic alterations are diverse and represent only small subsets of patients in certain tumor types, which limits testing their clinical impact in biomarker-driven clinical trials. Next-generation sequencing technologies are increasingly being implemented for molecular prescreening in clinical research, but issues regarding clinical interpretation of large genomic data make their wide clinical use difficult. Further, dealing with tumor heterogeneity and acquired resistance is probably the main limitation for the success of precision oncology. On the other hand, long-term survival benefits with immune checkpoint inhibitors (anti-programmed death cell protein-1/programmed death cell ligand-1[PD-1/L1] and anti-cytotoxic T lymphocyte antigen-4 monoclonal antibodies) are restricted to a minority of patients, and no predictive markers are yet robustly validated that could help us recognize these subsets and optimize treatment delivery and selection. To achieve long-term survival benefits, drug combinations targeting several molecular alterations or cancer hallmarks might be needed. This will probably be one of the most challenging but promising precision cancer treatment strategies in the future. Targeting single molecular

Programmable release of multiple protein drugs from aptamer-functionalized hydrogels via nucleic acid hybridization.

Science.gov (United States)

Battig, Mark R; Soontornworajit, Boonchoy; Wang, Yong

2012-08-01

Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer-protein interactions and aptamer-CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

Structure and Dynamic Properties of Membrane Proteins using NMR

DEFF Research Database (Denmark)

Rösner, Heike; Kragelund, Birthe

2012-01-01

conformational changes. Their structural and functional decoding is challenging and has imposed demanding experimental development. Solution nuclear magnetic resonance (NMR) spectroscopy is one of the techniques providing the capacity to make a significant difference in the deciphering of the membrane protein...... structure-function paradigm. The method has evolved dramatically during the last decade resulting in a plethora of new experiments leading to a significant increase in the scientific repertoire for studying membrane proteins. Besides solving the three-dimensional structures using state-of-the-art approaches......-populated states, this review seeks to introduce the vast possibilities solution NMR can offer to the study of membrane protein structure-function analyses with special focus on applicability. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012....

"Meatless days" or "less but better"? Exploring strategies to adapt Western meat consumption to health and sustainability challenges.

Science.gov (United States)

de Boer, Joop; Schösler, Hanna; Aiking, Harry

2014-05-01

Adapting Western meat consumption to health and sustainability challenges requires an overall reduction of industrially produced animal proteins plus a partial replacement by plant proteins. Combining insights on food, environment, and consumers, this paper aims to explore change strategies that may help to meet these challenges, such as promoting smaller portions of meat ("less"), smaller portions using meat raised in a more sustainable manner ("less but better"), smaller portions and eating more vegetable protein ("less and more varied"), and meatless meals with or without meat substitutes ("veggie-days"). The underlying logic of the strategies was clarified by analyzing dietary choices. A nationwide sample of 1083 Dutch consumers provided information on current eating practices and potential changes. The results show that strategies to change meat eating frequencies and meat portion sizes will appeal to overlapping but partly different segments of consumers and that these strategies can be applied to address consumers in terms of their own preferences. The strategies appeared to have different strengths and weaknesses, making them complementary pathways to facilitate step-by-step changes in the amounts and the sources of protein consumed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protein antigen adsorption to the DDA/TDB liposomal adjuvant

DEFF Research Database (Denmark)

Hamborg, Mette; Jorgensen, Lene; Bojsen, Anders Riber

2013-01-01

Understanding the nature of adjuvant-antigen interactions is important for the future design of efficient and safe subunit vaccines, but remains an analytical challenge. We studied the interactions between three model protein antigens and the clinically tested cationic liposomal adjuvant composed...... of dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB)....

Future Oceans: Meeting the Challenges of Securing Aquatic Food Resources

OpenAIRE

Dieckmann, U.

2012-01-01

Seafood is the primary source of animal protein for more than one billion people. Many economies and communities, in particular those in developing nations and coastal regions, depend on fisheries. Whereas the dire effects of overfishing on open-access ocean fisheries are already recognized, impacts of catches on freshwater systems are still underestimated. IIASA’s fisheries research elucidates how to secure and expand aquatic food resources, emphasizing three topical challenges. First, impro...

76 FR 41526 - Centennial Challenges 2011 Strong Tether Challenge

Science.gov (United States)

2011-07-14

... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION [Notice (11-063)] Centennial Challenges 2011 Strong... scheduled and teams that wish to compete may register. Centennial Challenges is a program of prize... NASA Centennial Challenges Program please visit: http://www.nasa.gov/challenges . General questions and...

Modulating the aggregation behaviour to restore the mechanical response of acid induced mixed gels of sodium caseinate and soy proteins

NARCIS (Netherlands)

Martin, Anneke H.; Los Reyes Jiménez, De Marta L.; Pouvreau, Laurice

2016-01-01

Partial replacement of milk proteins with plant proteins is a challenge due to the reported negative effect on physical and sensory properties. Understanding of how the mechanical properties of acidified milk gels can be restored when 30% casein is replaced with soy proteins is therefore

Modulating the aggregation behaviour to restore the mechanical response of acid induced mixed gels of sodium caseinate and soy proteins

NARCIS (Netherlands)

Martin, A.H.; Reyes Jiménez, M. L. de los; Pouvreau, L.

2016-01-01

Partial replacement of milk proteins with plant proteins is a challenge due to the reported negative effect on physical and sensory properties. Understanding of how the mechanical properties of acidified milk gels can be restored when 30% casein is replaced with soy proteins is therefore explored.

Plant Ribosome-Inactivating Proteins: Progesses, Challenges and Biotechnological Applications (and a Few Digressions

Directory of Open Access Journals (Sweden)

Maria Serena Fabbrini

2017-10-01

Full Text Available Plant ribosome-inactivating protein (RIP toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric and type II (dimeric as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA, they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications.

Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

Directory of Open Access Journals (Sweden)

Mie Kristensen

2016-01-01

Full Text Available The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB. CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies.

Towards data warehousing and mining of protein unfolding simulation data.

Science.gov (United States)

Berrar, Daniel; Stahl, Frederic; Silva, Candida; Rodrigues, J Rui; Brito, Rui M M; Dubitzky, Werner

2005-10-01

The prediction of protein structure and the precise understanding of protein folding and unfolding processes remains one of the greatest challenges in structural biology and bioinformatics. Computer simulations based on molecular dynamics (MD) are at the forefront of the effort to gain a deeper understanding of these complex processes. Currently, these MD simulations are usually on the order of tens of nanoseconds, generate a large amount of conformational data and are computationally expensive. More and more groups run such simulations and generate a myriad of data, which raises new challenges in managing and analyzing these data. Because the vast range of proteins researchers want to study and simulate, the computational effort needed to generate data, the large data volumes involved, and the different types of analyses scientists need to perform, it is desirable to provide a public repository allowing researchers to pool and share protein unfolding data. To adequately organize, manage, and analyze the data generated by unfolding simulation studies, we designed a data warehouse system that is embedded in a grid environment to facilitate the seamless sharing of available computer resources and thus enable many groups to share complex molecular dynamics simulations on a more regular basis. To gain insight into the conformational fluctuations and stability of the monomeric forms of the amyloidogenic protein transthyretin (TTR), molecular dynamics unfolding simulations of the monomer of human TTR have been conducted. Trajectory data and meta-data of the wild-type (WT) protein and the highly amyloidogenic variant L55P-TTR represent the test case for the data warehouse. Web and grid services, especially pre-defined data mining services that can run on or 'near' the data repository of the data warehouse, are likely to play a pivotal role in the analysis of molecular dynamics unfolding data.

Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

Science.gov (United States)

Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

2009-07-16

Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs.

Understanding the Added Value of G-Protein-Coupled Receptor Heteromers

Directory of Open Access Journals (Sweden)

Nuria Franco

2014-01-01

Full Text Available G-protein-coupled receptors (GPCRs constitute the most populated family of proteins within the human genome. Since the early sixties work on GPCRs and on GPCR-mediated signaling has led to a number of awards, the most recent being the Nobel Prize in Chemistry for 2012. The future of GPCRs research is surely based on their capacity for heteromerization. Receptor heteromers offer a series of challenges that will help in providing success in academic/basic research and translation into more effective and safer drugs.

78 FR 19742 - Centennial Challenges: 2014 Night Rover Challenge

Science.gov (United States)

2013-04-02

... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION [Notice 13-032] Centennial Challenges: 2014 Night... Centennial Challenges 2014 Night Rover Challenge. SUMMARY: This notice is issued in accordance with 51 U.S.C.... Centennial Challenges is a program of prize competitions to stimulate innovation in technologies of interest...

Blood proteins as carcinogen dosimeters

International Nuclear Information System (INIS)

Tannenbaum, S.R.; Skipper, P.L.

1986-01-01

The problem of quantifying exposure to genotoxins in a given individual represents a formidable challenge. In this paper methods which rely on the covalent binding of carcinogens and their metabolites to blood proteins are described. That carcinogens interact with proteins as well as with DNA has been established, although whether protein-carcinogen adducts can result in genetic damage has not been established. It has been shown, however, that the amount of a protein carcinogen adduct formed may be used as a quantitative measure of exposure to a carcinogen. Such a measure presumably is reflective of the absorption, metabolism, and excretion of the compound in an exposed individual. Protein adduction may reflect exposure in a time-frame of weeks to months. Thus, protein adduct measurement is a form of human chemical dosimetry. Hemoglobin and albumin are promising candidates for such dosimeters. Hemoglobin has a lifetime of about 120 days in humans; thus, circulating levels of carcinogen-modified hemoglobin will reflect the level of carcinogen exposure during a period of nearly four months. It also possesses some metabolic competence, particularly, the ability to oxidize aromatic hydroxylamines to nitroso compounds which react quite efficiently with sulfhydryl groups. Albumin has a half-life of 20 to 25 days in man. This protein does not possess metabolic capacity other than, perhaps, some esterase activity. In contrast to hemoglobin, though, it is not protected by the erythrocyte membrane and might be the target for a greater number of carcinogens. It is present and is synthesized in the same cells in which the reactive metabolic intermediates of carcinogens are mostly formed - the hepatocytes. Also, albumin has a number of high-affinity binding sites for a broad spectrum of xenobiotics and endobiotics. 25 refs., 1 tab

Protein engineering in designing tailored enzymes and microorganisms for biofuels production

Science.gov (United States)

Wen, Fei; Nair, Nikhil U; Zhao, Huimin

2009-01-01

Summary Lignocellulosic biofuels represent a sustainable, renewable, and the only foreseeable alternative energy source to transportation fossil fuels. However, the recalcitrant nature of lignocellulose poses technical hurdles to an economically viable biorefinery. Low enzymatic hydrolysis efficiency and low productivity, yield, and titer of biofuels are among the top cost contributors. Protein engineering has been used to improve the performances of lignocellulose-degrading enzymes, as well as proteins involved in biofuel synthesis pathways. Unlike its great success seen in other industrial applications, protein engineering has achieved only modest results in improving the lignocellulose-to-biofuels efficiency. This review will discuss the unique challenges that protein engineering faces in the process of converting lignocellulose to biofuels and how they are addressed by recent advances in this field. PMID:19660930

Intestinal infection following aerosol challenge of calves with Mycobacterium avium subspecies paratuberculosis

Directory of Open Access Journals (Sweden)

Eisenberg Susanne WF

2011-12-01

Full Text Available Abstract A challenge experiment was performed to investigate whether administration of Mycobacterium avium subsp. paratuberculosis (MAP via the respiratory route leads to MAP infection in calves. Eighteen calves from test negative dams were randomly allocated to four groups. Six calves were challenged with MAP nasally and six calves were challenged by transtracheal injection; three orally challenged calves served as positive controls, and three non challenged calves as negative controls. The challenge was performed as a nine-fold trickle dose, 107 CFU in total. Blood and faecal samples were collected frequently. Calves were euthanized three months post-challenge and extensively sampled. Blood samples were tested for the presence of antibodies and interferon gamma producing cells by ELISA. Faecal and tissue samples were cultured in a liquid culture system and the presence of MAP was confirmed by IS900 realtime PCR. Fourteen out of fifteen calves had no MAP antibody response. The negative controls remained negative; all positive controls became infected. Two nasally challenged calves showed a Purified Protein Derivative Avian (PPDA specific interferon gamma response. In all nasally challenged calves, MAP positive intestinal samples were detected. In three calves of the nasal group MAP positive retropharyngeal lymph nodes or tonsils were detected. In all calves of the transtracheal group MAP positive intestinal tissues were detected as well and three had a MAP positive tracheobronchial lymph node. These findings indicate that inhalation of MAP aerosols can result in infection. These experimental results may be relevant for transmission under field conditions since viable MAP has been detected in dust on commercial dairy farms.

Mixing Energy Models in Genetic Algorithms for On-Lattice Protein Structure Prediction

Directory of Open Access Journals (Sweden)

Mahmood A. Rashid

2013-01-01

Full Text Available Protein structure prediction (PSP is computationally a very challenging problem. The challenge largely comes from the fact that the energy function that needs to be minimised in order to obtain the native structure of a given protein is not clearly known. A high resolution 20×20 energy model could better capture the behaviour of the actual energy function than a low resolution energy model such as hydrophobic polar. However, the fine grained details of the high resolution interaction energy matrix are often not very informative for guiding the search. In contrast, a low resolution energy model could effectively bias the search towards certain promising directions. In this paper, we develop a genetic algorithm that mainly uses a high resolution energy model for protein structure evaluation but uses a low resolution HP energy model in focussing the search towards exploring structures that have hydrophobic cores. We experimentally show that this mixing of energy models leads to significant lower energy structures compared to the state-of-the-art results.

A unified conformational selection and induced fit approach to protein-peptide docking.

Directory of Open Access Journals (Sweden)

Mikael Trellet

Full Text Available Protein-peptide interactions are vital for the cell. They mediate, inhibit or serve as structural components in nearly 40% of all macromolecular interactions, and are often associated with diseases, making them interesting leads for protein drug design. In recent years, large-scale technologies have enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. Yet, the paucity of data regarding their molecular binding mechanisms together with their inherent flexibility makes the structural prediction of protein-peptide interactions very challenging. This leaves flexible docking as one of the few amenable computational techniques to model these complexes. We present here an ensemble, flexible protein-peptide docking protocol that combines conformational selection and induced fit mechanisms. Starting from an ensemble of three peptide conformations (extended, a-helix, polyproline-II, flexible docking with HADDOCK generates 79.4% of high quality models for bound/unbound and 69.4% for unbound/unbound docking when tested against the largest protein-peptide complexes benchmark dataset available to date. Conformational selection at the rigid-body docking stage successfully recovers the most relevant conformation for a given protein-peptide complex and the subsequent flexible refinement further improves the interface by up to 4.5 Å interface RMSD. Cluster-based scoring of the models results in a selection of near-native solutions in the top three for ∼75% of the successfully predicted cases. This unified conformational selection and induced fit approach to protein-peptide docking should open the route to the modeling of challenging systems such as disorder-order transitions taking place upon binding, significantly expanding the applicability limit of biomolecular interaction modeling by docking.

Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges

Science.gov (United States)

Habel, Joachim; Hansen, Michael; Kynde, Søren; Larsen, Nanna; Midtgaard, Søren Roi; Jensen, Grethe Vestergaard; Bomholt, Julie; Ogbonna, Anayo; Almdal, Kristoffer; Schulz, Alexander; Hélix-Nielsen, Claus

2015-01-01

In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs), block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes. PMID:26264033

77 FR 70835 - Centennial Challenges 2013 Sample Return Robot Challenge

Science.gov (United States)

2012-11-27

... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION Centennial Challenges 2013 Sample Return Robot... Challenge is scheduled and teams that wish to compete may register. Centennial Challenges is a program of... Challenge, please visit: http://challenge.wpi.edu . For general information on the NASA Centennial...

Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

Directory of Open Access Journals (Sweden)

Lihong Jiang

2018-06-01

Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

Lactoferrin, myeloperoxidase, lysozyme and eosinophil cationic protein in exudate in delayed type hypersensitivity

DEFF Research Database (Denmark)

Lerche, A; Bisgaard, H; Christensen, J D

1988-01-01

allergic patients with nickel challenge in the chamber medium showed a time-dependent increase of mononuclear cells, eosinophils and basophils and a concomitant decrease of polymorphonuclear granulocytes, characteristic of a combined specific and unspecific inflammation. The morphology of the exudate...... in contact allergic patients exposed to nickel showed a dominance of polymorphonuclear granulocytes throughout the study period, while mononuclear cells, eosinophils and basophils were detected at a much lower quantity and with a considerable delay. Further, we studied the kinetics of the leucocyte granule...... proteins: lactoferrin, myeloperoxidase, lysozyme and eosinophil cationic protein in exudate fluid in a parallel test. A significant higher flux was found for all during the second day of allergen exposure compared to contact allergic patients without allergen challenge as well as normal volunteers...

76 FR 56819 - Centennial Challenges 2012 Sample Return Robot Challenge

Science.gov (United States)

2011-09-14

... NATIONAL AERONAUTICS AND SPACE ADMINISTRATION [Notice (11-079)] Centennial Challenges 2012 Sample... Challenge is scheduled and teams that wish to compete may register. Centennial Challenges is a program of... Challenge, please visit: http://wp.wpi.edu/challenge/ . For general information on the NASA Centennial...

Protein misfolding and obstructive lung disease.

LENUS (Irish Health Repository)

Greene, Catherine M

2010-11-01

The endoplasmic reticulum has evolved a number of mechanisms to manage the accumulation of incorrectly folded proteins. This results in loss of function of these proteins, but occasionally, in conditions such as α-1 antitrpysin (A1AT) deficiency, the misfolded protein can acquire a toxic gain of function promoting exaggerated ER stress responses and inflammation. Mutations leading to deficiency in a second serine proteinase inhibitor, α-1 antichymotrpysin (ACT), can induce potentially similar consequences. A1AT and ACT deficiencies are associated with chronic obstructive lung disease. Until recently, it was thought that the lung diseases associated with these conditions were entirely due to loss of antiprotease protection in the lung (i.e., loss of function), whereas gain of function was the major cause of the liver disease associated with A1AT deficiency. This paradigm is being increasingly challenged because ER stress is being recognized in bronchial epithelial cells and inflammatory cells normally resident in the lung, giving rise to an inflammatory phenotype that adds to the proteolytic burden associated with these conditions. In this article, we describe the cellular mechanisms that are activated to cope with an increasing burden of misfolded proteins within the ER in A1AT and ACT deficiency, show how these events are linked to inflammation, and outline the therapeutic strategies that can potentially interfere with production of misfolded proteins.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

Science.gov (United States)

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

Ultrafiltration of pegylated proteins

Science.gov (United States)

Molek, Jessica R.

There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.

Science.gov (United States)

Lakbub, Jude C; Shipman, Joshua T; Desaire, Heather

2018-04-01

Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass

Finding the Subcellular Location of Barley, Wheat, Rice and Maize Proteins: The Compendium of Crop Proteins with Annotated Locations (cropPAL).

Science.gov (United States)

Hooper, Cornelia M; Castleden, Ian R; Aryamanesh, Nader; Jacoby, Richard P; Millar, A Harvey

2016-01-01

Barley, wheat, rice and maize provide the bulk of human nutrition and have extensive industrial use as agricultural products. The genomes of these crops each contains >40,000 genes encoding proteins; however, the major genome databases for these species lack annotation information of protein subcellular location for >80% of these gene products. We address this gap, by constructing the compendium of crop protein subcellular locations called crop Proteins with Annotated Locations (cropPAL). Subcellular location is most commonly determined by fluorescent protein tagging of live cells or mass spectrometry detection in subcellular purifications, but can also be predicted from amino acid sequence or protein expression patterns. The cropPAL database collates 556 published studies, from >300 research institutes in >30 countries that have been previously published, as well as compiling eight pre-computed subcellular predictions for all Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays protein sequences. The data collection including metadata for proteins and published studies can be accessed through a search portal http://crop-PAL.org. The subcellular localization information housed in cropPAL helps to depict plant cells as compartmentalized protein networks that can be investigated for improving crop yield and quality, and developing new biotechnological solutions to agricultural challenges. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transiently disordered tails accelerate folding of globular proteins.

Science.gov (United States)

Mallik, Saurav; Ray, Tanaya; Kundu, Sudip

2017-07-01

Numerous biological proteins exhibit intrinsic disorder at their termini, which are associated with multifarious functional roles. Here, we show the surprising result that an increased percentage of terminal short transiently disordered regions with enhanced flexibility (TstDREF) is associated with accelerated folding rates of globular proteins. Evolutionary conservation of predicted disorder at TstDREFs and drastic alteration of folding rates upon point-mutations suggest critical regulatory role(s) of TstDREFs in shaping the folding kinetics. TstDREFs are associated with long-range intramolecular interactions and the percentage of native secondary structural elements physically contacted by TstDREFs exhibit another surprising positive correlation with folding kinetics. These results allow us to infer probable molecular mechanisms behind the TstDREF-mediated regulation of folding kinetics that challenge protein biochemists to assess by direct experimental testing. © 2017 Federation of European Biochemical Societies.

HPSLPred: An Ensemble Multi-Label Classifier for Human Protein Subcellular Location Prediction with Imbalanced Source.

Science.gov (United States)

Wan, Shixiang; Duan, Yucong; Zou, Quan

2017-09-01

Predicting the subcellular localization of proteins is an important and challenging problem. Traditional experimental approaches are often expensive and time-consuming. Consequently, a growing number of research efforts employ a series of machine learning approaches to predict the subcellular location of proteins. There are two main challenges among the state-of-the-art prediction methods. First, most of the existing techniques are designed to deal with multi-class rather than multi-label classification, which ignores connections between multiple labels. In reality, multiple locations of particular proteins imply that there are vital and unique biological significances that deserve special focus and cannot be ignored. Second, techniques for handling imbalanced data in multi-label classification problems are necessary, but never employed. For solving these two issues, we have developed an ensemble multi-label classifier called HPSLPred, which can be applied for multi-label classification with an imbalanced protein source. For convenience, a user-friendly webserver has been established at http://server.malab.cn/HPSLPred. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A statistical view of protein chemical synthesis using NCL and extended methodologies.