Brachyury Protein: A Potential Target in Lung Cancer Therapy | Center for Cancer Research

Cancer.gov

Previous research has shown that Brachyury protein plays a role in initiating the processes that lead to the growth and spread of cancer. Now CCR scientists have for the first time demonstrated the expression of Brachyury protein in lung cancer tumors, as well as a correlation between the overexpression of Brachyury protein and drug resistance.

Tracking 20 Years of Compound-to-Target Output from Literature and Patents

PubMed Central

Southan, Christopher; Varkonyi, Peter; Boppana, Kiran; Jagarlapudi, Sarma A.R.P.; Muresan, Sorel

2013-01-01

The statistics of drug development output and declining yield of approved medicines has been the subject of many recent reviews. However, assessing research productivity that feeds development is more difficult. Here we utilise an extensive database of structure-activity relationships extracted from papers and patents. We have used this database to analyse published compounds cumulatively linked to nearly 4000 protein target identifiers from multiple species over the last 20 years. The compound output increases up to 2005 followed by a decline that parallels a fall in pharmaceutical patenting. Counts of protein targets have plateaued but not fallen. We extended these results by exploring compounds and targets for one large pharmaceutical company. In addition, we examined collective time course data for six individual protease targets, including average molecular weight of the compounds. We also tracked the PubMed profile of these targets to detect signals related to changes in compound output. Our results show that research compound output had decreased 35% by 2012. The major causative factor is likely to be a contraction in the global research base due to mergers and acquisitions across the pharmaceutical industry. However, this does not rule out an increasing stringency of compound quality filtration and/or patenting cost control. The number of proteins mapped to compounds on a yearly basis shows less decline, indicating the cumulative published target capacity of global research is being sustained in the region of 300 proteins for large companies. The tracking of six individual targets shows uniquely detailed patterns not discernible from cumulative snapshots. These are interpretable in terms of events related to validation and de-risking of targets that produce detectable follow-on surges in patenting. Further analysis of the type we present here can provide unique insights into the process of drug discovery based on the data it actually generates. PMID:24204758

Exploring the Therapeutic Mechanism of Desmodium styracifolium on Oxalate Crystal-Induced Kidney Injuries Using Comprehensive Approaches Based on Proteomics and Network Pharmacology.

PubMed

Hou, Jiebin; Chen, Wei; Lu, Hongtao; Zhao, Hongxia; Gao, Songyan; Liu, Wenrui; Dong, Xin; Guo, Zhiyong

2018-01-01

Purpose: As a Chinese medicinal herb, Desmodium styracifolium (Osb.) Merr (DS) has been applied clinically to alleviate crystal-induced kidney injuries, but its effective components and their specific mechanisms still need further exploration. This research first combined the methods of network pharmacology and proteomics to explore the therapeutic protein targets of DS on oxalate crystal-induced kidney injuries to provide a reference for relevant clinical use. Methods: Oxalate-induced kidney injury mouse, rat, and HK-2 cell models were established. Proteins differentially expressed between the oxalate and control groups were respectively screened using iTRAQ combined with MALDI-TOF-MS. The common differential proteins of the three models were further analyzed by molecular docking with DS compounds to acquire differential targets. The inverse docking targets of DS were predicted through the platform of PharmMapper. The protein-protein interaction (PPI) relationship between the inverse docking targets and the differential proteins was established by STRING. Potential targets were further validated by western blot based on a mouse model with DS treatment. The effects of constituent compounds, including luteolin, apigenin, and genistein, were investigated based on an oxalate-stimulated HK-2 cell model. Results: Thirty-six common differentially expressed proteins were identified by proteomic analysis. According to previous research, the 3D structures of 15 major constituents of DS were acquired. Nineteen differential targets, including cathepsin D (CTSD), were found using molecular docking, and the component-differential target network was established. Inverse-docking targets including p38 MAPK and CDK-2 were found, and the network of component-reverse docking target was established. Through PPI analysis, 17 inverse-docking targets were linked to differential proteins. The combined network of component-inverse docking target-differential proteins was then constructed. The expressions of CTSD, p-p38 MAPK, and p-CDK-2 were shown to be increased in the oxalate group and decreased in kidney tissue by the DS treatment. Luteolin, apigenin, and genistein could protect oxalate-stimulated tubular cells as active components of DS. Conclusion: The potential targets including the CTSD, p38 MAPK, and CDK2 of DS in oxalate-induced kidney injuries and the active components (luteolin, apigenin, and genistein) of DS were successfully identified in this study by combining proteomics analysis, network pharmacology prediction, and experimental validation.

Diversity-oriented synthetic strategy for developing a chemical modulator of protein-protein interaction

NASA Astrophysics Data System (ADS)

Kim, Jonghoon; Jung, Jinjoo; Koo, Jaeyoung; Cho, Wansang; Lee, Won Seok; Kim, Chanwoo; Park, Wonwoo; Park, Seung Bum

2016-10-01

Diversity-oriented synthesis (DOS) can provide a collection of diverse and complex drug-like small molecules, which is critical in the development of new chemical probes for biological research of undruggable targets. However, the design and synthesis of small-molecule libraries with improved biological relevance as well as maximized molecular diversity represent a key challenge. Herein, we employ functional group-pairing strategy for the DOS of a chemical library containing privileged substructures, pyrimidodiazepine or pyrimidine moieties, as chemical navigators towards unexplored bioactive chemical space. To validate the utility of this DOS library, we identify a new small-molecule inhibitor of leucyl-tRNA synthetase-RagD protein-protein interaction, which regulates the amino acid-dependent activation of mechanistic target of rapamycin complex 1 signalling pathway. This work highlights that privileged substructure-based DOS strategy can be a powerful research tool for the construction of drug-like compounds to address challenging biological targets.

Y-Trap Cancer Immunotherapy Drug Targets Two Proteins

Cancer.gov

Two groups of researchers, working independently, have fused a TGF-beta receptor to a monoclonal antibody that targets a checkpoint protein. The result, this Cancer Currents blog describes, is a single hybrid molecule called a Y-trap that blocks two pathways used by tumors to evade the immune system.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

PubMed

Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

2017-09-19

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK

PubMed Central

Bührmann, Mike; Wiedemann, Bianca M.; Müller, Matthias P.; Hardick, Julia; Ecke, Maria

2017-01-01

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. PMID:28892510

NCI Blog Post: CPTAC, the Complementary Sibling of TCGA (An Interview with Dr. Henry Rodriguez about NCI’s Proteomics Program) | Office of Cancer Clinical Proteomics Research

Cancer.gov

What is proteomics? Proteomics is a highly automated and rapid method for measuring all the proteins in a biological sample. Proteins are the molecules that actually do most of the work inside a cell. When researchers develop cancer drugs, those drugs typically target proteins, so scientists and clinicians really have to understand what the proteins are doing. Proteomics researchers are now able to measure up to 10,000 proteins per tumor sample.

[Techniques for rapid production of monoclonal antibodies for use with antibody technology].

PubMed

Kamada, Haruhiko

2012-01-01

A monoclonal antibody (Mab), due to its specific binding ability to a target protein, can potentially be one of the most useful tools for the functional analysis of proteins in recent proteomics-based research. However, the production of Mab is a very time-consuming and laborious process (i.e., preparation of recombinant antigens, immunization of animals, preparation of hybridomas), making it the rate-limiting step in using Mabs in high-throughput proteomics research, which heavily relies on comprehensive and rapid methods. Therefore, there is a great demand for new methods to efficiently generate Mabs against a group of proteins identified by proteome analysis. Here, we describe a useful method called "Antibody proteomic technique" for the rapid generations of Mabs to pharmaceutical target, which were identified by proteomic analyses of disease samples (ex. tumor tissue, etc.). We also introduce another method to find profitable targets on vasculature, which is called "Vascular proteomic technique". Our results suggest that this method for the rapid generation of Mabs to proteins may be very useful in proteomics-based research as well as in clinical applications.

Detecting protein-protein interactions using Renilla luciferase fusion proteins.

PubMed

Burbelo, Peter D; Kisailus, Adam E; Peck, Jeremy W

2002-11-01

We have developed a novel system designated the luciferase assay for protein detection (LAPD) to study protein-protein interactions. This method involves two protein fusions, a soluble reporter fusion and a fusion for immobilizing the target protein. The soluble reporter is an N-terminal Renilla luciferase fusion protein that exhibits high Renilla luciferase activity. Crude cleared lysates from transfected Cos1 cells that express the Renilla luciferase fusion protein can be used in binding assays with immobilized target proteins. Following incubation and washing, target-bound Renilla luciferase fusion proteins produce light from the coelenterazine substrate, indicating an interaction between the two proteins of interest. As proof of the principle, we reproduced known, transient protein-protein interactions between the Cdc42 GTPase and its effector proteins. GTPase Renilla fusion proteins produced in Cos1 cells were tested with immobilized recombinant GST-N-WASP and CEP5 effector proteins. Using this assay, we could detect specific interactions of Cdc42 with these effector proteins in approximately 50 min. The specificity of these interactions was demonstrated by showing that they were GTPase-specific and GTP-dependent and not seen with other unrelated target proteins. These results suggest that the LAPD method, which is both rapid and sensitive, may have research and practical applications.

Challenges in validating candidate therapeutic targets in cancer

PubMed Central

Sawyers, Charles L; Hunter, Tony

2018-01-01

More than 30 published articles have suggested that a protein kinase called MELK is an attractive therapeutic target in human cancer, but three recent reports describe compelling evidence that it is not. These reports highlight the caveats associated with some of the research tools that are commonly used to validate candidate therapeutic targets in cancer research. PMID:29417929

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

PubMed

Zhao, Jin-yao; Liu, Chun-qing; Zhao, He-nan; Ding, Yan-Fang; Bi, Tie; Wang, Bo; Lin, Xing-chi; Guo, Gordon; Cui, Shi-ying

2012-10-01

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained. Copyright © 2012 Elsevier Inc. All rights reserved.

Cell cycle proteins as promising targets in cancer therapy.

PubMed

Otto, Tobias; Sicinski, Piotr

2017-01-27

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Genome scale enzyme–metabolite and drug–target interaction predictions using the signature molecular descriptor

DOE PAGES

Faulon, Jean-Loup; Misra, Milind; Martin, Shawn; ...

2007-11-23

Motivation: Identifying protein enzymatic or pharmacological activities are important areas of research in biology and chemistry. Biological and chemical databases are increasingly being populated with linkages between protein sequences and chemical structures. Additionally, there is now sufficient information to apply machine-learning techniques to predict interactions between chemicals and proteins at a genome scale. Current machine-learning techniques use as input either protein sequences and structures or chemical information. We propose here a method to infer protein–chemical interactions using heterogeneous input consisting of both protein sequence and chemical information. Results: Our method relies on expressing proteins and chemicals with a common cheminformaticsmore » representation. We demonstrate our approach by predicting whether proteins can catalyze reactions not present in training sets. We also predict whether a given drug can bind a target, in the absence of prior binding information for that drug and target. Lastly, such predictions cannot be made with current machine-learning techniques requiring binding information for individual reactions or individual targets.« less

Beyond cysteine: recent developments in the area of targeted covalent inhibition.

PubMed

Mukherjee, Herschel; Grimster, Neil P

2018-05-29

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Orally active-targeted drug delivery systems for proteins and peptides.

PubMed

Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2014-09-01

In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

Retrieval of Enterobacteriaceae drug targets using singular value decomposition.

PubMed

Silvério-Machado, Rita; Couto, Bráulio R G M; Dos Santos, Marcos A

2015-04-15

The identification of potential drug target proteins in bacteria is important in pharmaceutical research for the development of new antibiotics to combat bacterial agents that cause diseases. A new model that combines the singular value decomposition (SVD) technique with biological filters composed of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of Escherichia coli (strain K12) has been created to predict potential antibiotic drug targets in the Enterobacteriaceae family. This model identified 99 potential drug target proteins in the studied family, which exhibit eight different functions and are protein-coding essential genes or similar to protein-coding essential genes of E.coli (strain K12), indicating that the disruption of the activities of these proteins is critical for cells. Proteins from bacteria with described drug resistance were found among the retrieved candidates. These candidates have no similarity to the human proteome, therefore exhibiting the advantage of causing no adverse effects or at least no known adverse effects on humans. rita_silverio@hotmail.com. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

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

Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2more » reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.« less

Crystal Structure of Protein Reveals Target for Drugs Against Lethal MERS Virus | FNLCR Staging

Cancer.gov

A research team of scientists from the National Cancer Institute and the Frederick National Laboratory for Cancer Research recently identified the structure of a key protein of the virus that causes the highly lethal Middle East Respiratory Syndrome.

A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

PubMed Central

Pullara, Filippo; Guerrero-Santoro, Jennifer; Calero, Monica; Zhang, Qiangmin; Peng, Ye; Spåhr, Henrik; Kornberg, Guy L.; Cusimano, Antonella; Stevenson, Hilary P.; Santamaria-Suarez, Hugo; Reynolds, Shelley L.; Brown, Ian S.; Monga, Satdarshan P.S.; Van Houten, Bennett; Rapić-Otrin, Vesna; Calero, Guillermo; Levine, Arthur S.

2014-01-01

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential “druggable” targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format. PMID:23137940

The identification of new protein kinase inhibitors as targets in modern drug discovery.

PubMed

Akritopoulou-Zanze, Irini

2006-07-01

In recent years there has been great interest in developing protein kinase inhibitors as therapeutic agents for a variety of diseases. This article provides an overview on the history, development and validity of kinases as drug targets, as well as a description of kinase research, including its limitations, challenges and successes.

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

Advances in targeted proteomics and applications to biomedical research

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

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

Small molecules targeting heterotrimeric G proteins.

PubMed

Ayoub, Mohammed Akli

2018-05-05

G protein-coupled receptors (GPCRs) represent the largest family of cell surface receptors regulating many human and animal physiological functions. Their implication in human pathophysiology is obvious with almost 30-40% medical drugs commercialized today directly targeting GPCRs as molecular entities. However, upon ligand binding GPCRs signal inside the cell through many key signaling, adaptor and regulatory proteins, including various classes of heterotrimeric G proteins. Therefore, G proteins are considered interesting targets for the development of pharmacological tools that are able to modulate their interaction with the receptors, as well as their activation/deactivation processes. In this review, old attempts and recent advances in the development of small molecules that directly target G proteins will be described with an emphasis on their utilization as pharmacological tools to dissect the mechanisms of activation of GPCR-G protein complexes. These molecules constitute a further asset for research in the "hot" areas of GPCR biology, areas such as multiple G protein coupling/signaling, GPCR-G protein preassembly, and GPCR functional selectivity or bias. Moreover, this review gives a particular focus on studies in vitro and in vivo supporting the potential applications of such small molecules in various GPCR/G protein-related diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

In Silico Investigation of the Pharmacological Mechanisms of Beneficial Effects of Ginkgo biloba L. on Alzheimer's Disease.

PubMed

Li, Hongxiang; Sun, Xiaoyuan; Yu, Fan; Xu, Lijia; Miu, Jianhua; Xiao, Peigen

2018-05-10

Based on compelling experimental and clinical evidence, Ginkgo biloba L. exerts a beneficial effect in ameliorating mild to moderate dementia in patients with Alzheimer’s disease (AD) and other neurological disorders, although the pharmacological mechanisms remain unknown. In the present study, compounds, their putative target proteins identified using an inverse docking approach, and clinically tested AD-related target proteins were systematically integrated together with applicable bioinformatics methods in silico . The results suggested that the beneficial effects of G. biloba on AD may be contributed by the regulation of hormone sensitivity, improvements in endocrine homeostasis, maintenance of endothelial microvascular integrity, and proteolysis of tau proteins, particularly prior to amyloid β-protein (Aβ) plaque formation. Moreover, we identified six putative protein targets that are significantly related to AD, but have not been researched or have had only preliminary studies conducted on the anti-AD effects of G. biloba . These mechanisms and protein targets are very significant for future scientific research. In addition, the existing mechanisms were also verified, such as the reduction of oxidative stress, anti-apoptotic effects, and protective effects against amyloidogenesis and Aβ aggregation. The discoveries summarized here may provide a macroscopic perspective that will improve our understanding of the molecular mechanism of medicinal plants or dietary supplements, as well as new clues for the future development of therapeutic strategies for AD.

Pharos: Collating protein information to shed light on the druggable genome.

PubMed

Nguyen, Dac-Trung; Mathias, Stephen; Bologa, Cristian; Brunak, Soren; Fernandez, Nicolas; Gaulton, Anna; Hersey, Anne; Holmes, Jayme; Jensen, Lars Juhl; Karlsson, Anneli; Liu, Guixia; Ma'ayan, Avi; Mandava, Geetha; Mani, Subramani; Mehta, Saurabh; Overington, John; Patel, Juhee; Rouillard, Andrew D; Schürer, Stephan; Sheils, Timothy; Simeonov, Anton; Sklar, Larry A; Southall, Noel; Ursu, Oleg; Vidovic, Dusica; Waller, Anna; Yang, Jeremy; Jadhav, Ajit; Oprea, Tudor I; Guha, Rajarshi

2017-01-04

The 'druggable genome' encompasses several protein families, but only a subset of targets within them have attracted significant research attention and thus have information about them publicly available. The Illuminating the Druggable Genome (IDG) program was initiated in 2014, has the goal of developing experimental techniques and a Knowledge Management Center (KMC) that would collect and organize information about protein targets from four families, representing the most common druggable targets with an emphasis on understudied proteins. Here, we describe two resources developed by the KMC: the Target Central Resource Database (TCRD) which collates many heterogeneous gene/protein datasets and Pharos (https://pharos.nih.gov), a multimodal web interface that presents the data from TCRD. We briefly describe the types and sources of data considered by the KMC and then highlight features of the Pharos interface designed to enable intuitive access to the IDG knowledgebase. The aim of Pharos is to encourage 'serendipitous browsing', whereby related, relevant information is made easily discoverable. We conclude by describing two use cases that highlight the utility of Pharos and TCRD. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

PubMed

Geel, T M; Ruiters, M H J; Cool, R H; Halby, L; Voshart, D C; Andrade Ruiz, L; Niezen-Koning, K E; Arimondo, P B; Rots, M G

2018-06-05

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.

Immunotherapy Targets Common Cancer Mutation

Cancer.gov

In a study of an immune therapy for colorectal cancer that involved a single patient, researchers identified a method for targeting the cancer-causing protein produced by a mutant form of the KRAS gene.

Cardioprotective role of G-Protein Coupled Estrogen Receptor 1 (GPER1).

PubMed

Koganti, Sivaramakrishna

2015-01-01

G-Protein Coupled Estrogen Receptor 1 (GPER1), also known as G-Protein Coupled Receptor 30 (GPR30) and initially considered an orphan receptor, has become one of the most important pharmacological targets in cardiovascular research. Since the gene encoding this putative receptor was cloned nearly 20 years ago, researchers have addressed its role in various aspects of physiology, including cardioprotection. Although extensive research has been carried out to understand the role of GPER1 as a pharmacological target to treat cardiovascular diseases, there are few current reviews addressing the overall cardioprotective benefits of this receptor and the signaling intermediates involved. This review considers the origins of GPER1, its cell biology, its physiological and pharmacological roles as a therapeutic target in cardiovascular disease, and what future research on GPER1 might entail. More specifically, the review focuses on GPER1 regulation of Angiotensin Type I Receptor (AT1R) and the role of estrogen receptors, epidermal growth factor receptor (EGFR) and matrix metalloproteinases (MMPs) in bringing about the cardioprotective effects of GPER1. Areas where improved knowledge of GPER1 biology is still needed to better understand the receptor's cardioprotective effects are also discussed.

Unexplored therapeutic opportunities in the human genome.

PubMed

Oprea, Tudor I; Bologa, Cristian G; Brunak, Søren; Campbell, Allen; Gan, Gregory N; Gaulton, Anna; Gomez, Shawn M; Guha, Rajarshi; Hersey, Anne; Holmes, Jayme; Jadhav, Ajit; Jensen, Lars Juhl; Johnson, Gary L; Karlson, Anneli; Leach, Andrew R; Ma'ayan, Avi; Malovannaya, Anna; Mani, Subramani; Mathias, Stephen L; McManus, Michael T; Meehan, Terrence F; von Mering, Christian; Muthas, Daniel; Nguyen, Dac-Trung; Overington, John P; Papadatos, George; Qin, Jun; Reich, Christian; Roth, Bryan L; Schürer, Stephan C; Simeonov, Anton; Sklar, Larry A; Southall, Noel; Tomita, Susumu; Tudose, Ilinca; Ursu, Oleg; Vidovic, Dušica; Waller, Anna; Westergaard, David; Yang, Jeremy J; Zahoránszky-Köhalmi, Gergely

2018-05-01

A large proportion of biomedical research and the development of therapeutics is focused on a small fraction of the human genome. In a strategic effort to map the knowledge gaps around proteins encoded by the human genome and to promote the exploration of currently understudied, but potentially druggable, proteins, the US National Institutes of Health launched the Illuminating the Druggable Genome (IDG) initiative in 2014. In this article, we discuss how the systematic collection and processing of a wide array of genomic, proteomic, chemical and disease-related resource data by the IDG Knowledge Management Center have enabled the development of evidence-based criteria for tracking the target development level (TDL) of human proteins, which indicates a substantial knowledge deficit for approximately one out of three proteins in the human proteome. We then present spotlights on the TDL categories as well as key drug target classes, including G protein-coupled receptors, protein kinases and ion channels, which illustrate the nature of the unexplored opportunities for biomedical research and therapeutic development.

Reminder: NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to improve rigor and reproducibility, the National Cancer Institute (NCI) Antibody Characterization Program requests cancer-related protein targets for monoclonal antibody production and distribution to the scientific community. The program from The Office of Cancer Clinical Proteomics Research provides well-characterized

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

Joubert, Fourie; Harrison, Claudia M; Koegelenberg, Riaan J; Odendaal, Christiaan J; de Beer, Tjaart AP

2009-01-01

Background Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties. Methods A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available. Results An in silico system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from Plasmodium falciparum. Conclusion The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties. PMID:19642978

Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

PubMed Central

Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

2016-01-01

Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biological background, 1 PPDT-Module and 22 PCOS potential drug targets were identified, 21 of which were verified by literatures to be associated with the pathogenesis of PCOS. 42 drugs targeting to 13 PCOS potential drug targets were investigated experimentally or clinically for PCOS. Evaluated by independent datasets, the whole PPDT-Module and 22 PCOS potential drug targets could not only reveal the drug response, but also distinguish the statuses between normal and disease. Our identified PPDT-Module and PCOS potential drug targets would shed light on the treatment of PCOS. And our approach would provide valuable insights to research on the pathogenesis and drug response of other diseases. PMID:27191267

Changes in miRNAs Signal High-Risk HPV Infections | Center for Cancer Research

Cancer.gov

microRNAs (miRNAs) are approximately 21 nucleotide long, non-coding RNAs that regulate the expression of certain proteins. As part of the RNA-induced silencing complex or RISC, miRNAs bind to complementary sequences in the 3’ untranslated regions of target messenger RNAs, blocking protein synthesis and sometimes leading to the destruction of the target RNA. Numerous studies

CPTAC Investigators Identify Rogue Breast Tumor Proteins That Point To Potential Drug Therapies | Office of Cancer Clinical Proteomics Research

Cancer.gov

For patients with difficult-to-treat cancers, doctors increasingly rely on genomic testing of tumors to identify errors in the DNA that indicate a tumor can be targeted by existing therapies. But this approach overlooks another potential marker — rogue proteins — that may be driving cancer cells and also could be targeted with existing treatments.

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

PubMed

Prieto, DaRue A; Chan, King C; Johann, Donald J; Ye, Xiaoying; Whitely, Gordon; Blonder, Josip

2017-01-01

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

[Research progress on ebola virus glycoprotein].

PubMed

Ding, Guo-Yong; Wang, Zhi-Yu; Gao, Lu; Jiang, Bao-Fa

2013-03-01

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.

Immunohistochemical Evaluation of AKT Protein Activation in Canine Mast Cell Tumours

PubMed Central

Rodriguez, S.; Fadlalla, K.; Graham, T.; Tameru, B.; Fermin, C. D.; Samuel, T.

2011-01-01

Summary The pathogenesis of canine mast cell tumour (MCT) remains unknown. Moreover, therapeutic options are limited and resistance to targeted drugs and recurrences are common, necessitating the identification of additional cellular targets for therapy. In this study we investigated the expression of phosphorylated AKT protein in 25 archival canine MCT samples by immunohistochemistry and examined the correlation between the immunohistochemical scores and histopathological tumour grades. AKT protein was detected in all of the samples and 24 of the 25 samples expressed the phosphorylated form of the protein, albeit with variable intensity. However, when the immunohistochemical scores of weak, intermediate and strong labelling were compared with the histopathological grades of I to III, there was no strong correlation. This study suggests that canine MCT cells have activated AKT and indicates the need for further research on the role of the AKT protein and the possibility of targeting the AKT signalling pathway in MCTs. PMID:22289273

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula.

PubMed

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling; Wang, Yun

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components.

GEPSI: A Gene Expression Profile Similarity-Based Identification Method of Bioactive Components in Traditional Chinese Medicine Formula

PubMed Central

Zhang, Baixia; He, Shuaibing; Lv, Chenyang; Zhang, Yanling

2018-01-01

The identification of bioactive components in traditional Chinese medicine (TCM) is an important part of the TCM material foundation research. Recently, molecular docking technology has been extensively used for the identification of TCM bioactive components. However, target proteins that are used in molecular docking may not be the actual TCM target. For this reason, the bioactive components would likely be omitted or incorrect. To address this problem, this study proposed the GEPSI method that identified the target proteins of TCM based on the similarity of gene expression profiles. The similarity of the gene expression profiles affected by TCM and small molecular drugs was calculated. The pharmacological action of TCM may be similar to that of small molecule drugs that have a high similarity score. Indeed, the target proteins of the small molecule drugs could be considered TCM targets. Thus, we identified the bioactive components of a TCM by molecular docking and verified the reliability of this method by a literature investigation. Using the target proteins that TCM actually affected as targets, the identification of the bioactive components was more accurate. This study provides a fast and effective method for the identification of TCM bioactive components. PMID:29692857

Assay Development Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

Typical steps involved in the development of a  mass spectrometry-based targeted assay include: (1) selection of surrogate or signature peptides corresponding to the targeted protein or modification of interest; (2) iterative optimization of instrument and method parameters for optimal detection of the selected peptide; (3) method development for protein extraction from biological matrices such as tissue, whole cell lysates, or blood plasma/serum and proteolytic digestion of proteins (usually with trypsin); (4) evaluation of the assay in the intended biological matrix to determine if e

The Emory Chemical Biology Discovery Center: leveraging academic innovation to advance novel targets through HTS and beyond.

PubMed

Johns, Margaret A; Meyerkord-Belton, Cheryl L; Du, Yuhong; Fu, Haian

2014-03-01

The Emory Chemical Biology Discovery Center (ECBDC) aims to accelerate high throughput biology and translation of biomedical research discoveries into therapeutic targets and future medicines by providing high throughput research platforms to scientific collaborators worldwide. ECBDC research is focused at the interface of chemistry and biology, seeking to fundamentally advance understanding of disease-related biology with its HTS/HCS platforms and chemical tools, ultimately supporting drug discovery. Established HTS/HCS capabilities, university setting, and expertise in diverse assay formats, including protein-protein interaction interrogation, have enabled the ECBDC to contribute to national chemical biology efforts, empower translational research, and serve as a training ground for young scientists. With these resources, the ECBDC is poised to leverage academic innovation to advance biology and therapeutic discovery.

Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

PubMed

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

Inhibitors of Protein Methyltransferases and Demethylases

PubMed Central

2017-01-01

Post-translational modifications of histones by protein methyltransferases (PMTs) and histone demethylases (KDMs) play an important role in the regulation of gene expression and transcription and are implicated in cancer and many other diseases. Many of these enzymes also target various nonhistone proteins impacting numerous crucial biological pathways. Given their key biological functions and implications in human diseases, there has been a growing interest in assessing these enzymes as potential therapeutic targets. Consequently, discovering and developing inhibitors of these enzymes has become a very active and fast-growing research area over the past decade. In this review, we cover the discovery, characterization, and biological application of inhibitors of PMTs and KDMs with emphasis on key advancements in the field. We also discuss challenges, opportunities, and future directions in this emerging, exciting research field. PMID:28338320

NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, the National Cancer Institute (NCI) Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance

PubMed Central

Lee, Samuel; Kim, Soo Min

2013-01-01

Abstract The thioredoxin (Trx) system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through Trx reductase to Trx, which reduces its target proteins using highly conserved thiol groups. While the importance of protecting cells from the detrimental effects of reactive oxygen species is clear, decades of research in this field revealed that there is a network of redox-sensitive proteins forming redox-dependent signaling pathways that are crucial for fundamental cellular processes, including metabolism, proliferation, differentiation, migration, and apoptosis. Trx participates in signaling pathways interacting with different proteins to control their dynamic regulation of structure and function. In this review, we focus on Trx target proteins that are involved in redox-dependent signaling pathways. Specifically, Trx-dependent reductive enzymes that participate in classical redox reactions and redox-sensitive signaling molecules are discussed in greater detail. The latter are extensively discussed, as ongoing research unveils more and more details about the complex signaling networks of Trx-sensitive signaling molecules such as apoptosis signal-regulating kinase 1, Trx interacting protein, and phosphatase and tensin homolog, thus highlighting the potential direct and indirect impact of their redox-dependent interaction with Trx. Overall, the findings that are described here illustrate the importance and complexity of Trx-dependent, redox-sensitive signaling in the cell. Our increasing understanding of the components and mechanisms of these signaling pathways could lead to the identification of new potential targets for the treatment of diseases, including cancer and diabetes. Antioxid. Redox Signal. 18, 1165–1207. PMID:22607099

Advanced and recurring thymic carcinoma is target of new clinical trial | Center for Cancer Research

Cancer.gov

Adults diagnosed with thymic carcinoma who overexpress the protein mesothelin may be eligible to participate in a new clinical trial at the NIH Clinical Center. The study will look at the safety and effectiveness of an investigational drug, anetumab ravtansine, developed by Bayer HealthCare Pharmaceuticals. The drug works by binding to mesothelin, therefore overexpression of the protein could be useful for targeting cancer cells. Read more...

A protein in neuroblastoma could be a target of immunotoxins or immunotherapy | Center for Cancer Research

Cancer.gov

A cell surface protein, glycoprotein glypican-2 (GPC2), has been found to be an effective therapeutic target in cell cultures and mouse models that mimic childhood neuroblastoma.  The CCR scientists who made this discovery, reported July 24, 2017, in PNAS, have also produced immunotoxins and chimeric antigen receptor (CAR) T cells, a type of immunotherapy, that have shown promise against this solid tumor. Read more...

Liver proteomics for therapeutic drug discovery: inhibition of the cyclophilin receptor CD147 attenuates sepsis-induced acute renal failure

PubMed Central

Dear, James W.; Leelahavanichkul, Asada; Aponte, Angel; Hu, Xuzhen; Constant, Stephanie L.; Hewitt, Stephen M.; Yuen, Peter S.T.; Star, Robert A.

2008-01-01

Objective Sepsis-induced multi-organ failure continues to have a high mortality. The liver is an organ central to the disease pathogenesis. The objective of this study was to identify the liver proteins that change in abundance with sepsis and, therefore, identify new drug targets. Design Proteomic discovery study and drug target validation Setting Research institute laboratory Subjects Three month old C57BL/6 mice Interventions We used a mouse model of sepsis based on cecal ligation and puncture (CLP) but with fluid and antibiotic resuscitation. Liver proteins that changed in abundance were identified by difference in-gel electrophoresis (DIGE). We compared liver proteins from 6 hr post-CLP to sham-operated mice (‘early proteins’) and 24 hr post-CLP with 6 hr post-CLP (‘late proteins’). Proteins that changed in abundance were identified by tandem mass spectrometry. We then inhibited the receptor for one protein and determined the effect on sepsis-induced organ dysfunction. Results The liver proteins that changed in abundance after sepsis had a range of functions such as acute phase proteins, coagulation, ER stress, oxidative stress, apoptosis, mitochondrial proteins and nitric oxide metabolism. We found that cyclophilin increased in abundance after CLP. When the receptor for this protein, CD147, was inhibited sepsis-induced renal dysfunction was reduced. There was also a significant reduction in serum cytokine production when CD147 was inhibited. Conclusion By applying proteomics to a clinically relevant mouse model of sepsis we identified a number of novel proteins that changed in abundance. The inhibition of the receptor for one of these proteins, cyclophilin, attenuated sepsis-induced acute renal failure. The application of proteomics to sepsis research can facilitate the discovery of new therapeutic targets. PMID:17944020

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

DOE PAGES

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.; ...

2017-04-19

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Leucine-rich-repeat-containing variable lymphocyte receptors as modules to target plant-expressed proteins

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

Velásquez, André C.; Nomura, Kinya; Cooper, Max D.

The ability to target and manipulate protein-based cellular processes would accelerate plant research; yet, the technology to specifically and selectively target plant-expressed proteins is still in its infancy. Leucine-rich repeats (LRRs) are ubiquitously present protein domains involved in mediating protein–protein interactions. LRRs confer the binding specificity to the highly diverse variable lymphocyte receptor (VLR) antibodies (including VLRA, VLRB and VLRC types) that jawless vertebrates make as the functional equivalents of jawed vertebrate immunoglobulin-based antibodies. Here, VLRBs targeting an effector protein from a plant pathogen, HopM1, were developed by immunizing lampreys and using yeast surface display to select for high-affinity VLRBs.more » HopM1-specific VLRBs (VLRM1) were expressed in planta in the cytosol, the trans-Golgi network, and the apoplast. Expression of VLRM1 was higher when the protein localized to an oxidizing environment that would favor disulfide bridge formation (when VLRM1 was not localized to the cytoplasm), as disulfide bonds are necessary for proper VLR folding. VLRM1 specifically interacted in planta with HopM1 but not with an unrelated bacterial effector protein while HopM1 failed to interact with a non-specific VLRB. Later, VLRs may be used as flexible modules to bind proteins or carbohydrates of interest in planta, with broad possibilities for their use by binding directly to their targets and inhibiting their action, or by creating chimeric proteins with new specificities in which endogenous LRR domains are replaced by those present in VLRs.« less

Target research on tumor biology characteristics of mir-155-5p regulation on gastric cancer cell.

PubMed

Feng, Jun-an

2016-03-01

After the mir-155-5p over expressed in gastric cancer cells, the expression profile chip was adopted to screen its target genes. Some of the intersection of target genes were selected based on the bioinformatics prediction, in order to study the mechanism of its function and role of research. Affymetrix eukaryotic gene expression spectrum was conducted to screen mir-155-5p regulated genetic experiment. Western blot technique was employed to detect and screen the protein expression of target genes. Mimics was transfected in BGC-823 of gastric cancer cells. Compared with mimics-nc group and mock group, the mRNA expression quantities of SMAD1, STAT1, CAB39, CXCR4 and CA9 were significantly lower. After the gastric cancer cells BGC-823 and MKN-45 had been transfected by mimics, compared with mimics-nc (MNC) group and mock (MOCK) group, it was decreased for the protein expression of SMAD1, STAT1 and CAB39 in mimics (MIMICS) group. The verification of qRT-PCR demonstrated that SMAD1, STAT1, CAB39, CXCR4 and CA9 were the predicted target genes and target proteins of mir-155-5p, the over expression of mir-155-5p could enable the decreasing of its expression level in gastric cancer cells MKN-45 and BGC-823.

PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.

PubMed

Mohammed, Yassene; Domański, Dominik; Jackson, Angela M; Smith, Derek S; Deelder, André M; Palmblad, Magnus; Borchers, Christoph H

2014-06-25

One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day. Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM-MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

PubMed Central

Unzueta, Ugutz; Céspedes, María Virtudes; Ferrer-Miralles, Neus; Casanova, Isolda; Cedano, Juan; Corchero, José Luis; Domingo-Espín, Joan; Villaverde, Antonio; Mangues, Ramón; Vázquez, Esther

2012-01-01

Background Cell-targeting peptides or proteins are appealing tools in nanomedicine and innovative medicines because they increase the local drug concentration and reduce potential side effects. CXC chemokine receptor 4 (CXCR4) is a cell surface marker associated with several severe human pathologies, including colorectal cancer, for which intracellular targeting agents are currently missing. Results Four different peptides that bind CXCR4 were tested for their ability to internalize a green fluorescent protein-based reporter nanoparticle into CXCR4+ cells. Among them, only the 18 mer peptide T22, an engineered segment derivative of polyphemusin II from the horseshoe crab, efficiently penetrated target cells via a rapid, receptor-specific endosomal route. This resulted in accumulation of the reporter nanoparticle in a fully fluorescent and stable form in the perinuclear region of the target cells, without toxicity either in cell culture or in an in vivo model of metastatic colorectal cancer. Conclusion Given the urgent demand for targeting agents in the research, diagnosis, and treatment of CXCR4-linked diseases, including colorectal cancer and human immunodeficiency virus infection, T22 appears to be a promising tag for the intracellular delivery of protein drugs, nanoparticles, and imaging agents. PMID:22923991

False-positive rate determination of protein target discovery using a covalent modification- and mass spectrometry-based proteomics platform.

PubMed

Strickland, Erin C; Geer, M Ariel; Hong, Jiyong; Fitzgerald, Michael C

2014-01-01

Detection and quantitation of protein-ligand binding interactions is important in many areas of biological research. Stability of proteins from rates of oxidation (SPROX) is an energetics-based technique for identifying the proteins targets of ligands in complex biological mixtures. Knowing the false-positive rate of protein target discovery in proteome-wide SPROX experiments is important for the correct interpretation of results. Reported here are the results of a control SPROX experiment in which chemical denaturation data is obtained on the proteins in two samples that originated from the same yeast lysate, as would be done in a typical SPROX experiment except that one sample would be spiked with the test ligand. False-positive rates of 1.2-2.2% and <0.8% are calculated for SPROX experiments using Q-TOF and Orbitrap mass spectrometer systems, respectively. Our results indicate that the false-positive rate is largely determined by random errors associated with the mass spectral analysis of the isobaric mass tag (e.g., iTRAQ®) reporter ions used for peptide quantitation. Our results also suggest that technical replicates can be used to effectively eliminate such false positives that result from this random error, as is demonstrated in a SPROX experiment to identify yeast protein targets of the drug, manassantin A. The impact of ion purity in the tandem mass spectral analyses and of background oxidation on the false-positive rate of protein target discovery using SPROX is also discussed.

Phase I clinical trial will test multi-targeted immunotherapy in common childhood cancer | Center for Cancer Research

Cancer.gov

Chimeric antigen receptor (CAR) T-cell immunotherapy targeting the protein CD19 has shown promise in treating acute lymphoblastic leukemia (ALL). CD22-CAR T-cell therapy has yielded similarly encouraging results, but many patients relapse after either therapy. In an upcoming phase I clinical trial, Center for Cancer Research investigators will test a new strategy—treating patients with a CAR T-cell therapy that targets CD19 and CD22 simultaneously.

Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity.

PubMed

Abdeeva, Inna A; Pogorelko, Gennady V; Maloshenok, Liliya G; Mokrykova, Maria V; Fursova, Oksana V; Bruskin, Sergey A

2018-04-19

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes ( AtCYP19-1/ROC3 , AtFKBP65/ROF2 , and AtCYP57 ) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein⁻protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3 , ROF2 , and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

Sirtuins in dermatology: applications for future research and therapeutics.

PubMed

Serravallo, Melissa; Jagdeo, Jared; Glick, Sharon A; Siegel, Daniel M; Brody, Neil I

2013-05-01

Sirtuins are a family of seven proteins in humans (SIRT1-SIRT7) that are involved in multiple cellular processes relevant to dermatology. The role of sirtuins in other organ systems is established. However, the importance of these proteins in dermatology is less defined. Recently, sirtuins gained international attention because of their role as "longevity proteins" that may extend and enhance human life. Sirtuins function in the cell via histone deacetylase and/or adenosine diphosphate ribosyltransferase enzymatic activity that target histone and non-histone substrates, including transcription regulators, tumor suppressors, structural proteins, DNA repair proteins, cell signaling proteins, transport proteins, and enzymes. Sirtuins are involved in cellular pathways related to skin structure and function, including aging, ultraviolet-induced photoaging, inflammation, epigenetics, cancer, and a variety of cellular functions including cell cycle, DNA repair and proliferation. This review highlights sirtuin-related cellular pathways, therapeutics and pharmacological targets in atopic dermatitis, bullous dermatoses, collagen vascular disorders, psoriasis, systemic lupus erythematosus, hypertrophic and keloid scars, cutaneous infections, and non-melanoma and melanoma skin cancer. Also discussed is the role of sirtuins in the following genodermatoses: ataxia telangiectasia, Cowden's syndrome, dyskeratosis congenita, Rubenstein-Taybi, Werner syndrome, and xeroderma pigmentosum. The pathophysiology of these inherited diseases is not well understood, and sirtuin-related processes represent potential therapeutic targets for diseases lacking suitable alternative treatments. The goal of this review is to bring attention to the dermatology community, physicians, and scientists, the importance of sirtuins in dermatology and provide a foundation and impetus for future discussion, research and pharmacologic discovery.

CCProf: exploring conformational change profile of proteins

PubMed Central

Chang, Che-Wei; Chou, Chai-Wei; Chang, Darby Tien-Hao

2016-01-01

In many biological processes, proteins have important interactions with various molecules such as proteins, ions or ligands. Many proteins undergo conformational changes upon these interactions, where regions with large conformational changes are critical to the interactions. This work presents the CCProf platform, which provides conformational changes of entire proteins, named conformational change profile (CCP) in the context. CCProf aims to be a platform where users can study potential causes of novel conformational changes. It provides 10 biological features, including conformational change, potential binding target site, secondary structure, conservation, disorder propensity, hydropathy propensity, sequence domain, structural domain, phosphorylation site and catalytic site. All these information are integrated into a well-aligned view, so that researchers can capture important relevance between different biological features visually. The CCProf contains 986 187 protein structure pairs for 3123 proteins. In addition, CCProf provides a 3D view in which users can see the protein structures before and after conformational changes as well as binding targets that induce conformational changes. All information (e.g. CCP, binding targets and protein structures) shown in CCProf, including intermediate data are available for download to expedite further analyses. Database URL: http://zoro.ee.ncku.edu.tw/ccprof/ PMID:27016699

Metformin targets multiple signaling pathways in cancer.

PubMed

Lei, Yong; Yi, Yanhua; Liu, Yang; Liu, Xia; Keller, Evan T; Qian, Chao-Nan; Zhang, Jian; Lu, Yi

2017-01-26

Metformin, an inexpensive and well-tolerated oral agent commonly used in the first-line treatment of type 2 diabetes, has become the focus of intense research as a candidate anticancer agent. Here, we discuss the potential of metformin in cancer therapeutics, particularly its functions in multiple signaling pathways, including AMP-activated protein kinase, mammalian target of rapamycin, insulin-like growth factor, c-Jun N-terminal kinase/mitogen-activated protein kinase (p38 MAPK), human epidermal growth factor receptor-2, and nuclear factor kappaB pathways. In addition, cutting-edge targeting of cancer stem cells by metformin is summarized.

Characterizing and Targeting Replication Stress Response Defects in Breast Cancer

DTIC Science & Technology

2013-08-01

This project is to use cutting-edge technologies to characterize novel RSR genes and their functions in tumor suppression; identify gene signature...and membrane proteins associated with defective RSR; identify drugs that target these defects; and develop RSR-defect-targeting nanoparticles for...screening and validation of drugs that target RSR-defect cells. The progress of our third year research is described below. BODY The tasks

Targeting Common but Complex Proteoglycans on Breast Cancer Cells and Stem Cells Using Evolutionary Refined Malaria Proteins

DTIC Science & Technology

2014-09-01

chondroitin sulfate A proteoglycans present on all tested breast cancer cells and the vast majority of tested tissue biopsies. Using pull down assays...Invited, Daugaard. C) 2014. Gordon research conference, July 6-11; Targeting of cancer-specific chondroitin sulfate on circulating tumor cells using...now successfully identified a number of proteoglycans that interact with the recombinant malaria protein VAR2CSA when sulfated on carbon 4 of the CS

Phase I clinical trial will test multi-targeted immunotherapy in common childhood cancer | Center for Cancer Research

Cancer.gov

Chimeric antigen receptor (CAR) T-cell immunotherapy targeting the protein CD19 has shown promise in treating acute lymphoblastic leukemia (ALL). CD22-CAR T-cell therapy has yielded similarly encouraging results, but many patients relapse after either therapy. In an upcoming phase I clinical trial, Center for Cancer Research investigators will test a new strategy—treating

Male contraception: past, present and future.

PubMed

Payne, Christopher; Goldberg, Erwin

2014-01-01

Current contraceptive options available to men include withdrawal, condoms, and vasectomy, each of which has its own drawbacks. In this chapter we will describe the pros and cons for each, as well as methodological and product updates. Statistics from the U.S. Centers for Disease Control on acceptance and satisfaction will be included. Advances in vasectomy and reversal will be presented. Methods to develop new contraceptive technologies fall into two categories: hormonal and non-hormonal. Many targets and strategies have been proposed for non-hormonal male contraception within the testis. Targets include structural components in the testis, as well as enzymes, ion channels and other proteins specific to spermatozoa. Here we provide an overview of the spermatogenic mechanisms and proteins that have received research interest to date. We also discuss potential novel targets, such as ubiquitin specific proteases, that warrant greater research emphasis.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 9, 2012.

Chemical probes targeting epigenetic proteins: Applications beyond oncology

PubMed Central

Ackloo, Suzanne; Brown, Peter J.; Müller, Susanne

2017-01-01

ABSTRACT Epigenetic chemical probes are potent, cell-active, small molecule inhibitors or antagonists of specific domains in a protein; they have been indispensable for studying bromodomains and protein methyltransferases. The Structural Genomics Consortium (SGC), comprising scientists from academic and pharmaceutical laboratories, has generated most of the current epigenetic chemical probes. Moreover, the SGC has shared about 4 thousand aliquots of these probes, which have been used primarily for phenotypic profiling or to validate targets in cell lines or primary patient samples cultured in vitro. Epigenetic chemical probes have been critical tools in oncology research and have uncovered mechanistic insights into well-established targets, as well as identify new therapeutic starting points. Indeed, the literature primarily links epigenetic proteins to oncology, but applications in inflammation, viral, metabolic and neurodegenerative diseases are now being reported. We summarize the literature of these emerging applications and provide examples where existing probes might be used. PMID:28080202

[Crystal structure of SMU.2055 protein from Streptococcus mutans and its small molecule inhibitors design and selection].

PubMed

Xiaodan, Chen; Xiurong, Zhan; Xinyu, Wu; Chunyan, Zhao; Wanghong, Zhao

2015-04-01

The aim of this study is to analyze the three-dimensional crystal structure of SMU.2055 protein, a putative acetyltransferase from the major caries pathogen Streptococcus mutans (S. mutans). The design and selection of the structure-based small molecule inhibitors are also studied. The three-dimensional crystal structure of SMU.2055 protein was obtained by structural genomics research methods of gene cloning and expression, protein purification with Ni²⁺-chelating affinity chromatography, crystal screening, and X-ray diffraction data collection. An inhibitor virtual model matching with its target protein structure was set up using computer-aided drug design methods, virtual screening and fine docking, and Libdock and Autodock procedures. The crystal of SMU.2055 protein was obtained, and its three-dimensional crystal structure was analyzed. This crystal was diffracted to a resolution of 0.23 nm. It belongs to orthorhombic space group C222(1), with unit cell parameters of a = 9.20 nm, b = 9.46 nm, and c = 19.39 nm. The asymmetric unit contained four molecules, with a solvent content of 56.7%. Moreover, five small molecule compounds, whose structure matched with that of the target protein in high degree, were designed and selected. Protein crystallography research of S. mutans SMU.2055 helps to understand the structures and functions of proteins from S. mutans at the atomic level. These five compounds may be considered as effective inhibitors to SMU.2055. The virtual model of small molecule inhibitors we built will lay a foundation to the anticaries research based on the crystal structure of proteins.

Drug Target Mining and Analysis of the Chinese Tree Shrew for Pharmacological Testing

PubMed Central

Liu, Jie; Lee, Wen-hui; Zhang, Yun

2014-01-01

The discovery of new drugs requires the development of improved animal models for drug testing. The Chinese tree shrew is considered to be a realistic candidate model. To assess the potential of the Chinese tree shrew for pharmacological testing, we performed drug target prediction and analysis on genomic and transcriptomic scales. Using our pipeline, 3,482 proteins were predicted to be drug targets. Of these predicted targets, 446 and 1,049 proteins with the highest rank and total scores, respectively, included homologs of targets for cancer chemotherapy, depression, age-related decline and cardiovascular disease. Based on comparative analyses, more than half of drug target proteins identified from the tree shrew genome were shown to be higher similarity to human targets than in the mouse. Target validation also demonstrated that the constitutive expression of the proteinase-activated receptors of tree shrew platelets is similar to that of human platelets but differs from that of mouse platelets. We developed an effective pipeline and search strategy for drug target prediction and the evaluation of model-based target identification for drug testing. This work provides useful information for future studies of the Chinese tree shrew as a source of novel targets for drug discovery research. PMID:25105297

BCL-2: Long and winding path from discovery to therapeutic target

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

Schenk, Robyn L.; Strasser, Andreas; Department of Medical Biology, University of Melbourne, Parkville, Melbourne, Victoria 3010

In 1988, the BCL-2 protein was found to promote cancer by limiting cell death rather than enhancing proliferation. This discovery set the wheels in motion for an almost 30 year journey involving many international research teams that has recently culminated in the approval for a drug, ABT-199/venetoclax/Venclexta that targets this protein in the treatment of cancer. This review will describe the long and winding path from the discovery of this protein and understanding the fundamental process of apoptosis that BCL-2 and its numerous homologues control, through to its exploitation as a drug target that is set to have significant benefitmore » for cancer patients. - Highlights: • BCL-2 proteins control the intrinsic or mitochondrial pathway of apoptosis. • Defective apoptosis is a hallmark of cancer. • BH3-mimetics inhibit pro-survival BCL-2 proteins to induce cancer cell death. • ABT-199/venetoclax is approved for treatment of chronic lymphocytic leukaemia.« less

Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis.

PubMed

Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan Mhai; Mondol, Sobuj; Ahmed, C M Sabbir

2014-01-01

Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim-sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein-protein interaction. In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to develop and discover more effective and specific therapeutic agents against S. sanguinis.

Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

PubMed

Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

2017-01-01

A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment.

PubMed

Kintzing, James R; Filsinger Interrante, Maria V; Cochran, Jennifer R

2016-12-01

Protein-based therapeutics have been revolutionizing the oncology space since they first appeared in the clinic two decades ago. Unlike traditional small-molecule chemotherapeutics, protein biologics promote active targeting of cancer cells by binding to cell-surface receptors and other markers specifically associated with or overexpressed on tumors versus healthy tissue. While the first approved cancer biologics were monoclonal antibodies, the burgeoning field of protein engineering is spawning research on an expanded range of protein formats and modifications that allow tuning of properties such as target-binding affinity, serum half-life, stability, and immunogenicity. In this review we highlight some of these strategies and provide examples of modified and engineered proteins under development as preclinical and clinical-stage drug candidates for the treatment of cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

An integrative system biology approach to unravel potential drug candidates for multiple age related disorders.

PubMed

Srivastava, Isha; Khurana, Pooja; Yadav, Mohini; Hasija, Yasha

2017-12-01

Aging, though an inevitable part of life, is becoming a worldwide social and economic problem. Healthy aging is usually marked by low probability of age related disorders. Good therapeutic approaches are still in need to cure age related disorders. Occurrence of more than one ARD in an individual, expresses the need of discovery of such target proteins, which can affect multiple ARDs. Advanced scientific and medical research technologies throughout last three decades have arrived to the point where lots of key molecular determinants affect human disorders can be examined thoroughly. In this study, we designed and executed an approach to prioritize drugs that may target multiple age related disorders. Our methodology, focused on the analysis of biological pathways and protein protein interaction networks that may contribute to the pharmacology of age related disorders, included various steps such as retrieval and analysis of data, protein-protein interaction network analysis, and statistical and comparative analysis of topological coefficients, pathway, and functional enrichment analysis, and identification of drug-target proteins. We assume that the identified molecular determinants may be prioritized for further screening as novel drug targets to cure multiple ARDs. Based on the analysis, an online tool named as 'ARDnet' has been developed to construct and demonstrate ARD interactions at the level of PPI, ARDs and ARDs protein interaction, ARDs pathway interaction and drug-target interaction. The tool is freely made available at http://genomeinformatics.dtu.ac.in/ARDNet/Index.html. Copyright © 2017 Elsevier B.V. All rights reserved.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through February 5, 2016.

NCI Requests Cancer Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 11, 2014.

NCI Requests Targets for Monoclonal Antibody Production and Characterization | Office of Cancer Clinical Proteomics Research

Cancer.gov

In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution. Submissions will be accepted through July 12, 2013.

Human, vector and parasite Hsp90 proteins: A comparative bioinformatics analysis.

PubMed

Faya, Ngonidzashe; Penkler, David L; Tastan Bishop, Özlem

2015-01-01

The treatment of protozoan parasitic diseases is challenging, and thus identification and analysis of new drug targets is important. Parasites survive within host organisms, and some need intermediate hosts to complete their life cycle. Changing host environment puts stress on parasites, and often adaptation is accompanied by the expression of large amounts of heat shock proteins (Hsps). Among Hsps, Hsp90 proteins play an important role in stress environments. Yet, there has been little computational research on Hsp90 proteins to analyze them comparatively as potential parasitic drug targets. Here, an attempt was made to gain detailed insights into the differences between host, vector and parasitic Hsp90 proteins by large-scale bioinformatics analysis. A total of 104 Hsp90 sequences were divided into three groups based on their cellular localizations; namely cytosolic, mitochondrial and endoplasmic reticulum (ER). Further, the parasitic proteins were divided according to the type of parasite (protozoa, helminth and ectoparasite). Primary sequence analysis, phylogenetic tree calculations, motif analysis and physicochemical properties of Hsp90 proteins suggested that despite the overall structural conservation of these proteins, parasitic Hsp90 proteins have unique features which differentiate them from human ones, thus encouraging the idea that protozoan Hsp90 proteins should be further analyzed as potential drug targets.

Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

PubMed Central

Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan MHAI; Mondol, Sobuj; Ahmed, C M Sabbir

2014-01-01

Purpose Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Conclusion Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to develop and discover more effective and specific therapeutic agents against S. sanguinis. PMID:25473301

Utilization of protein intrinsic disorder knowledge in structural proteomics

PubMed Central

Oldfield, Christopher J.; Xue, Bin; Van, Ya-Yue; Ulrich, Eldon L.; Markley, John L.; Dunker, A. Keith; Uversky, Vladimir N.

2014-01-01

Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed. PMID:23232152

Clinical trial uses combination therapy for certain types of non-Hodgkin lymphoma | Center for Cancer Research

Cancer.gov

Researchers are testing the safety of the combination of an experimental drug with rituximab, a standard treatment, for patients with indolent or diffuse large B-cell lymphoma. The antibody is designed to target and block a protein that is present on cancer cells and is used by those cells to protect themselves from your body’s immune system. Blocking the protein may enable

Hedgehog Protein Cholesterolysis: A New Therapeutic Target for Advanced Prostate Cancer

DTIC Science & Technology

2016-10-01

research group page, with links to publicationshttp://www.binghamton.edu/chemistry/people/callahan/callahan.html s 5. PARTICIPANTS & OTHER COLLABORATING...which changes the side chain by only a methylene group . All four expressed as soluble proteins in E. coli and exhibited strong FRET signals consistent...Acknowledgements We thank members of the Callahan research group for technical assistance and helpful comments on the manuscript. Special thanks to Dr

A Proteomics View of the Molecular Mechanisms and Biomarkers of Glaucomatous Neurodegeneration

PubMed Central

Tezel, Gülgün

2013-01-01

Despite improving understanding of glaucoma, key molecular players of neurodegeneration that can be targeted for treatment of glaucoma, or molecular biomarkers that can be useful for clinical testing, remain unclear. Proteomics technology offers a powerful toolbox to accomplish these important goals of the glaucoma research and is increasingly being applied to identify molecular mechanisms and biomarkers of glaucoma. Recent studies of glaucoma using proteomics analysis techniques have resulted in the lists of differentially expressed proteins in human glaucoma and animal models. The global analysis of protein expression in glaucoma has been followed by cell-specific proteome analysis of retinal ganglion cells and astrocytes. The proteomics data have also guided targeted studies to identify post-translational modifications and protein-protein interactions during glaucomatous neurodegeneration. In addition, recent applications of proteomics have provided a number of potential biomarker candidates. Proteomics technology holds great promise to move glaucoma research forward toward new treatment strategies and biomarker discovery. By reviewing the major proteomics approaches and their applications in the field of glaucoma, this article highlights the power of proteomics in translational and clinical research related to glaucoma and also provides a framework for future research to functionally test the importance of specific molecular pathways and validate candidate biomarkers. PMID:23396249

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cullin 5: A Destabilizing Force for Some Oncogenes | Center for Cancer Research

Cancer.gov

Cancer can result when cellular processes such as proliferation and cell death go haywire. Among the many mechanisms in place to regulate these critical processes are molecular chaperones, which help proteins attain their proper functional shape and also regulate protein degradation through the cell’s recycling program, called the ubiquitin/proteasome system. One molecular chaperone, heat shock protein 90 (Hsp90), is of particular interest to cancer researchers because many of its target proteins—sometimes called client proteins—have been implicated in the maintenance and progression of a number of cancers.

Identification and validation nucleolin as a target of curcumol in nasopharyngeal carcinoma cells.

PubMed

Wang, Juan; Wu, Jiacai; Li, Xumei; Liu, Haowei; Qin, Jianli; Bai, Zhun; Chi, Bixia; Chen, Xu

2018-06-30

Identification of the specific protein target(s) of a drug is a critical step in unraveling its mechanisms of action (MOA) in many natural products. Curcumol, isolated from well known Chinese medicinal plant Curcuma zedoary, has been shown to possess multiple biological activities. It can inhibit nasopharyngeal carcinoma (NPC) proliferation and induce apoptosis, but its target protein(s) in NPC cells remains unclear. In this study, we employed a mass spectrometry-based chemical proteomics approach reveal the possible protein targets of curcumol in NPC cells. Cellular thermal shift assay (CETSA), molecular docking and cell-based assay was used to validate the binding interactions. Chemical proteomics capturing uncovered that NCL is a target of curcumol in NPC cells, Molecular docking showed that curcumol bound to NCL with an -7.8 kcal/mol binding free energy. Cell function analysis found that curcumol's treatment leads to a degradation of NCL in NPC cells, and it showed slight effects on NP69 cells. In conclusion, our results providing evidences that NCL is a target protein of curcumol. We revealed that the anti-cancer effects of curcumol in NPC cells are mediated, at least in part, by NCL inhibition. Many natural products showed high bioactivity, while their mechanisms of action (MOA) are very poor or completely missed. Understanding the MOA of natural drugs can thoroughly exploit their therapeutic potential and minimize their adverse side effects. Identification of the specific protein target(s) of a drug is a critical step in unraveling its MOA. Compound-centric chemical proteomics is a classic chemical proteomics approach which integrates chemical synthesis with cell biology and mass spectrometry (MS) to identify protein targets of natural products determine the drug mechanism of action, describe its toxicity, and figure out the possible cause of off-target. It is an affinity-based chemical proteomics method to identify small molecule-protein interactions through affinity chromatography approach coupled with mass spectrometry, has been conventionally used to identify target proteins and has yielded good results. Curcumol, has shown effective inhibition on Nasopharyngeal Carcinoma (NPC) Cells, interacted with NCL and then initiated the anti-tumor biological effect. This research demonstrated the effectiveness of chemical proteomics approaches in natural drugs molecular target identification, revealing and understanding of the novel mechanism of actions of curcumol is crucial for cancer prevention and treatment in nasopharynx cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Unbiased and targeted mass spectrometry for the HDL proteome.

PubMed

Singh, Sasha A; Aikawa, Masanori

2017-02-01

Mass spectrometry is an ever evolving technology that is equipped with a variety of tools for protein research. Some lipoprotein studies, especially those pertaining to HDL biology, have been exploiting the versatility of mass spectrometry to understand HDL function through its proteome. Despite the role of mass spectrometry in advancing research as a whole, however, the technology remains obscure to those without hands on experience, but still wishing to understand it. In this review, we walk the reader through the coevolution of common mass spectrometry workflows and HDL research, starting from the basic unbiased mass spectrometry methods used to profile the HDL proteome to the most recent targeted methods that have enabled an unprecedented view of HDL metabolism. Unbiased global proteomics have demonstrated that the HDL proteome is organized into subgroups across the HDL size fractions providing further evidence that HDL functional heterogeneity is in part governed by its varying protein constituents. Parallel reaction monitoring, a novel targeted mass spectrometry method, was used to monitor the metabolism of HDL apolipoproteins in humans and revealed that apolipoproteins contained within the same HDL size fraction exhibit diverse metabolic properties. Mass spectrometry provides a variety of tools and strategies to facilitate understanding, through its proteins, the complex biology of HDL.

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

PubMed Central

Prosser, Haydn M.; Koike-Yusa, Hiroko; Cooper, James D.; Law, Frances C.; Bradley, Allan

2011-01-01

The 21-23 nucleotide single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in a wide range of cellular and developmental processes. miRNAs regulate protein expression through sequence-specific base pairing with target messenger RNAs (mRNA) reducing both their stability and the process of protein translation1, 2. At least 30% of protein coding genes appear to be conserved targets for miRNAs1. In contrast to the protein coding genes3, 4, no public resource of miRNA mouse mutant alleles exists. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with targeted deletions for the majority of miRNA genes currently annotated within the miRBase registry5. These alleles have been designed to be highly adaptable research tools that can be efficiently altered to create reporter, conditional and other allelic variants. This ES cell resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community6. PMID:21822254

Tissue-Specific Analysis of Pharmacological Pathways.

PubMed

Hao, Yun; Quinnies, Kayla; Realubit, Ronald; Karan, Charles; Tatonetti, Nicholas P

2018-06-19

Understanding the downstream consequences of pharmacologically targeted proteins is essential to drug design. Current approaches investigate molecular effects under tissue-naïve assumptions. Many target proteins, however, have tissue-specific expression. A systematic study connecting drugs to target pathways in in vivo human tissues is needed. We introduced a data-driven method that integrates drug-target relationships with gene expression, protein-protein interaction, and pathway annotation data. We applied our method to four independent genomewide expression datasets and built 467,396 connections between 1,034 drugs and 954 pathways in 259 human tissues or cell lines. We validated our results using data from L1000 and Pharmacogenomics Knowledgebase (PharmGKB), and observed high precision and recall. We predicted and tested anticoagulant effects of 22 compounds experimentally that were previously unknown, and used clinical data to validate these effects retrospectively. Our systematic study provides a better understanding of the cellular response to drugs and can be applied to many research topics in systems pharmacology. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer

PubMed Central

Mehra, Ranee; Serebriiskii, Ilya G.; Dunbrack, Roland L.; Robinson, Matthew K.; Burtness, Barbara; Golemis, Erica A.

2011-01-01

Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-β, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors. PMID:21920801

A Network Approach to Rare Disease Modeling

NASA Astrophysics Data System (ADS)

Ghiassian, Susan; Rabello, Sabrina; Sharma, Amitabh; Wiest, Olaf; Barabasi, Albert-Laszlo

2011-03-01

Network approaches have been widely used to better understand different areas of natural and social sciences. Network Science had a particularly great impact on the study of biological systems. In this project, using biological networks, candidate drugs as a potential treatment of rare diseases were identified. Developing new drugs for more than 2000 rare diseases (as defined by ORPHANET) is too expensive and beyond expectation. Disease proteins do not function in isolation but in cooperation with other interacting proteins. Research on FDA approved drugs have shown that most of the drugs do not target the disease protein but a protein which is 2 or 3 steps away from the disease protein in the Protein-Protein Interaction (PPI) network. We identified the already known drug targets in the disease gene's PPI subnetwork (up to the 3rd neighborhood) and among them those in the same sub cellular compartment and higher coexpression coefficient with the disease gene are expected to be stronger candidates. Out of 2177 rare diseases, 1092 were found not to have any drug target. Using the above method, we have found the strongest candidates among the rest in order to further experimental validations.

[Expression and Preliminary Research on the Soluble Domain of EV-D68 3A Protein].

PubMed

Li, Ting; Kong, Jia; Yu, Xiao-fang; Han, Xue

2015-11-01

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.

Drug target ontology to classify and integrate drug discovery data.

PubMed

Lin, Yu; Mehta, Saurabh; Küçük-McGinty, Hande; Turner, John Paul; Vidovic, Dusica; Forlin, Michele; Koleti, Amar; Nguyen, Dac-Trung; Jensen, Lars Juhl; Guha, Rajarshi; Mathias, Stephen L; Ursu, Oleg; Stathias, Vasileios; Duan, Jianbin; Nabizadeh, Nooshin; Chung, Caty; Mader, Christopher; Visser, Ubbo; Yang, Jeremy J; Bologa, Cristian G; Oprea, Tudor I; Schürer, Stephan C

2017-11-09

One of the most successful approaches to develop new small molecule therapeutics has been to start from a validated druggable protein target. However, only a small subset of potentially druggable targets has attracted significant research and development resources. The Illuminating the Druggable Genome (IDG) project develops resources to catalyze the development of likely targetable, yet currently understudied prospective drug targets. A central component of the IDG program is a comprehensive knowledge resource of the druggable genome. As part of that effort, we have developed a framework to integrate, navigate, and analyze drug discovery data based on formalized and standardized classifications and annotations of druggable protein targets, the Drug Target Ontology (DTO). DTO was constructed by extensive curation and consolidation of various resources. DTO classifies the four major drug target protein families, GPCRs, kinases, ion channels and nuclear receptors, based on phylogenecity, function, target development level, disease association, tissue expression, chemical ligand and substrate characteristics, and target-family specific characteristics. The formal ontology was built using a new software tool to auto-generate most axioms from a database while supporting manual knowledge acquisition. A modular, hierarchical implementation facilitate ontology development and maintenance and makes use of various external ontologies, thus integrating the DTO into the ecosystem of biomedical ontologies. As a formal OWL-DL ontology, DTO contains asserted and inferred axioms. Modeling data from the Library of Integrated Network-based Cellular Signatures (LINCS) program illustrates the potential of DTO for contextual data integration and nuanced definition of important drug target characteristics. DTO has been implemented in the IDG user interface Portal, Pharos and the TIN-X explorer of protein target disease relationships. DTO was built based on the need for a formal semantic model for druggable targets including various related information such as protein, gene, protein domain, protein structure, binding site, small molecule drug, mechanism of action, protein tissue localization, disease association, and many other types of information. DTO will further facilitate the otherwise challenging integration and formal linking to biological assays, phenotypes, disease models, drug poly-pharmacology, binding kinetics and many other processes, functions and qualities that are at the core of drug discovery. The first version of DTO is publically available via the website http://drugtargetontology.org/ , Github ( http://github.com/DrugTargetOntology/DTO ), and the NCBO Bioportal ( http://bioportal.bioontology.org/ontologies/DTO ). The long-term goal of DTO is to provide such an integrative framework and to populate the ontology with this information as a community resource.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

PubMed

Cai, Dongbo; Wang, Hao; He, Penghui; Zhu, Chengjun; Wang, Qin; Wei, Xuetuan; Nomura, Christopher T; Chen, Shouwen

2017-04-24

Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Development of aptamers against unpurified proteins.

PubMed

Goto, Shinichi; Tsukakoshi, Kaori; Ikebukuro, Kazunori

2017-12-01

SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples. © 2017 Wiley Periodicals, Inc.

In Silico Identification of Proteins Associated with Drug-induced Liver Injury Based on the Prediction of Drug-target Interactions.

PubMed

Ivanov, Sergey; Semin, Maxim; Lagunin, Alexey; Filimonov, Dmitry; Poroikov, Vladimir

2017-07-01

Drug-induced liver injury (DILI) is the leading cause of acute liver failure as well as one of the major reasons for drug withdrawal from clinical trials and the market. Elucidation of molecular interactions associated with DILI may help to detect potentially hazardous pharmacological agents at the early stages of drug development. The purpose of our study is to investigate which interactions with specific human protein targets may cause DILI. Prediction of interactions with 1534 human proteins was performed for the dataset with information about 699 drugs, which were divided into three categories of DILI: severe (178 drugs), moderate (310 drugs) and without DILI (211 drugs). Based on the comparison of drug-target interactions predicted for different drugs' categories and interpretation of those results using clustering, Gene Ontology, pathway and gene expression analysis, we identified 61 protein targets associated with DILI. Most of the revealed proteins were linked with hepatocytes' death caused by disruption of vital cellular processes, as well as the emergence of inflammation in the liver. It was found that interaction of a drug with the identified targets is the essential molecular mechanism of the severe DILI for the most of the considered pharmaceuticals. Thus, pharmaceutical agents interacting with many of the identified targets may be considered as candidates for filtering out at the early stages of drug research. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Focus on flaviviruses: current and future drug targets.

PubMed

Geiss, Brian J; Stahla, Hillary; Hannah, Amanda M; Gari, Amanda M; Keenan, Susan M

2009-05-01

Infection by mosquito-borne flaviviruses (family Flaviviridae) is increasing in prevalence worldwide. The vast global, social and economic impact due to the morbidity and mortality associated with the diseases caused by these viruses necessitates therapeutic intervention. There is currently no effective clinical treatment for any flaviviral infection. Therefore, there is a great need for the identification of novel inhibitors to target the virus life cycle. In this article, we discuss structural and nonstructural viral proteins that are the focus of current target validation and drug discovery efforts. Both inhibition of essential enzymatic activities and disruption of necessary protein–protein interactions are considered. In addition, we address promising new targets for future research. As our molecular and biochemical understanding of the flavivirus life cycle increases, the number of targets for antiviral therapeutic discovery grows and the possibility for novel drug discovery continues to strengthen.

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in

Protein Neddylation: Beyond Cullin-RING Ligases

PubMed Central

Enchev, Radoslav I.; Schulman, Brenda A.; Peter, Matthias

2016-01-01

NEDD8 is a ubiquitin-like protein that activates the largest ubiquitin E3 ligase family, the cullin RING ligases. Many non-cullin neddylation targets have been proposed in recent years. However, overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery, which makes validating potential NEDD8 targets challenging. Here we re-evaluate these studies in light of the current understanding of the neddylation pathway, and suggest criteria for the identification of genuine neddylation substrates under homeostatic conditions. We describe the biological processes that might be regulated by non-cullin neddylation, and the utility of neddylation inhibitors for research and as potential therapies. Understanding the biological significance of non-cullin neddylation is an exciting research prospect primed to reveal fundamental insights. PMID:25531226

Controlling protein release using biodegradable microparticles

NASA Astrophysics Data System (ADS)

Kline, Benjamin Patrick

Research in the field of protein therapeutics has exploded over the past decade and continues to grow in both academia and in industry. Protein drugs have advantages of being highly specific and highly active making them coveted targets for high profile disease states like cancer and multiple sclerosis. Unfortunately, their many advantages are complemented by their obstacles. Because proteins are highly active and highly specific, the window between efficacy and toxicity is very narrow and drug development can be long and arduous. In addition, protein activity is dependent on its specific folding conformation that is easily disrupted by a variety of development processes. This research aimed to identify microparticle formulations to control protein release and also to determine which formulation parameters affected burst release, encapsulation, and steady-state release the most. It was found that polymer type and composition were two of the most important factors. Long-term controlled release of bovine serum albumin (BSA) was achieved as well as a wide variety of release profiles. A method was identified for micronizing protein at low cost to retain activity and coacervation was evaluated as a method for preparing protein loaded microspheres. This research provides a basis from which researchers can create better controlled release formulations for future protein therapeutics.

Target identification for small bioactive molecules: finding the needle in the haystack.

PubMed

Ziegler, Slava; Pries, Verena; Hedberg, Christian; Waldmann, Herbert

2013-03-04

Identification and confirmation of bioactive small-molecule targets is a crucial, often decisive step both in academic and pharmaceutical research. Through the development and availability of several new experimental techniques, target identification is, in principle, feasible, and the number of successful examples steadily grows. However, a generic methodology that can successfully be applied in the majority of the cases has not yet been established. Herein we summarize current methods for target identification of small molecules, primarily for a chemistry audience but also the biological community, for example, the chemist or biologist attempting to identify the target of a given bioactive compound. We describe the most frequently employed experimental approaches for target identification and provide several representative examples illustrating the state-of-the-art. Among the techniques currently available, protein affinity isolation using suitable small-molecule probes (pulldown) and subsequent mass spectrometric analysis of the isolated proteins appears to be most powerful and most frequently applied. To provide guidance for rapid entry into the field and based on our own experience we propose a typical workflow for target identification, which centers on the application of chemical proteomics as the key step to generate hypotheses for potential target proteins. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. Electronic supplementary information (ESI) available: Additional circular dichroism data and nanoparticle tracking analysis trace. See DOI: 10.1039/c6nr02009g

Molecular pathways: targeting RAC-p21-activated serine-threonine kinase signaling in RAS-driven cancers.

PubMed

Baker, Nicole M; Yee Chow, Hoi; Chernoff, Jonathan; Der, Channing J

2014-09-15

Cancers driven by oncogenic Ras proteins encompass some of the most deadly human cancer types, and there is a pressing need to develop therapies for these diseases. Although recent studies suggest that mutant Ras proteins may yet be druggable, the most promising and advanced efforts involve inhibitors of Ras effector signaling. Most efforts to target Ras signaling have been aimed at the ERK mitogen-activated protein kinase and the phosphoinositide 3-kinase signaling networks. However, to date, no inhibitors of these Ras effector pathways have been effective against RAS-mutant cancers. This ineffectiveness is due, in part, to the involvement of additional effectors in Ras-dependent cancer growth, such as the Rac small GTPase and the p21-activated serine-threonine kinases (PAK). PAK proteins are involved in many survival, cell motility, and proliferative pathways in the cell and may present a viable new target in Ras-driven cancers. In this review, we address the role and therapeutic potential of Rac and group I PAK proteins in driving mutant Ras cancers. ©2014 American Association for Cancer Research.

Emulsomes Meet S-layer Proteins: An Emerging Targeted Drug Delivery System

PubMed Central

Ucisik, Mehmet H.; Sleytr, Uwe B.; Schuster, Bernhard

2015-01-01

Here, the use of emulsomes as a drug delivery system is reviewed and compared with other similar lipidic nanoformulations. In particular, we look at surface modification of emulsomes using S-layer proteins, which are self-assembling proteins that cover the surface of many prokaryotic organisms. It has been shown that covering emulsomes with a crystalline S-layer lattice can protect cells from oxidative stress and membrane damage. In the future, the capability to recrystallize S-layer fusion proteins on lipidic nanoformulations may allow the presentation of binding functions or homing protein domains to achieve highly specific targeted delivery of drug-loaded emulsomes. Besides the discussion on several designs and advantages of composite emulsomes, the success of emulsomes for the delivery of drugs to fight against viral and fungal infections, dermal therapy, cancer, and autoimmunity is summarized. Further research might lead to smart, biocompatible emulsomes, which are able to protect and reduce the side effects caused by the drug, but at the same time are equipped with specific targeting molecules to find the desired site of action. PMID:25697368

A New Way to Treat Brain Tumors: Targeting Proteins Coded by Microcephaly Genes?: Brain tumors and microcephaly arise from opposing derangements regulating progenitor growth. Drivers of microcephaly could be attractive brain tumor targets.

PubMed

Lang, Patrick Y; Gershon, Timothy R

2018-05-01

New targets for brain tumor therapies may be identified by mutations that cause hereditary microcephaly. Brain growth depends on the repeated proliferation of stem and progenitor cells. Microcephaly syndromes result from mutations that specifically impair the ability of brain progenitor or stem cells to proliferate, by inducing either premature differentiation or apoptosis. Brain tumors that derive from brain progenitor or stem cells may share many of the specific requirements of their cells of origin. These tumors may therefore be susceptible to disruptions of the protein products of genes that are mutated in microcephaly. The potential for the products of microcephaly genes to be therapeutic targets in brain tumors are highlighted hereby reviewing research on EG5, KIF14, ASPM, CDK6, and ATR. Treatments that disrupt these proteins may open new avenues for brain tumor therapy that have increased efficacy and decreased toxicity. © 2018 WILEY Periodicals, Inc.

In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein-protein interaction networks.

PubMed

Folador, Edson Luiz; de Carvalho, Paulo Vinícius Sanches Daltro; Silva, Wanderson Marques; Ferreira, Rafaela Salgado; Silva, Artur; Gromiha, Michael; Ghosh, Preetam; Barh, Debmalya; Azevedo, Vasco; Röttger, Richard

2016-11-04

Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing according wet-lab studies. The non-host homologous essential proteins are attractive targets for therapeutic and diagnostic proposes. They allow for searching of small molecule inhibitors of binding interactions enabling modern drug discovery. Overall, the predicted Cp PPI networks form a valuable and versatile tool for researchers interested in Corynebacterium pseudotuberculosis.

The Structural Biology Knowledgebase: a portal to protein structures, sequences, functions, and methods.

PubMed

Gabanyi, Margaret J; Adams, Paul D; Arnold, Konstantin; Bordoli, Lorenza; Carter, Lester G; Flippen-Andersen, Judith; Gifford, Lida; Haas, Juergen; Kouranov, Andrei; McLaughlin, William A; Micallef, David I; Minor, Wladek; Shah, Raship; Schwede, Torsten; Tao, Yi-Ping; Westbrook, John D; Zimmerman, Matthew; Berman, Helen M

2011-07-01

The Protein Structure Initiative's Structural Biology Knowledgebase (SBKB, URL: http://sbkb.org ) is an open web resource designed to turn the products of the structural genomics and structural biology efforts into knowledge that can be used by the biological community to understand living systems and disease. Here we will present examples on how to use the SBKB to enable biological research. For example, a protein sequence or Protein Data Bank (PDB) structure ID search will provide a list of related protein structures in the PDB, associated biological descriptions (annotations), homology models, structural genomics protein target status, experimental protocols, and the ability to order available DNA clones from the PSI:Biology-Materials Repository. A text search will find publication and technology reports resulting from the PSI's high-throughput research efforts. Web tools that aid in research, including a system that accepts protein structure requests from the community, will also be described. Created in collaboration with the Nature Publishing Group, the Structural Biology Knowledgebase monthly update also provides a research library, editorials about new research advances, news, and an events calendar to present a broader view of structural genomics and structural biology.

Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

PubMed

Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

2013-12-01

The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers*

PubMed Central

Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-01-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. PMID:28235782

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

PubMed

Song, Jun; Du, Lina; Li, Li; Kalt, Wilhelmina; Palmer, Leslie Campbell; Fillmore, Sherry; Zhang, Ying; Zhang, ZhaoQi; Li, XiHong

2015-06-03

To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides and proteins provided reliable design and validation of quantitative approaches using SRM on targeted proteins proposed involved in strawberry fruit. Our data revealed the identifying candidate proteins and their quantitative changes in relation to fruit ripening and flavonoids and anthocyanin biosynthesis and regulation. More importantly, this quantitative proteomic data is also compared with chemical analysis to reveal possible control levels of this important quality trait. Although, MRM approach is not new in plant biology research, the application has been very rare. This is the first systematic multi-targeted interrogation of the possible regulation of entire pathway of flavonoids and anthocyanin biosynthesis in strawberry fruit at different ripening stages using quantitative MRM technique on mass spectrometry. Our results demonstrate the power of targeted quantitative mass spectrometry data for analysis of proteins in biological regulation. These results indicate that distinct and diverse control of flavonoids and anthocyanin biosynthesis mechanisms at metabolism and proteins levels. This important and complementary knowledge will be useful for systematically characterizing the flavonoids and anthocyanin biosynthesis pathway of any fruit/plant species. Copyright © 2015. Published by Elsevier B.V.

The Impact of CRISPR/Cas9-Based Genomic Engineering on Biomedical Research and Medicine.

PubMed

Go, D E; Stottmann, R W

2016-01-01

There has been prolonged and significant interest in manipulating the genome for a wide range of applications in biomedical research and medicine. An existing challenge in realizing this potential has been the inability to precisely edit specific DNA sequences. Past efforts to generate targeted double stranded DNA cleavage have fused DNA-targeting elements such as zinc fingers and DNA-binding proteins to endonucleases. However, these approaches are limited by both design complexity and inefficient, costineffective operation. The discovery of CRISPR/Cas9, a branch of the bacterial adaptive immune system, as a potential genomic editing tool holds the promise of facile targeted cleavage. Its novelty lies in its RNA-guided endonuclease activity, which enhances its efficiency, scalability, and ease of use. The only necessary components are a Cas9 endonuclease protein and an RNA molecule tailored to the gene of interest. This lowbarrier of adoption has facilitated a plethora of advances in just the past three years since its discovery. In this review, we will discuss the impact of CRISPR/Cas9 on biomedical research and its potential implications in medicine.

Changes in miRNAs Signal High-Risk HPV Infections | Center for Cancer Research

Cancer.gov

microRNAs (miRNAs) are approximately 21 nucleotide long, non-coding RNAs that regulate the expression of certain proteins. As part of the RNA-induced silencing complex or RISC, miRNAs bind to complementary sequences in the 3’ untranslated regions of target messenger RNAs, blocking protein synthesis and sometimes leading to the destruction of the target RNA. Numerous studies have shown that the levels of cellular miRNAs can be altered in diseased tissues, and these changes potentially could be used for diagnosis or disease monitoring.

Internalization and Recycling of the HER2 Receptor on Human Breast Adenocarcinoma Cells Treated with Targeted Phototoxic Protein DARPinminiSOG

PubMed Central

Shilova, O. N.; Proshkina, G. M.; Lebedenko, E. N.; Deyev, S. M.

2015-01-01

Design and evaluation of new high-affinity protein compounds that can selectively and efficiently destroy human cancer cells are a priority research area in biomedicine. In this study we report on the ability of the recombinant phototoxic protein DARPin-miniSOG to interact with breast adenacarcinoma human cells overexpressing the extracellular domain of human epidermal growth factor receptor 2 (HER2). It was found that the targeted phototoxin DARPin-miniSOG specifically binds to the HER2 with following internalization and slow recycling back to the cell membrane. An insight into the role of DARPin-miniSOG in HER2 internalization could contribute to the treatment of HER2-positive cancer using this phototoxic protein. PMID:26483969

Assays without Borders | Office of Cancer Clinical Proteomics Research

Cancer.gov

CPTAC researchers, partner with international labs to demonstrate the ability of targeted mass spectrometry–based assays to reproducibly quantify human proteins across labs, countries and continents in a recently published journal article. In a landmark paper appearing in the Dec.

Fusion proteins in head and neck neoplasms: Clinical implications, genetics, and future directions for targeting

PubMed Central

Escalante, Derek A.; Wang, He; Fundakowski, Christopher E.

2016-01-01

ABSTRACT Fusion proteins resulting from chromosomal rearrangements are known to drive the pathogenesis of a variety of hematological and solid neoplasms such as chronic myeloid leukemia and non-small-cell lung cancer. Efforts to elucidate the role they play in these malignancies have led to important diagnostic and therapeutic triumphs, including the famous development of the tyrosine kinase inhibitor dasatinib targeting the BCR-ABL fusion. Until recently, there has been a paucity of research investigating fusion proteins harbored by head and neck neoplasms. The discovery and characterization of novel fusion proteins in neoplasms originating from the thyroid, nasopharynx, salivary glands, and midline head and neck structures offer substantial contributions to our understanding of the pathogenesis and biological behavior of these neoplasms, while raising new therapeutic and diagnostic opportunities. Further characterization of these fusion proteins promises to facilitate advances on par with those already achieved with regard to hematologic malignancies in the precise, molecularly guided diagnosis and treatment of head and neck neoplasms. The following is a subsite specific review of the clinical implications of fusion proteins in head and neck neoplasms and the future potential for diagnostic targeting. PMID:27636353

Polymerase Acidic Protein-Basic Protein 1 (PA-PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics.

PubMed

Massari, Serena; Goracci, Laura; Desantis, Jenny; Tabarrini, Oriana

2016-09-08

The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent RNA polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunit interface has become an active field of research following the publication of PA-PB1 crystal structures. In this Perspective, we briefly discuss the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy, including a comprehensive analysis of available PA-PB1 structures. An overview of all of the reported PA-PB1 complex formation inhibitors is provided, and approaches used for identification of the inhibitors, the hit-to-lead studies, and the emerged structure-activity relationship are described. In addition to highlighting the strengths and weaknesses of all of the PA-PB1 heterodimerization inhibitors, we analyze their hypothesized binding modes and alignment with a pharmacophore model that we have developed.

Legume Lectins: Proteins with Diverse Applications

PubMed Central

Lagarda-Diaz, Irlanda; Guzman-Partida, Ana Maria; Vazquez-Moreno, Luz

2017-01-01

Lectins are a diverse class of proteins distributed extensively in nature. Among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. Because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. This review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. PMID:28604616

[Prokaryotic expression, purification and biological activity analysis of recombinant β-Lactamase protein].

PubMed

Zhou, Xiao-liang; Shi, Pei-ji; Wang, Hao

2011-01-01

To prepare RGD4CβL fusion protein using prokaryotic expression system and evaluate the biological activity of the RGD4CβL. RGD4CβL gene was cloned into pColdII to contruct β-Lactamase prokaryotic expression vector. After transformation, the recombinant vector was induced to express recombinant protein RGD4CβL by IPTG in E.coli BL(DE3). The recombinant protein was purified by Ni-NTA resin under denaturing condition and then dialyzed to renature. The tumor cell targeting ability of the recombinant protein was analyzed by flow cytometric analysis. After cleavage and purification, β-Lactamase moiety showed the expected size of 42 000 on Tricine-SDS-PAGE, and was further confirmed by Western blotting. Based on flow cytometric analysis, the purified protein specially targeted breast cancer cell line MCF-7. This research successfully estiblished a method for prokaryotic expression and purification of β-lactamase. These results suggest the potential use of the protein as an agent for ADEPT.

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

CAPSAICIN: ITS BIOLOGICAL ACTIVITIES AND IN SILICO TARGET FISHING.

PubMed

Akhtar, Fahad; Muhammad Sharif, Hajra; Arshad Mallick, Muhammad; Zahoor, Fareeha; Abdulmalik, Attiya; Baig, Warda; Shujaat, Nodia; Gul, Sundas; Bibi, Gulfam; Ramzan, Rahdia; Murtaza, Ghulam

2017-03-01

Capsicum annuum L. is a rich source of capsaicin, an alkaloid, which is a very pungent compound. Due to ever growing need of capsaicin, an extensive research on its efficient cultivation as well as chemical synthesis is underway. Owing to the pungent nature of capsaicin, its analogous molecules without pungent effect are being explored. The objective of this descriptive review is to comprehensively present the updates on the bioactivities of capsaicin. Additionally, the in silico target fishing approach has been used to identify the possible protein targets of capsaicin. This article will definitely provide future perspectives of research on capsaicin.

RNA therapeutics: RNAi and antisense mechanisms and clinical applications.

PubMed

Chery, Jessica

2016-07-01

RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology 1 . Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials 2 . Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs.

Targeting Epigenetics to Prevent Obesity Promoted Cancers.

PubMed

Berger, Nathan A; Scacheri, Peter C

2018-03-01

Epigenetic changes in DNA and associated chromatin proteins are increasingly being considered as important mediators of the linkage between obesity and cancer. Although multiple agents, targeted at epigenetic changes, are being tested for therapy of established cancers, this issue of Cancer Prevention Research carries two articles demonstrating that the bromodomain inhibitor I-BET-762 can attenuate adipose tissue-promoted cancers. Although I-BET-762 significantly delayed, rather than completely prevented, the onset of adiposity-promoted transformation and malignancy, these experiments provide important proof of principle for the strategies of targeting epigenetic changes to disrupt the obesity-cancer linkage. Because bromodomain proteins represent only one of multiple epigenetic mediators, it is probable that targeting other epigenetic processes, alone or in combination, may serve to even more effectively disrupt the obesity promotion of cancer. Given the magnitude of the current obesity pandemic and its impact on cancer, preventive measures to disrupt this linkage are critically important. Cancer Prev Res; 11(3); 125-8. ©2018 AACR See related article by Chakraborty et al., p. 129 . ©2018 American Association for Cancer Research.

Target identification in Fusobacterium nucleatum by subtractive genomics approach and enrichment analysis of host-pathogen protein-protein interactions.

PubMed

Kumar, Amit; Thotakura, Pragna Lakshmi; Tiwary, Basant Kumar; Krishna, Ramadas

2016-05-12

Fusobacterium nucleatum, a well studied bacterium in periodontal diseases, appendicitis, gingivitis, osteomyelitis and pregnancy complications has recently gained attention due to its association with colorectal cancer (CRC) progression. Treatment with berberine was shown to reverse F. nucleatum-induced CRC progression in mice by balancing the growth of opportunistic pathogens in tumor microenvironment. Intestinal microbiota imbalance and the infections caused by F. nucleatum might be regulated by therapeutic intervention. Hence, we aimed to predict drug target proteins in F. nucleatum, through subtractive genomics approach and host-pathogen protein-protein interactions (HP-PPIs). We also carried out enrichment analysis of host interacting partners to hypothesize the possible mechanisms involved in CRC progression due to F. nucleatum. In subtractive genomics approach, the essential, virulence and resistance related proteins were retrieved from RefSeq proteome of F. nucleatum by searching against Database of Essential Genes (DEG), Virulence Factor Database (VFDB) and Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) tool respectively. A subsequent hierarchical screening to identify non-human homologous, metabolic pathway-independent/pathway-specific and druggable proteins resulted in eight pathway-independent and 27 pathway-specific druggable targets. Co-aggregation of F. nucleatum with host induces proinflammatory gene expression thereby potentiates tumorigenesis. Hence, proteins from IBDsite, a database for inflammatory bowel disease (IBD) research and those involved in colorectal adenocarcinoma as interpreted from The Cancer Genome Atlas (TCGA) were retrieved to predict drug targets based on HP-PPIs with F. nucleatum proteome. Prediction of HP-PPIs exhibited 186 interactions contributed by 103 host and 76 bacterial proteins. Bacterial interacting partners were accounted as putative targets. And enrichment analysis of host interacting partners showed statistically enriched terms that were in positive correlation with CRC, atherosclerosis, cardiovascular, osteoporosis, Alzheimer's and other diseases. Subtractive genomics analysis provided a set of target proteins suggested to be indispensable for survival and pathogenicity of F. nucleatum. These target proteins might be considered for designing potent inhibitors to abrogate F. nucleatum infections. From enrichment analysis, it was hypothesized that F. nucleatum infection might enhance CRC progression by simultaneously regulating multiple signaling cascades which could lead to up-regulation of proinflammatory responses, oncogenes, modulation of host immune defense mechanism and suppression of DNA repair system.

Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

PubMed

Luo, Wentian; Galvan, Daniel L; Woodard, Lauren E; Dorset, Dan; Levy, Shawn; Wilson, Matthew H

2017-08-21

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Potential role of DNA methylation as a facilitator of target search processes for transcription factors through interplay with methyl-CpG-binding proteins.

PubMed

Kemme, Catherine A; Marquez, Rolando; Luu, Ross H; Iwahara, Junji

2017-07-27

Eukaryotic genomes contain numerous non-functional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences similar to the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. In this study, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescence-labeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (∼10-30-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in non-functional locations. This effect may occur in vivo for DNA methylation outside CpG islands (CGIs) and could facilitate localization of some transcription factors within regulatory CGIs, where DNA methylation is rare. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Progress and future opportunities in the development of vaccines against atherosclerosis.

PubMed

Govea-Alonso, Dania O; Beltrán-López, Josué; Salazar-González, Jorge A; Vargas-Morales, Juan; Rosales-Mendoza, Sergio

2017-04-01

Atherosclerosis represents a serious global health problem that demands new therapeutic and prophylactic interventions. Considering that atherosclerosis has autoimmune and inflammatory components, immunotherapy is a possible focus to treat this disease. Areas covered: Based on the analysis of the current biomedical literature, this review describes the status on the development of vaccines against atherosclerosis. Several targets have been identified including sequences of apolipoprotein B100 (ApoB100), cholesteryl ester transfer protein (CETP), heat shock proteins (HSP), extracellular matrix proteins, T cell receptor β chain variable region 31 (TRBV31), the major outer membrane protein (MOMP), and the outer membrane protein 5 (Pomp5) from Chlamydia pneumoniae. Humoral and cellular immunities to these targets have been associated with therapeutic effects in murine models and humans. The evaluation of some candidates in clinical trials is ongoing. Expert commentary: New research paths based on the use of next generation vaccine production platforms are envisioned.

Insecticidal activity of plant lectins and potential application in crop protection.

PubMed

Macedo, Maria Lígia R; Oliveira, Caio F R; Oliveira, Carolina T

2015-01-27

Lectins constitute a complex group of proteins found in different organisms. These proteins constitute an important field for research, as their structural diversity and affinity for several carbohydrates makes them suitable for numerous biological applications. This review addresses the classification and insecticidal activities of plant lectins, providing an overview of the applicability of these proteins in crop protection. The likely target sites in insect tissues, the mode of action of these proteins, as well as the use of lectins as biotechnological tools for pest control are also described. The use of initial bioassays employing artificial diets has led to the most recent advances in this field, such as plant breeding and the construction of fusion proteins, using lectins for targeting the delivery of toxins and to potentiate expected insecticide effects. Based on the data presented, we emphasize the contribution that plant lectins may make as tools for the development of integrated insect pest control strategies.

Molecular Targets for Antiepileptic Drug Development

PubMed Central

Meldrum, Brian S.; Rogawski, Michael A.

2007-01-01

Summary This review considers how recent advances in the physiology of ion channels and other potential molecular targets, in conjunction with new information on the genetics of idiopathic epilepsies, can be applied to the search for improved antiepileptic drugs (AEDs). Marketed AEDs predominantly target voltage-gated cation channels (the α subunits of voltage-gated Na+ channels and also T-type voltage-gated Ca2+ channels) or influence GABA-mediated inhibition. Recently, α2–δ voltage-gated Ca2+ channel subunits and the SV2A synaptic vesicle protein have been recognized as likely targets. Genetic studies of familial idiopathic epilepsies have identified numerous genes associated with diverse epilepsy syndromes, including genes encoding Na+ channels and GABAA receptors, which are known AED targets. A strategy based on genes associated with epilepsy in animal models and humans suggests other potential AED targets, including various voltage-gated Ca2+ channel subunits and auxiliary proteins, A- or M-type voltage-gated K+ channels, and ionotropic glutamate receptors. Recent progress in ion channel research brought about by molecular cloning of the channel subunit proteins and studies in epilepsy models suggest additional targets, including G-protein-coupled receptors, such as GABAB and metabotropic glutamate receptors; hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits, responsible for hyperpolarization-activated current Ih; connexins, which make up gap junctions; and neurotransmitter transporters, particularly plasma membrane and vesicular transporters for GABA and glutamate. New information from the structural characterization of ion channels, along with better understanding of ion channel function, may allow for more selective targeting. For example, Na+ channels underlying persistent Na+ currents or GABAA receptor isoforms responsible for tonic (extrasynaptic) currents represent attractive targets. The growing understanding of the pathophysiology of epilepsy and the structural and functional characterization of the molecular targets provide many opportunities to create improved epilepsy therapies. PMID:17199015

A Framework of Knowledge Integration and Discovery for Supporting Pharmacogenomics Target Predication of Adverse Drug Events: A Case Study of Drug-Induced Long QT Syndrome.

PubMed

Jiang, Guoqian; Wang, Chen; Zhu, Qian; Chute, Christopher G

2013-01-01

Knowledge-driven text mining is becoming an important research area for identifying pharmacogenomics target genes. However, few of such studies have been focused on the pharmacogenomics targets of adverse drug events (ADEs). The objective of the present study is to build a framework of knowledge integration and discovery that aims to support pharmacogenomics target predication of ADEs. We integrate a semantically annotated literature corpus Semantic MEDLINE with a semantically coded ADE knowledgebase known as ADEpedia using a semantic web based framework. We developed a knowledge discovery approach combining a network analysis of a protein-protein interaction (PPI) network and a gene functional classification approach. We performed a case study of drug-induced long QT syndrome for demonstrating the usefulness of the framework in predicting potential pharmacogenomics targets of ADEs.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

PubMed

Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

2016-06-07

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Identification of Tengfu Jiangya Tablet Target Biomarkers with Quantitative Proteomic Technique

PubMed Central

Xu, Jingwen; Zhang, Shijun; Jiang, Haiqiang; Wang, Nan; Lin, Haiqing

2017-01-01

Tengfu Jiangya Tablet (TJT) is a well accepted antihypertension drug in China and its major active components were Uncaria total alkaloids and Semen Raphani soluble alkaloid. To further explore treatment effects mechanism of TJT on essential hypertension, a serum proteomic study was performed. Potential biomarkers were quantified in serum of hypertension individuals before and after taking TJT with isobaric tags for relative and absolute quantitation (iTRAQ) coupled two-dimensional liquid chromatography followed electrospray ionization-tandem mass spectrometry (2D LC-MS/MS) proteomics technique. Among 391 identified proteins with high confidence, 70 proteins were differentially expressed (fold variation criteria, >1.2 or <0.83) between two groups (39 upregulated and 31 downregulated). Combining with Gene Ontology annotation, KEGG pathway analysis, and literature retrieval, 5 proteins were chosen as key target biomarkers during TJT therapeutic process. And the alteration profiles of these 5 proteins were verified by ELISA and Western Blot. Proteins Kininogen 1 and Keratin 1 are members of Kallikrein system, while Myeloperoxidase, Serum Amyloid protein A, and Retinol binding protein 4 had been reported closely related to vascular endothelial injury. Our study discovered 5 target biomarkers of the compound Chinese medicine TJT. Secondly, this research initially revealed the antihypertension therapeutic mechanism of this drug from a brand-new aspect. PMID:28408942

Novel Methods for Mosquito Control using RNAi.

USDA-ARS?s Scientific Manuscript database

The discovery and development of novel insecticides for vector control is a primary focus of toxicology research conducted at the Mosquito and Fly Research Unit, Gainesville, FL. Targeting critical genes/proteins in mosquitoes using RNA interference (RNAi) is being investigated as a method to devel...

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets.

PubMed

Quick, Quincy A

2018-01-26

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Review: biosafety assessment of Bt rice and other Bt crops using spiders as example for non-target arthropods in China.

PubMed

Yang, Huilin; Peng, Yuande; Tian, Jianxiang; Wang, Juan; Hu, Jilin; Song, Qisheng; Wang, Zhi

2017-04-01

Since the birth of transgenic crops expressing Bacillus thuringiensis (Bt) toxin for pest control, the public debate regarding ecological and environmental risks as well as benefits of Bt crops has continued unabated. The impact of Bt crops, especially on non-target invertebrates, has received particular attention. In this review, we summarize and analyze evidences for non-target effects of Bt rice on spiders, major predators in rice fields. Bt rice has been genetically modified to express the Bt protein, which has been shown to be transferred and accumulate in spiders as part of their food chain. Moreover, the Bt protein exhibits unintended effects on the physiology of spiders and spreads to higher trophic levels. Spiders possess unique physiological and ecological characteristics, revealing traits of surrogate species, and are thus considered to be excellent non-target arthropod model systems for study of Bt protein impacts. Due to the complexities of Bt protein transfer and accumulation mechanisms, as well as the apparent lack of information about resulting physiological, biochemical, and ecological effects on spiders, we raise questions and provide recommendations for promising further research.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

PubMed Central

Quick, Quincy A.

2018-01-01

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents. PMID:29373494

Targeting the GPI biosynthetic pathway.

PubMed

Yadav, Usha; Khan, Mohd Ashraf

2018-02-27

The GPI (Glycosylphosphatidylinositol) biosynthetic pathway is a multistep conserved pathway in eukaryotes that culminates in the generation of GPI glycolipid which in turn anchors many proteins (GPI-APs) to the cell surface. In spite of the overall conservation of the pathway, there still exist subtle differences in the GPI pathway of mammals and other eukaryotes which holds a great promise so far as the development of drugs/inhibitors against specific targets in the GPI pathway of pathogens is concerned. Many of the GPI structures and their anchored proteins in pathogenic protozoans and fungi act as pathogenicity factors. Notable examples include GPI-anchored variant surface glycoprotein (VSG) in Trypanosoma brucei, GPI-anchored merozoite surface protein 1 (MSP1) and MSP2 in Plasmodium falciparum, protein-free GPI related molecules like lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) in Leishmania spp., GPI-anchored Gal/GalNAc lectin and proteophosphoglycans in Entamoeba histolytica or the GPI-anchored mannoproteins in pathogenic fungi like Candida albicans. Research in this active area has already yielded encouraging results in Trypanosoma brucei by the development of parasite-specific inhibitors of GlcNCONH 2 -β-PI, GlcNCONH 2 -(2-O-octyl)-PI and salicylic hydroxamic acid (SHAM) targeting trypanosomal GlcNAc-PI de-N-acetylase as well as the development of antifungal inhibitors like BIQ/E1210/gepinacin/G365/G884 and YW3548/M743/M720 targeting the GPI specific fungal inositol acyltransferase (Gwt1) and the phosphoethanolamine transferase-I (Mcd4), respectively. These confirm the fact that the GPI pathway continues to be the focus of researchers, given its implications for the betterment of human life.

The emergence of top-down proteomics in clinical research

PubMed Central

2013-01-01

Proteomic technology has advanced steadily since the development of 'soft-ionization' techniques for mass-spectrometry-based molecular identification more than two decades ago. Now, the large-scale analysis of proteins (proteomics) is a mainstay of biological research and clinical translation, with researchers seeking molecular diagnostics, as well as protein-based markers for personalized medicine. Proteomic strategies using the protease trypsin (known as bottom-up proteomics) were the first to be developed and optimized and form the dominant approach at present. However, researchers are now beginning to understand the limitations of bottom-up techniques, namely the inability to characterize and quantify intact protein molecules from a complex mixture of digested peptides. To overcome these limitations, several laboratories are taking a whole-protein-based approach, in which intact protein molecules are the analytical targets for characterization and quantification. We discuss these top-down techniques and how they have been applied to clinical research and are likely to be applied in the near future. Given the recent improvements in mass-spectrometry-based proteomics and stronger cooperation between researchers, clinicians and statisticians, both peptide-based (bottom-up) strategies and whole-protein-based (top-down) strategies are set to complement each other and help researchers and clinicians better understand and detect complex disease phenotypes. PMID:23806018

The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

PubMed

Amarasinghe, Chinthaka; Jin, Jian-Ping

2015-01-01

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

A novel tumor-activated ALA fusion protein for specific inhibition on the growth and invasion of breast cancer cells MDA-MB-231.

PubMed

Liu, Xiufeng; Liu, Xintong; Sunchen, Suwen; Liu, Meixia; Shen, Chen; Wu, Juanjuan; Zhao, Wanli; Yu, Boyang; Liu, Jihua

2017-11-01

The aim of this research was to develop a novel ALA fusion protein for target to the malignant cells surface with high uPAR expression and locally release of the scorpion toxin AGAP in an uPA-cleavable manner. It will provide an effective approach for controlled release of the peptide toxins to treat cancerous cells. The ALA fusion proteins were expressed in pichia pastoris, and the recombinant proteins were purified by Ni-NTA affinity chromatography. The proteins were added to human breast cancer cells (MDA-MB-231) and human embryonic kidney cells (HEK-293) in order to investigate the characteristic of selective targeting and releasing of scorpion toxin AGAP in cancer cells with high uPAR expression. The inhibitory effect of ALA on MDA-MB-231, MCF7, LO2 and HEK-293 was evaluated by MTT assay. Moreover, the antiproliferation mechanism of ALA was determined by flow cytometric and western blot analysis. The results showed that ALA could target MDA-MB-231 cells and the scorpion toxin AGAP could be released with high efficiency and selectivity. ALA inhibited the growth and invasion of breast cancer cells MDA-MB231. Also, cell apoptosis pathway was found to be associated with the inhibition mechanism of ALA according to the data of flow cytometric and western blot analysis. Therefore, ALA could be a novel antitumor candidate for targeting treatment of malignant cell. This study successfully demonstrated that fusion of biotoxins with tumor target domain could provide a simple yet effective way to delivery of peptide or protein drugs.

Sys-BodyFluid: a systematical database for human body fluid proteome research

PubMed Central

Li, Su-Jun; Peng, Mao; Li, Hong; Liu, Bo-Shu; Wang, Chuan; Wu, Jia-Rui; Li, Yi-Xue; Zeng, Rong

2009-01-01

Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10 000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/. PMID:18978022

Sys-BodyFluid: a systematical database for human body fluid proteome research.

PubMed

Li, Su-Jun; Peng, Mao; Li, Hong; Liu, Bo-Shu; Wang, Chuan; Wu, Jia-Rui; Li, Yi-Xue; Zeng, Rong

2009-01-01

Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10,000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/.

N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

PubMed

Shaikh, Saame Raza; Teague, Heather

2012-12-01

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

Clinical trial uses combination therapy for certain types of non-Hodgkin lymphoma | Center for Cancer Research

Cancer.gov

Researchers are testing the safety of the combination of an experimental drug with rituximab, a standard treatment, for patients with indolent or diffuse large B-cell lymphoma. The antibody is designed to target and block a protein that is present on cancer cells and is used by those cells to protect themselves from your body’s immune system. Blocking the protein may enable your body’s immune system to find and destroy the cancer cells. Read more…

Characterizing protein domain associations by Small-molecule ligand binding

PubMed Central

Li, Qingliang; Cheng, Tiejun; Wang, Yanli; Bryant, Stephen H.

2012-01-01

Background Protein domains are evolutionarily conserved building blocks for protein structure and function, which are conventionally identified based on protein sequence or structure similarity. Small molecule binding domains are of great importance for the recognition of small molecules in biological systems and drug development. Many small molecules, including drugs, have been increasingly identified to bind to multiple targets, leading to promiscuous interactions with protein domains. Thus, a large scale characterization of the protein domains and their associations with respect to small-molecule binding is of particular interest to system biology research, drug target identification, as well as drug repurposing. Methods We compiled a collection of 13,822 physical interactions of small molecules and protein domains derived from the Protein Data Bank (PDB) structures. Based on the chemical similarity of these small molecules, we characterized pairwise associations of the protein domains and further investigated their global associations from a network point of view. Results We found that protein domains, despite lack of similarity in sequence and structure, were comprehensively associated through binding the same or similar small-molecule ligands. Moreover, we identified modules in the domain network that consisted of closely related protein domains by sharing similar biochemical mechanisms, being involved in relevant biological pathways, or being regulated by the same cognate cofactors. Conclusions A novel protein domain relationship was identified in the context of small-molecule binding, which is complementary to those identified by traditional sequence-based or structure-based approaches. The protein domain network constructed in the present study provides a novel perspective for chemogenomic study and network pharmacology, as well as target identification for drug repurposing. PMID:23745168

CPTAC Team Releases Targeted Proteomic Assays for Ovarian Cancer | Office of Cancer Clinical Proteomics Research

Cancer.gov

Pacific Northwest National Laboratory (PNNL) investigators in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), announces the public release of 98 targeted mass spectrometry-based assays for ovarian cancer research studies.  Chosen based on proteogenomic observations from the recently published multi-institutional collaborative project between PNNL and Johns Hopkins University that comprehensively examined the collections of proteins in the tumors of ovarian cancer patients (highlighted in a paper in

Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation.

PubMed

Watanabe, Daisuke; Takagi, Hiroshi

2017-06-01

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G 2 /M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.

Proteomic Approaches and Identification of Novel Therapeutic Targets for Alcoholism

PubMed Central

Gorini, Giorgio; Adron Harris, R; Dayne Mayfield, R

2014-01-01

Recent studies have shown that gene regulation is far more complex than previously believed and does not completely explain changes at the protein level. Therefore, the direct study of the proteome, considerably different in both complexity and dynamicity to the genome/transcriptome, has provided unique insights to an increasing number of researchers. During the past decade, extraordinary advances in proteomic techniques have changed the way we can analyze the composition, regulation, and function of protein complexes and pathways underlying altered neurobiological conditions. When combined with complementary approaches, these advances provide the contextual information for decoding large data sets into meaningful biologically adaptive processes. Neuroproteomics offers potential breakthroughs in the field of alcohol research by leading to a deeper understanding of how alcohol globally affects protein structure, function, interactions, and networks. The wealth of information gained from these advances can help pinpoint relevant biomarkers for early diagnosis and improved prognosis of alcoholism and identify future pharmacological targets for the treatment of this addiction. PMID:23900301

Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics.

PubMed

Puré, Ellen; Blomberg, Rachel

2018-05-03

Fibroblast activation protein (FAP) is a cell-surface serine protease that acts on various hormones and extracellular matrix components. FAP is highly upregulated in a wide variety of cancers, and is often used as a marker for pro-tumorigenic stroma. It has also been proposed as a molecular target of cancer therapies, and, especially in recent years, a great deal of research has gone into design and testing of diverse FAP-targeted treatments. Yet despite this growing field of research, our knowledge of FAP's basic biology and functional roles in various cancers has lagged behind its use as a tumor-stromal marker. In this review, we summarize and analyze recent advances in understanding the functions of FAP in cancer, most notably its prognostic value in various tumor types, cellular effects on various cell types, and potential as a therapeutic target. We highlight outstanding questions in the field, the answers to which could shape preclinical and clinical studies of FAP.

Tes, a potential Mena-related cancer therapy target.

PubMed

Li, X

2008-02-01

Cancer remains one of the world's most prominent causes of human morbidity and mortality, particularly in developing countries. According to 2005 statistics from the WHO, approximately 7.6 million people died of cancer out of 58 million deaths worldwide, with 9 million people estimated to die from cancer in 2015 and 11.4 million to die in 2030 (http://www.who.int/mediacentre/factsheets/fs297/en/index.html). The principal and internationally recognized methods of cancer treatment are surgery, radiotherapy, chemotherapy, or multimodality therapy. With the recent development of cancer biology, more and more tumor-related targets have been identified, ushering in a new era for target therapy. Every possible step that causes cellular cancer, such as signal transduction pathways, oncogenes and anti-oncogenes, cytokines and receptors, antiangiogenesis, suicide genes, and telomerase (Shay JW, Keith WN. Br J Cancer 2008), that is biologically relevant, reproducibly measurable, and definably correlated with clinical benefit represents a target for target therapies like targeting gene-virotherapy and monoclonal antibody-directed therapy. These therapies can specifically inhibit the growth of tumor cells at the molecular level and even kill them. Generally speaking, cancer-related targets should be crucial to the tumor's malignant phenotype, easily measurable in readily obtained clinical samples, and yield a significant clinical response. Since tumorigenesis is a very complex process involving the interaction of multiple factors and pathways, target treatment offers hopes to maximize efficacy while minimizing toxicity and specificity. More importantly, treatment should have little or no toxicity on normal cells, thus representing the most promising aspect of cancer research (Friday BB, Adjei AA. Clin Cancer Res 2008; 14:342-346). A recent cancer study has provided exciting information. According to Xinhua News from London, Michael Way and fellow researchers from Cancer Research UK, have found a specific tumor-related protein, "Tes," that can prevent the diffusion of cancer cells through a Mena-dependent mechanism (http://news.xinhuanet.com/newscenter/2007-12/29/content_7337328.htm, available as of December 28,2007). Research has found that a large amount of "Mena" protein is expressed in tumor tissues, helping cancer cells to diffuse throughout the body. Nevertheless, the protein "Tes" adheres to Mena, preventing it from reacting with another specific substance and rendering it ineffective, thus stopping Mena from helping cancer cells to diffuse somewhere else. However, there are large amounts of Mena in a tumor, so Tes is usually unable to stop the diffusion of cancer cells. In light of other research, Way explained that new study results will open the door to new directions in cancer therapy research. Way also noted that if drugs containing large amounts of the protein Tes are developed in the future, they could stop Mena's action in the body, and thus prevent the massive diffusion of cancer cells. Results of the study by Way and colleagues have been published in a recent issue of the journal Molecular Cell (Boëda B, Briggs DC, Higgins T, et al. Mol Cell 2007; 28:1071-1082).

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

PubMed

Chen, Yi-Ting; Chen, Hsiao-Wei; Wu, Chun-Feng; Chu, Lichieh Julie; Chiang, Wei-Fang; Wu, Chih-Ching; Yu, Jau-Song; Tsai, Cheng-Han; Liang, Kung-Hao; Chang, Yu-Sun; Wu, Maureen; Ou Yang, Wei-Ting

2017-05-01

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Affimer proteins are versatile and renewable affinity reagents

PubMed Central

Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

2017-01-01

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

Argonaute Proteins and Mechanisms of RNA Interference in Eukaryotes and Prokaryotes.

PubMed

Olina, A V; Kulbachinskiy, A V; Aravin, A A; Esyunina, D M

2018-05-01

Noncoding RNAs play essential roles in genetic regulation in all organisms. In eukaryotic cells, many small noncoding RNAs act in complex with Argonaute proteins and regulate gene expression by recognizing complementary RNA targets. The complexes of Argonaute proteins with small RNAs also play a key role in silencing of mobile genetic elements and, in some cases, viruses. These processes are collectively called RNA interference. RNA interference is a powerful tool for specific gene silencing in both basic research and therapeutic applications. Argonaute proteins are also found in prokaryotic organisms. Recent studies have shown that prokaryotic Argonautes can also cleave their target nucleic acids, in particular DNA. This activity of prokaryotic Argonautes might potentially be used to edit eukaryotic genomes. However, the molecular mechanisms of small nucleic acid biogenesis and the functions of Argonaute proteins, in particular in bacteria and archaea, remain largely unknown. Here we briefly review available data on the RNA interference processes and Argonaute proteins in eukaryotes and prokaryotes.

WRKY Proteins: Signaling and Regulation of Expression during Abiotic Stress Responses

PubMed Central

Banerjee, Aditya

2015-01-01

WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research. PMID:25879071

WRKY proteins: signaling and regulation of expression during abiotic stress responses.

PubMed

Banerjee, Aditya; Roychoudhury, Aryadeep

2015-01-01

WRKY proteins are emerging players in plant signaling and have been thoroughly reported to play important roles in plants under biotic stress like pathogen attack. However, recent advances in this field do reveal the enormous significance of these proteins in eliciting responses induced by abiotic stresses. WRKY proteins act as major transcription factors, either as positive or negative regulators. Specific WRKY factors which help in the expression of a cluster of stress-responsive genes are being targeted and genetically modified to induce improved abiotic stress tolerance in plants. The knowledge regarding the signaling cascade leading to the activation of the WRKY proteins, their interaction with other proteins of the signaling pathway, and the downstream genes activated by them are altogether vital for justified targeting of the WRKY genes. WRKY proteins have also been considered to generate tolerance against multiple abiotic stresses with possible roles in mediating a cross talk between abiotic and biotic stress responses. In this review, we have reckoned the diverse signaling pattern and biological functions of WRKY proteins throughout the plant kingdom along with the growing prospects in this field of research.

Identification and characterization of potential druggable targets among hypothetical proteins of extensively drug resistant Mycobacterium tuberculosis (XDR KZN 605) through subtractive genomics approach.

PubMed

Uddin, Reaz; Siddiqui, Quratulain Nehal; Azam, Syed Sikander; Saima, Bibi; Wadood, Abdul

2018-03-01

Among the resistant isolates of tuberculosis (TB), the multidrug resistance tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) are the areas of growing concern for which the front-line antibiotics are no more effective. As a result, the search of new therapeutic targets against TB is an imperative need of time. On the other hand, the target identification is an a priori step in drug discovery based research. Furthermore, the availability of the complete proteomic data of extensively drug resistant Mycobacterium tuberculosis (XDR-MTB) made it possible to carry out in silico analysis for the discovery of new drug targets. In the current study, we aimed to prioritize the potential drug targets among the hypothetical proteins of XDR-TB via subtractive genomics approach. In the subtractive genomics, we stepwise reduced the complete proteome of XDR-MTB to only two hypothetical proteins and evidently proposed them as new therapeutic targets. The 3D structure of one of the two target proteins was predicted via homology modeling and later on, validated by various analysis tools. Our study suggested that the domains identified and the motif hits found in the sequences of the shortlisted drug targets are crucial for the survival of the XDR-MTB. To the best of our knowledge, the current study is the first attempt in which the complete proteomic data of XDR-MTB was subjected to the computational subtractive genomics approach and therefore, would provide an opportunity to identify the unique therapeutic targets against deadly XDR-MTB. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target.

PubMed

Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M Michael; Taguchi, Y-H; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A Mary; Velmurugan, D; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

2015-11-26

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective.

Identification of potential inhibitors based on compound proposal contest: Tyrosine-protein kinase Yes as a target

PubMed Central

Chiba, Shuntaro; Ikeda, Kazuyoshi; Ishida, Takashi; Gromiha, M. Michael; Taguchi, Y-h.; Iwadate, Mitsuo; Umeyama, Hideaki; Hsin, Kun-Yi; Kitano, Hiroaki; Yamamoto, Kazuki; Sugaya, Nobuyoshi; Kato, Koya; Okuno, Tatsuya; Chikenji, George; Mochizuki, Masahiro; Yasuo, Nobuaki; Yoshino, Ryunosuke; Yanagisawa, Keisuke; Ban, Tomohiro; Teramoto, Reiji; Ramakrishnan, Chandrasekaran; Thangakani, A. Mary; Velmurugan, D.; Prathipati, Philip; Ito, Junichi; Tsuchiya, Yuko; Mizuguchi, Kenji; Honma, Teruki; Hirokawa, Takatsugu; Akiyama, Yutaka; Sekijima, Masakazu

2015-01-01

A search of broader range of chemical space is important for drug discovery. Different methods of computer-aided drug discovery (CADD) are known to propose compounds in different chemical spaces as hit molecules for the same target protein. This study aimed at using multiple CADD methods through open innovation to achieve a level of hit molecule diversity that is not achievable with any particular single method. We held a compound proposal contest, in which multiple research groups participated and predicted inhibitors of tyrosine-protein kinase Yes. This showed whether collective knowledge based on individual approaches helped to obtain hit compounds from a broad range of chemical space and whether the contest-based approach was effective. PMID:26607293

The drug target genes show higher evolutionary conservation than non-target genes.

PubMed

Lv, Wenhua; Xu, Yongdeng; Guo, Yiying; Yu, Ziqi; Feng, Guanglong; Liu, Panpan; Luan, Meiwei; Zhu, Hongjie; Liu, Guiyou; Zhang, Mingming; Lv, Hongchao; Duan, Lian; Shang, Zhenwei; Li, Jin; Jiang, Yongshuai; Zhang, Ruijie

2016-01-26

Although evidence indicates that drug target genes share some common evolutionary features, there have been few studies analyzing evolutionary features of drug targets from an overall level. Therefore, we conducted an analysis which aimed to investigate the evolutionary characteristics of drug target genes. We compared the evolutionary conservation between human drug target genes and non-target genes by combining both the evolutionary features and network topological properties in human protein-protein interaction network. The evolution rate, conservation score and the percentage of orthologous genes of 21 species were included in our study. Meanwhile, four topological features including the average shortest path length, betweenness centrality, clustering coefficient and degree were considered for comparison analysis. Then we got four results as following: compared with non-drug target genes, 1) drug target genes had lower evolutionary rates; 2) drug target genes had higher conservation scores; 3) drug target genes had higher percentages of orthologous genes and 4) drug target genes had a tighter network structure including higher degrees, betweenness centrality, clustering coefficients and lower average shortest path lengths. These results demonstrate that drug target genes are more evolutionarily conserved than non-drug target genes. We hope that our study will provide valuable information for other researchers who are interested in evolutionary conservation of drug targets.

The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development.

PubMed

Luo, Dahai; Vasudevan, Subhash G; Lescar, Julien

2015-06-01

The flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein. The multiple roles played by the NS2B-NS3 protein in the virus life cycle makes it an attractive target for antiviral drug discovery. The N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA triphosphatase activities and is involved both in viral RNA replication and virus particle formation. In addition, NS2B-NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response. Here, we review biochemical and structural advances on the NS2B-NS3 protein, including the network of interactions it forms with NS5 and NS4B and highlight recent drug development efforts targeting this protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.

Aptamers as inhibitors of target proteins.

PubMed

Missailidis, S; Hardy, A

2009-08-01

Aptamers as inhibitors of proteins in therapeutic applications offer great advantages over their antibody counterparts and the promise to be developed into the next generation therapeutic agents. However, the control of aptamer intellectual property (IP) by two major players has made aptamers an area difficult to operate and often off-putting for academic and commercial organisations. Yet, their great potential is keeping aptamers at the research forefront, with one aptamer in the clinic and various at different stages of clinical trials. To provide a comprehensive review of the aptamer IP landscape and the issues associated with aptamer therapeutics against protein targets. Extensive review of the scientific and patent literature. Following our experience in developing, patenting and commercialising our aptamers against MUC1 and an extensive review of the literature, we have identified a variety of issues pertaining to the development of aptamers against protein targets for therapeutic applications, their patenting and granting of patents, the original IP holders and their policy, as well as the current market and traits. Despite a slow start, aptamers have been developed against various therapeutic proteins and offer the promise of providing a novel generation of therapeutic entities with a variety of applications.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

Cas9, Cpf1 and C2c1/2/3―What's next?

PubMed Central

Yamamoto, Takashi; Sakuma, Tetsushi

2017-01-01

ABSTRACT Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012–2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond. PMID:28140746

Cas9, Cpf1 and C2c1/2/3-What's next?

PubMed

Nakade, Shota; Yamamoto, Takashi; Sakuma, Tetsushi

2017-05-04

Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

Discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction as potential therapeutic and preventive agents.

PubMed

Abed, Dhulfiqar Ali; Goldstein, Melanie; Albanyan, Haifa; Jin, Huijuan; Hu, Longqin

2015-07-01

The Keap1-Nrf2-ARE pathway is an important antioxidant defense mechanism that protects cells from oxidative stress and the Keap1-Nrf2 protein-protein interaction (PPI) has become an important drug target to upregulate the expression of ARE-controlled cytoprotective oxidative stress response enzymes in the development of therapeutic and preventive agents for a number of diseases and conditions. However, most known Nrf2 activators/ARE inducers are indirect inhibitors of Keap1-Nrf2 PPI and they are electrophilic species that act by modifying the sulfhydryl groups of Keap1׳s cysteine residues. The electrophilicity of these indirect inhibitors may cause "off-target" side effects by reacting with cysteine residues of other important cellular proteins. Efforts have recently been focused on the development of direct inhibitors of Keap1-Nrf2 PPI. This article reviews these recent research efforts including the development of high throughput screening assays, the discovery of peptide and small molecule direct inhibitors, and the biophysical characterization of the binding of these inhibitors to the target Keap1 Kelch domain protein. These non-covalent direct inhibitors of Keap1-Nrf2 PPI could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions.

In silico investigation of potential mTOR inhibitors from traditional Chinese medicine for treatment of Leigh syndrome.

PubMed

Chen, Kuan-Chung; Lee, Wen-Yuan; Chen, Hsin-Yi; Chen, Calvin Yu-Chian

2014-01-01

A recent research demonstrates that the inhibition of mammalian target of rapamycin (mTOR) improves survival and health for patients with Leigh syndrome. mTOR proteins can be treated as drug target proteins against Leigh syndrome and other mitochondrial disorders. In this study, we aim to identify potent TCM compounds from the TCM Database@Taiwan as lead compounds of mTOR inhibitors. PONDR-Fit protocol was employed to predict the disordered disposition in mTOR protein before virtual screening. After virtual screening, the MD simulation was employed to validate the stability of interactions between each ligand and mTOR protein in the docking poses from docking simulation. The top TCM compounds, picrasidine M and acerosin, have higher binding affinities with target protein in docking simulation than control. There have H-bonds with residues Val2240 and π interactions with common residue Trp2239. After MD simulation, the top TCM compounds maintain similar docking poses under dynamic conditions. The top two TCM compounds, picrasidine M and acerosin, were extracted from Picrasma quassioides (D. Don) Benn. and Vitex negundo L. Hence, we propose the TCM compounds, picrasidine M and acerosin, as potential candidates as lead compounds for further study in drug development process with the mTOR protein against Leigh syndrome and other mitochondrial disorders.

The Controversy Surrounding Bone Morphogenetic Proteins in the Spine: A Review of Current Research

PubMed Central

Hustedt, Joshua W.; Blizzard, Daniel J.

2014-01-01

Bone morphogenetic proteins have been in use in spinal surgery since 2002. These proteins are members of the TGF-beta superfamily and guide mesenchymal stem cells to differentiate into osteoblasts to form bone in targeted tissues. Since the first commercial BMP became available in 2002, a host of research has supported BMPs and they have been rapidly incorporated in spinal surgeries in the United States. However, recent controversy has arisen surrounding the ethical conduct of the research supporting the use of BMPs. Yale University Open Data Access (YODA) recently teamed up with Medtronic to offer a meta-analysis of the effectiveness of BMPs in spinal surgery. This review focuses on the history of BMPs and examines the YODA research to guide spine surgeons in their use of BMP in spinal surgery. PMID:25506287

Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science.

PubMed

Giessen, Tobias W

2016-10-01

Compartmentalization is one of the defining features of life. Cells use protein compartments to exert spatial control over their metabolism, store nutrients and create unique microenvironments needed for essential physiological processes. Encapsulins are a recently discovered class of protein nanocompartments found in bacteria and archaea that naturally encapsulate cargo proteins. A short C-terminal targeting sequence directs the highly specific encapsulation process in vivo. Here, I will initially discuss the properties, diversity and putative function of encapsulins. The unique characteristics and potential uses of the self-sorting cargo-packaging process found in encapsulin systems will then be highlighted. Examples for the application of encapsulins as cell-specific optical nanoprobes and targeted therapeutic delivery systems will be discussed with an emphasis on the ability to integrate multiple functionalities within a single nanodevice. By fusing targeting sequences to non-native proteins, encapsulins can also be used as specific nanocontainers and enzymatic nanoreactors in vivo. I will end by briefly discussing future avenues for encapsulin research related to both basic microbial metabolism and applications in biomedicine, catalysis and materials science. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using Functional Signature Ontology (FUSION) to Identify Mechanisms of Action for Natural Products

PubMed Central

Potts, Malia B.; Kim, Hyun Seok; Fisher, Kurt W.; Hu, Youcai; Carrasco, Yazmin P.; Bulut, Gamze Betul; Ou, Yi-Hung; Herrera-Herrera, Mireya L.; Cubillos, Federico; Mendiratta, Saurabh; Xiao, Guanghua; Hofree, Matan; Ideker, Trey; Xie, Yang; Huang, Lily Jun-shen; Lewis, Robert E.; MacMillan, John B.; White, Michael A.

2014-01-01

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by siRNA and synthetic microRNA libraries. With this strategy, we matched proteins and microRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected short-interfering RNAs, microRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets. PMID:24129700

Recent trends in drug delivery system using protein nanoparticles.

PubMed

Sripriyalakshmi, S; Jose, Pinkybel; Ravindran, Aswathy; Anjali, C H

2014-09-01

Engineered nanoparticles that can facilitate drug formulation and passively target tumours have been under extensive research in recent years. These successes have driven a new wave of significant innovation in the generation of advanced particles. The fate and transport of diagnostic nanoparticles would significantly depend on nonselective drug delivery, and hence the use of high drug dosage is implemented. In this perspective, nanocarrier-based drug targeting strategies can be used which improve the selective delivery of drugs to the site of action, i.e. drug targeting. Pharmaceutical industries majorly focus on reducing the toxicity and side effects of drugs but only recently it has been realised that carrier systems themselves may pose risks to the patient. Proteins are compatible with biological systems and they are biodegradable. They offer a multitude of moieties for modifications to tailor drug binding, imaging or targeting entities. Thus, protein nanoparticles provide outstanding contributions as a carrier for drug delivery systems. This review summarises recent progress in particle-based therapeutic delivery and discusses important concepts in particle design and biological barriers for developing the next generation of particles drug delivery systems.

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

PubMed

Lim, Wooi F; Burdach, Jon; Funnell, Alister P W; Pearson, Richard C M; Quinlan, Kate G R; Crossley, Merlin

2016-04-20

Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA bindingin vitro, it appears thatin vivoFDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to theVEGF-Apromoter as predicted, but was also found to occupy approximately 25,000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50,000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Essential proteins and possible therapeutic targets of Wolbachia endosymbiont and development of FiloBase-a comprehensive drug target database for Lymphatic filariasis

NASA Astrophysics Data System (ADS)

Sharma, Om Prakash; Kumar, Muthuvel Suresh

2016-01-01

Lymphatic filariasis (Lf) is one of the oldest and most debilitating tropical diseases. Millions of people are suffering from this prevalent disease. It is estimated to infect over 120 million people in at least 80 nations of the world through the tropical and subtropical regions. More than one billion people are in danger of getting affected with this life-threatening disease. Several studies were suggested its emerging limitations and resistance towards the available drugs and therapeutic targets for Lf. Therefore, better medicine and drug targets are in demand. We took an initiative to identify the essential proteins of Wolbachia endosymbiont of Brugia malayi, which are indispensable for their survival and non-homologous to human host proteins. In this current study, we have used proteome subtractive approach to screen the possible therapeutic targets for wBm. In addition, numerous literatures were mined in the hunt for potential drug targets, drugs, epitopes, crystal structures, and expressed sequence tag (EST) sequences for filarial causing nematodes. Data obtained from our study were presented in a user friendly database named FiloBase. We hope that information stored in this database may be used for further research and drug development process against filariasis. URL: http://filobase.bicpu.edu.in.

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

PubMed

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

PubMed

Csősz, É; Emri, G; Kalló, G; Tsaprailis, G; Tőzsér, J

2015-10-01

The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries. Our aim was to provide information about the normal composition of the sweat, and to study the chemical barrier found at the surface of skin. Sweat samples from healthy individuals were collected during sauna bathing, and the global protein panel was analysed by label-free mass spectrometry. SRM-based targeted proteomic methods were designed and stable isotope labelled reference peptides were used for method validation. Ninety-five sweat proteins were identified, 20 of them were novel proteins. It was shown that dermcidin is the most abundant sweat protein, and along with apolipoprotein D, clusterin, prolactin-inducible protein and serum albumin, they make up 91% of secreted sweat proteins. The roles of these highly abundant proteins were reviewed; all of which have protective functions, highlighting the importance of sweat glands in composing the first line of innate immune defense system, and maintaining the epidermal barrier integrity. Our findings with regard to the proteins forming the chemical barrier of the skin as determined by label-free quantification and targeted proteomics methods are in accordance with previous studies, and can be further used as a starting point for non-invasive sweat biomarker research. © 2015 European Academy of Dermatology and Venereology.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Newer antiatherosclerosis treatment strategies.

PubMed

Aggarwal, Amitesh; Singh, Safal

2011-01-01

Atherosclerosis has been a target of much clinical and molecular research. As a result of this extensive research, it is amply clear that atherogenesis is a multifactorial process involving an interplay of metabolic, immune and inflammatory mechanisms. Antiatherosclerotic strategies are today aiming for a multipronged approach targeting each arm of this multifactorial process. The newer agents under development can be divided into three broad categories: anti-inflammatory agents, modulators of intermediary metabolism and antiatherosclerosis vaccines. Potential targets for anti-inflammatory agents include inhibition of conversion of low-density lipoprotein (LDL) to oxidised LDL, blocking or downregulation of cell adhesion molecules, chemokine modulation and macrophage receptor blockade. Beyond inhibition of plaque formation, efforts are also ongoing to develop agents which stabilise the plaque by increasing its fibrous content and inhibiting its disruption. So far as research in the sphere of intermediary metabolism is concerned, the focus is now primarily on raising high-density lipoprotein and promoting reverse cholesterol transport; potential targets include cholesteryl ester transfer protein, liver X-receptor, lecithin cholesterol acyltransferase and high-density lipoprotein mimetics. Acyl-coenzymeA: cholesterol acyltransferase is another enzyme whose selective and differential inhibition is under active investigation. The concept of immunisation against a non-communicable disease such as atherosclerosis is still in its nascent stages. However, with increasing evidence to suggest the role of antigen-specific T-cell-mediated immunity in atherogenesis, this approach is potentially promising. Possible antigens under evaluation include oxidised LDL and its subparticles, heat-shock proteins and cholesteryl ester transfer protein. With cardiovascular disease being the single leading cause of death worldwide, the development of a safe and successful antiatherosclerosis strategy (possibly employing a combination of agents acting at various levels) will indeed be a major 21st-century achievement.

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

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

Shi, Tujin; Qian, Weijun

2013-02-01

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

F-box proteins involved in cancer-associated drug resistance.

PubMed

Gong, Jian; Zhou, Yuqian; Liu, Deliang; Huo, Jirong

2018-06-01

The ubiquitin proteasome system (UPS) regulated human biological processes through the appropriate and efficient proteolysis of cellular proteins. F-box proteins are the vital components of SKP1-CUL1-FBP (SCF)-type E3 ubiquitin ligases that determine substrate specificity. As F-box proteins have the ability to control the degradation of several crucial protein targets associated with drug resistance, the dysregulation of these proteins may lead to induction of chemoresistance in cancer cells. Chemotherapy is one of the most conventional therapeutic approaches of treatment of patients with cancer. However, its exclusive application in clinical settings is restricted due to the development of chemoresistance, which typically results treatment failure. Therefore, overcoming drug resistance is considered as one of the most critical issues that researchers and clinician associated with oncology face. The present review serves to provide a comprehensive overview of F-box proteins and their possible targets as well as their correlation with the chemoresistance and chemosensitization of cancer cells. The article also presents an integrated representation of the complex regulatory mechanisms responsible for chemoresistance, which may lay the foundation to explore sensible candidate drugs for therapeutic intervention.

Isotope labeling of proteins in insect cells.

PubMed

Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

2015-01-01

Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies. © 2015 Elsevier Inc. All rights reserved.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

PubMed

Zhao, Li-Hui; Yu, Xiang-Hui; Jiang, Chun-Lai; Wu, Yong-Ge; Shen, Jia-Cong; Kong, Wei

2007-05-01

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

Development and characterization of polyclonal peptide antibodies for the detection of Yellow fever virus proteins.

PubMed

Stock, N K; Escadafal, C; Achazi, K; Cissé, M; Niedrig, M

2015-09-15

There is still a considerable need for development of new tools and methods detecting specific viral proteins for the diagnosis and pathogenesis study of the Yellow fever virus (YFV). This study aimed to develop and characterize polyclonal peptide antisera for detection of YFV-C and -NS1 proteins. The antisera were used further to investigate NS1 protein expression during YFV infection in mammalian cells. YFV target proteins were detected by all antisera in western blot and immunofluorescence assays. No cross-reactivity was observed with Dengue virus, West Nile virus, Tick-borne encephalitis virus and Japanese encephalitis virus. Nuclear localization of the YFV-C protein was demonstrated for the first time. Experiments investigating NS1 expression suggested a potential use of the YFV-NS1 antisera for development of diagnostic approaches targeting the secreted form of the NS1 protein. The antisera described in this study offer new possibilities for use in YFV research and for the development of novel diagnostic tests. Copyright © 2015 Elsevier B.V. All rights reserved.

DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations.

PubMed

Chakraborty, Sandeep

2014-01-01

The ability to accurately and effectively predict the interaction between proteins and small drug-like compounds has long intrigued researchers for pedagogic, humanitarian and economic reasons. Protein docking methods (AutoDock, GOLD, DOCK, FlexX and Glide to name a few) rank a large number of possible conformations of protein-ligand complexes using fast algorithms. Previously, it has been shown that structural congruence leading to the same enzymatic function necessitates the congruence of electrostatic properties (CLASP). The current work presents a methodology for docking a ligand into a target protein, provided that there is at least one known holoenzyme with ligand bound - DOCLASP (Docking using CLASP). The contact points of the ligand in the holoenzyme defines a motif, which is used to query the target enzyme using CLASP. If there are significant matches, the holoenzyme and the target protein are superimposed based on congruent atoms. The same linear and rotational transformations are also applied to the ligand, thus creating a unified coordinate framework having the holoenzyme, the ligand and the target enzyme. In the current work, the dipeptidyl peptidase-IV inhibitor vildagliptin was docked to the PI-PLC structure complexed with myo-inositol using DOCLASP. Also, corroboration of the docking of phenylthiourea to the modelled structure of polyphenol oxidase (JrPPO1) from walnut is provided based on the subsequently solved structure of JrPPO1 (PDBid:5CE9). Analysis of the binding of the antitrypanosomial drug suramin to nine non-homologous proteins in the PDB database shows a diverse set of binding motifs, and multiple binding sites in the phospholipase A2-likeproteins from the Bothrops genus of pitvipers. The conformational changes in the suramin molecule on binding highlights the challenges in docking flexible ligands into an already 'plastic' binding site. Thus, DOCLASP presents a method for 'soft docking' ligands to proteins with low computational requirements.

Bridging the gap to therapeutic strategies based on connexin/pannexin biology.

PubMed

Naus, Christian C; Giaume, Christian

2016-11-29

A unique workshop was recently held focusing on enhancing collaborations leading to identify and update the development of therapeutic strategies targeting connexin/pannexin large pore channels. Basic scientists exploring the functions of these channels in various pathologies gathered together with leading pharma companies which are targeting gap junction proteins for specific therapeutic applications. This highlights how paths of discovery research can converge with therapeutic strategies in innovative ways to enhance target identification and validation.

Understanding the Role of Intrinsic Disorder of Viral Proteins in the Oncogenicity of Different Types of HPV.

PubMed

Tamarozzi, Elvira Regina; Giuliatti, Silvana

2018-01-09

Intrinsic disorder is very important in the biological function of several proteins, and is directly linked to their foldability during interaction with their targets. There is a close relationship between the intrinsically disordered proteins and the process of carcinogenesis involving viral pathogens. Among these pathogens, we have highlighted the human papillomavirus (HPV) in this study. HPV is currently among the most common sexually transmitted infections, besides being the cause of several types of cancer. HPVs are divided into two groups, called high- and low-risk, based on their oncogenic potential. The high-risk HPV E6 protein has been the target of much research, in seeking treatments against HPV, due to its direct involvement in the process of cell cycle control. To understand the role of intrinsic disorder of the viral proteins in the oncogenic potential of different HPV types, the structural characteristics of intrinsically disordered regions of high and low-risk HPV E6 proteins were analyzed. In silico analyses of primary sequences, prediction of tertiary structures, and analyses of molecular dynamics allowed the observation of the behavior of such disordered regions in these proteins, thereby proving a direct relationship of structural variation with the degree of oncogenicity of HPVs. The results obtained may contribute to the development of new therapies, targeting the E6 oncoprotein, for the treatment of HPV-associated diseases.

The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes

PubMed Central

Rigden, Daniel J

2017-01-01

Abstract This year's Database Issue of Nucleic Acids Research contains 152 papers that include descriptions of 54 new databases and update papers on 98 databases, of which 16 have not been previously featured in NAR. As always, these databases cover a broad range of molecular biology subjects, including genome structure, gene expression and its regulation, proteins, protein domains, and protein–protein interactions. Following the recent trend, an increasing number of new and established databases deal with the issues of human health, from cancer-causing mutations to drugs and drug targets. In accordance with this trend, three recently compiled databases that have been selected by NAR reviewers and editors as ‘breakthrough’ contributions, denovo-db, the Monarch Initiative, and Open Targets, cover human de novo gene variants, disease-related phenotypes in model organisms, and a bioinformatics platform for therapeutic target identification and validation, respectively. We expect these databases to attract the attention of numerous researchers working in various areas of genetics and genomics. Looking back at the past 12 years, we present here the ‘golden set’ of databases that have consistently served as authoritative, comprehensive, and convenient data resources widely used by the entire community and offer some lessons on what makes a successful database. The Database Issue is freely available online at the https://academic.oup.com/nar web site. An updated version of the NAR Molecular Biology Database Collection is available at http://www.oxfordjournals.org/nar/database/a/. PMID:28053160

Advances in recombinant protein expression for use in pharmaceutical research.

PubMed

Assenberg, Rene; Wan, Paul T; Geisse, Sabine; Mayr, Lorenz M

2013-06-01

Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells. Copyright © 2013. Published by Elsevier Ltd.

Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

PubMed Central

2014-01-01

While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

LIBP-Pred: web server for lipid binding proteins using structural network parameters; PDB mining of human cancer biomarkers and drug targets in parasites and bacteria.

PubMed

González-Díaz, Humberto; Munteanu, Cristian R; Postelnicu, Lucian; Prado-Prado, Francisco; Gestal, Marcos; Pazos, Alejandro

2012-03-01

Lipid-Binding Proteins (LIBPs) or Fatty Acid-Binding Proteins (FABPs) play an important role in many diseases such as different types of cancer, kidney injury, atherosclerosis, diabetes, intestinal ischemia and parasitic infections. Thus, the computational methods that can predict LIBPs based on 3D structure parameters became a goal of major importance for drug-target discovery, vaccine design and biomarker selection. In addition, the Protein Data Bank (PDB) contains 3000+ protein 3D structures with unknown function. This list, as well as new experimental outcomes in proteomics research, is a very interesting source to discover relevant proteins, including LIBPs. However, to the best of our knowledge, there are no general models to predict new LIBPs based on 3D structures. We developed new Quantitative Structure-Activity Relationship (QSAR) models based on 3D electrostatic parameters of 1801 different proteins, including 801 LIBPs. We calculated these electrostatic parameters with the MARCH-INSIDE software and they correspond to the entire protein or to specific protein regions named core, inner, middle, and surface. We used these parameters as inputs to develop a simple Linear Discriminant Analysis (LDA) classifier to discriminate 3D structure of LIBPs from other proteins. We implemented this predictor in the web server named LIBP-Pred, freely available at , along with other important web servers of the Bio-AIMS portal. The users can carry out an automatic retrieval of protein structures from PDB or upload their custom protein structural models from their disk created with LOMETS server. We demonstrated the PDB mining option performing a predictive study of 2000+ proteins with unknown function. Interesting results regarding the discovery of new Cancer Biomarkers in humans or drug targets in parasites have been discussed here in this sense.

Mitotic Vulnerability in Triple-Negative Breast Cancer Associated with LIN9 Is Targetable with BET Inhibitors.

PubMed

Sahni, Jennifer M; Gayle, Sylvia S; Webb, Bryan M; Weber-Bonk, Kristen L; Seachrist, Darcie D; Singh, Salendra; Sizemore, Steven T; Restrepo, Nicole A; Bebek, Gurkan; Scacheri, Peter C; Varadan, Vinay; Summers, Matthew K; Keri, Ruth A

2017-10-01

Triple-negative breast cancers (TNBC) are highly aggressive, lack FDA-approved targeted therapies, and frequently recur, making the discovery of novel therapeutic targets for this disease imperative. Our previous analysis of the molecular mechanisms of action of bromodomain and extraterminal protein inhibitors (BETi) in TNBC revealed these drugs cause multinucleation, indicating BET proteins are essential for efficient mitosis and cytokinesis. Here, using live cell imaging, we show that BET inhibition prolonged mitotic progression and induced mitotic cell death, both of which are indicative of mitotic catastrophe. Mechanistically, the mitosis regulator LIN9 was a direct target of BET proteins that mediated the effects of BET proteins on mitosis in TNBC. Although BETi have been proposed to function by dismantling super-enhancers (SE), the LIN9 gene lacks an SE but was amplified or overexpressed in the majority of TNBCs. In addition, its mRNA expression predicted poor outcome across breast cancer subtypes. Together, these results provide a mechanism for cancer selectivity of BETi that extends beyond modulation of SE-associated genes and suggest that cancers dependent upon LIN9 overexpression may be particularly vulnerable to BETi. Cancer Res; 77(19); 5395-408. ©2017 AACR . ©2017 American Association for Cancer Research.

Cellular and molecular mechanisms of HIV-1 integration targeting.

PubMed

Engelman, Alan N; Singh, Parmit K

2018-07-01

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Identification of Direct Protein Targets of Small Molecules

PubMed Central

2010-01-01

Small-molecule target identification is a vital and daunting task for the chemical biology community as well as for researchers interested in applying the power of chemical genetics to impact biology and medicine. To overcome this “target ID” bottleneck, new technologies are being developed that analyze protein–drug interactions, such as drug affinity responsive target stability (DARTS), which aims to discover the direct binding targets (and off targets) of small molecules on a proteome scale without requiring chemical modification of the compound. Here, we review the DARTS method, discuss why it works, and provide new perspectives for future development in this area. PMID:21077692

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food.

PubMed

Agyei, Dominic; Acquah, Caleb; Tan, Kei Xian; Hii, Hieng Kok; Rajendran, Subin R C K; Udenigwe, Chibuike C; Danquah, Michael K

2018-01-01

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed. Graphical abstract Advancing bioactive proteins and peptide functionality via aptameric ligands.

Closing the door on flaviviruses: entry as a target for antiviral drug design.

PubMed

Perera, Rushika; Khaliq, Mansoora; Kuhn, Richard J

2008-10-01

With the emergence and rapid spread of West Nile virus in the United States since 1999, and the 50-100 million infections per year caused by dengue virus globally, the threat of flaviviruses as re-emerging human pathogens has become a reality. To support the efforts that are currently being pursued to develop effective vaccines against these viruses, researchers are also actively pursuing the development of small molecule compounds that target various aspects of the virus life cycle. Recent advances in the structural characterization of the flaviviruses have provided a strong foundation towards these efforts. These studies have provided the pseudo-atomic structures of virions from several members of the genus as well as atomic resolution structures of several viral proteins. Most importantly, these studies have highlighted specific structural rearrangements that occur within the virion that are necessary for the virus to complete its life cycle. These rearrangements occur when the virus must transition from immature, to mature, to fusion-active states and rely heavily on the conformational flexibility of the envelope (E) protein that forms the outer glycoprotein shell of the virus. Analysis of these conformational changes can suggest promising targets for structure-based antiviral design. For instance, by targeting the flexibility of the E protein, it might be possible to inhibit required rearrangements of this protein and trap the virus in a specific state. This would interfere with a productive flaviviral infection. This review presents a structural perspective of the flavivirus life cycle and focuses on the role of the E protein as an opportune target for structure-based antiviral drug design.

Acquisition, Conservation, and Loss of Dual-Targeted Proteins in Land Plants1[W][OA

PubMed Central

Xu, Lin; Carrie, Chris; Law, Simon R.; Murcha, Monika W.; Whelan, James

2013-01-01

The dual-targeting ability of a variety of proteins from Physcomitrella patens, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) was tested to determine when dual targeting arose and to what extent it was conserved in land plants. Overall, the targeting ability of over 80 different proteins from rice and P. patens, representing 42 dual-targeted proteins in Arabidopsis, was tested. We found that dual targeting arose early in land plant evolution, as it was evident in many cases with P. patens proteins that were conserved in rice and Arabidopsis. Furthermore, we found that the acquisition of dual-targeting ability is still occurring, evident in P. patens as well as rice and Arabidopsis. The loss of dual-targeting ability appears to be rare, but does occur. Ascorbate peroxidase represents such an example. After gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Although we found that dual targeting was generally conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore, it appears that small changes in the targeting signal can result in a loss (or gain) of dual-targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual-targeting ability. The acquisition of dual-targeting ability also appears to be coordinated between proteins. Mitochondrial intermembrane space import and assembly protein40, a protein involved in oxidative folding in mitochondria and peroxisomes, provides an example where acquisition of dual targeting is accompanied by the dual targeting of substrate proteins. PMID:23257241

Cloning, expression and crystallisation of SGT1 co-chaperone protein from Glaciozyma antarctica

NASA Astrophysics Data System (ADS)

Yusof, Nur Athirah; Bakar, Farah Diba Abu; Beddoe, Travis; Murad, Abdul Munir Abdul

2013-11-01

Studies on psycrophiles are now in the limelight of today's post genomic era as they fascinate the research and development industries. The discovery from Glaciozyma antarctica, an extreme cold adapted yeast from Antarctica shows promising future to provide cost effective natural sustainable energy and create wider understanding of the property that permits this organisms to adapt to extreme temperature downshift. In plants and yeast, studies show the interaction between SGT1 and HSP90 are essential for disease resistance and heat stress by activating a number of resistance proteins. Here we report for the first time cloning, expression and crystallization of the recombinant SGT1 protein of G. antarctica (rGa_SGT1), a highly conserved eukaryotic protein that interacts with the molecular chaperones HSP90 (heat shock protein 90) apparently associated in a role of co-chaperone that may play important role in cold adaptation. The sequence analysis of rGa_SGT1 revealed the presence of all the characteristic features of SGT1 protein. In this study, we present the outlines and results of protein structural study of G. antarctica SGT1 protein. We validate this approach by starting with cloning the target insert into Ligation Independent Cloning system proceeded with expression using E. coli system, and crystallisation of the target rGA_SGT1 protein. The work is still on going with the target subunit of the complex proteins yielded crystals. These results, still ongoing, open a platform for better understanding of the uniqueness of this crucial molecular machine function in cold adaptation.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

PubMed Central

Ghisaidoobe, Amar B. T.; Chung, Sang J.

2014-01-01

Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λEX ∼ 280 nm, λEM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins. PMID:25490136

Fred Hutch's Paulovich Laboratory to Lead Protein Assay Work for National Beau Biden Cancer Moonshot | Office of Cancer Clinical Proteomics Research

Cancer.gov

The Applied Proteogenomics OrganizationaL Learning and Outcomes (APOLLO) network, which is a partnership among the National Cancer Institute, Department of Defense, and Department of Veterans Affairs, has tapped the Paulovich Laboratory at Fred Hutchinson Cancer Research Center to create a panel of tests to measure key proteins that can serve as markers for tumors. The effort could ultimately lead to treatments that are more specifically targeted to a patient’s distinct type of cancer.  

Diagnostic Marker for Improving Treatment Outcomes of Hepatitis C | NCI Technology Transfer Center | TTC

Cancer.gov

NCI Researchers have discovered Interferon-lambda 4 (IFNL4), a protein found through analysis of genomic data. Preliminary studies indicate that this protein may play a role in the clearance of HCV and may be a new target for diagnosing and treating HCV infection. The National Cancer Institute (NCI) Division of Cancer Epidemiology and Genetics (DCEG) Immunoepidemiology Branch is seeking statements of capability or interest from parties interested in in-licensing or collaborative research to further co-develop a gene-based diagnostic for Hepatitis C virus (HepC, HCV).

Protein detection by Simple Western™ analysis.

PubMed

Harris, Valerie M

2015-01-01

Protein Simple© has taken a well-known protein detection method, the western blot, and revolutionized it. The Simple Western™ system uses capillary electrophoresis to identify and quantitate a protein of interest. Protein Simple© provides multiple detection apparatuses (Wes, Sally Sue, or Peggy Sue) that are suggested to save scientists valuable time by allowing the researcher to prepare the protein sample, load it along with necessary antibodies and substrates, and walk away. Within 3-5 h the protein will be separated by size, or charge, immuno-detection of target protein will be accurately quantitated, and results will be immediately made available. Using the Peggy Sue instrument, one study recently examined changes in MAPK signaling proteins in the sex-determining stage of gonadal development. Here the methodology is described.

Efficient visualization of high-throughput targeted proteomics experiments: TAPIR.

PubMed

Röst, Hannes L; Rosenberger, George; Aebersold, Ruedi; Malmström, Lars

2015-07-15

Targeted mass spectrometry comprises a set of powerful methods to obtain accurate and consistent protein quantification in complex samples. To fully exploit these techniques, a cross-platform and open-source software stack based on standardized data exchange formats is required. We present TAPIR, a fast and efficient Python visualization software for chromatograms and peaks identified in targeted proteomics experiments. The input formats are open, community-driven standardized data formats (mzML for raw data storage and TraML encoding the hierarchical relationships between transitions, peptides and proteins). TAPIR is scalable to proteome-wide targeted proteomics studies (as enabled by SWATH-MS), allowing researchers to visualize high-throughput datasets. The framework integrates well with existing automated analysis pipelines and can be extended beyond targeted proteomics to other types of analyses. TAPIR is available for all computing platforms under the 3-clause BSD license at https://github.com/msproteomicstools/msproteomicstools. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Functions of bromodomain-containing proteins and their roles in homeostasis and cancer.

PubMed

Fujisawa, Takao; Filippakopoulos, Panagis

2017-04-01

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease.

Protein targets for anticancer gold compounds: mechanistic inferences.

PubMed

Gabbiani, Chiara; Messori, Luigi

2011-12-01

Gold compounds form an interesting class of antiproliferative agents of potential pharmacological use in cancer treatment. Indeed, a number of gold compounds, either gold(III) or gold(I), were recently described and characterised that manifested remarkable cytotoxic properties in vitro against cultured cancer cells; for some of them encouraging in vivo results were also reported toward a few relevant animal models of cancer. The molecular mechanisms through which gold compounds exert their biological effects are still largely unknown and the subject of intense investigations. Recent studies point out that the modes of action of cytotoxic gold compounds are essentially DNA-independent and cisplatin-unrelated, relying -most likely- on gold interactions with a variety of protein targets. Notably, a few cellular proteins playing relevant functional roles were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses need adequate validation. The state of the art of this research area and the perspectives for future studies are herein critically analysed and discussed.

Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

DOE PAGES

Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

2017-07-19

Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

PubMed Central

Raff, Adam B.; Gray, Andrew; Kast, W. Martin

2009-01-01

The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

PubMed

Hengel, Sarah R; Spies, M Ashley; Spies, Maria

2017-09-21

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

Brandon Knott | NREL

Science.gov Websites

-6223 Research Interests Molecular mechanisms of cellulose-degrading enzymes Structure-function relationships of biomass-derived polymers Structure-function relationships in glycoside hydrolases Methane potential protein engineering targets. Structure-Function Relationships of Biomass-Derived Polymers

Analysis of in vitro bioactivity data extracted from drug discovery literature and patents: Ranking 1654 human protein targets by assayed compounds and molecular scaffolds

PubMed Central

2011-01-01

Background Since the classic Hopkins and Groom druggable genome review in 2002, there have been a number of publications updating both the hypothetical and successful human drug target statistics. However, listings of research targets that define the area between these two extremes are sparse because of the challenges of collating published information at the necessary scale. We have addressed this by interrogating databases, populated by expert curation, of bioactivity data extracted from patents and journal papers over the last 30 years. Results From a subset of just over 27,000 documents we have extracted a set of compound-to-target relationships for biochemical in vitro binding-type assay data for 1,736 human proteins and 1,654 gene identifiers. These are linked to 1,671,951 compound records derived from 823,179 unique chemical structures. The distribution showed a compounds-per-target average of 964 with a maximum of 42,869 (Factor Xa). The list includes non-targets, failed targets and cross-screening targets. The top-278 most actively pursued targets cover 90% of the compounds. We further investigated target ranking by determining the number of molecular frameworks and scaffolds. These were compared to the compound counts as alternative measures of chemical diversity on a per-target basis. Conclusions The compounds-per-protein listing generated in this work (provided as a supplementary file) represents the major proportion of the human drug target landscape defined by published data. We supplemented the simple ranking by the number of compounds assayed with additional rankings by molecular topology. These showed significant differences and provide complementary assessments of chemical tractability. PMID:21569515

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

2008-05-13

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

Antiadhesion agents against Gram-positive pathogens.

PubMed

Cascioferro, Stella; Cusimano, Maria Grazia; Schillaci, Domenico

2014-01-01

A fundamental step of Gram-positive pathogenesis is the bacterial adhesion to the host tissue involving interaction between bacterial surface molecules and host ligands. This review is focused on antivirulence compounds that target Gram-positive adhesins and on their potential development as therapeutic agents alternative or complementary to conventional antibiotics in the contrast of pathogens. In particular, compounds that target the sortase A, wall theicoic acid inhibitors, carbohydrates able to bind bacterial proteins and proteins capable of influencing the bacterial adhesion, were described. We further discuss the advantages and disadvantages of this strategy in the development of novel antimicrobials and the future perspective of this research field still at its first steps.

ProTSAV: A protein tertiary structure analysis and validation server.

PubMed

Singh, Ankita; Kaushik, Rahul; Mishra, Avinash; Shanker, Asheesh; Jayaram, B

2016-01-01

Quality assessment of predicted model structures of proteins is as important as the protein tertiary structure prediction. A highly efficient quality assessment of predicted model structures directs further research on function. Here we present a new server ProTSAV, capable of evaluating predicted model structures based on some popular online servers and standalone tools. ProTSAV furnishes the user with a single quality score in case of individual protein structure along with a graphical representation and ranking in case of multiple protein structure assessment. The server is validated on ~64,446 protein structures including experimental structures from RCSB and predicted model structures for CASP targets and from public decoy sets. ProTSAV succeeds in predicting quality of protein structures with a specificity of 100% and a sensitivity of 98% on experimentally solved structures and achieves a specificity of 88%and a sensitivity of 91% on predicted protein structures of CASP11 targets under 2Å.The server overcomes the limitations of any single server/method and is seen to be robust in helping in quality assessment. ProTSAV is freely available at http://www.scfbio-iitd.res.in/software/proteomics/protsav.jsp. Copyright © 2015 Elsevier B.V. All rights reserved.

In silico re-identification of properties of drug target proteins.

PubMed

Kim, Baeksoo; Jo, Jihoon; Han, Jonghyun; Park, Chungoo; Lee, Hyunju

2017-05-31

Computational approaches in the identification of drug targets are expected to reduce time and effort in drug development. Advances in genomics and proteomics provide the opportunity to uncover properties of druggable genomes. Although several studies have been conducted for distinguishing drug targets from non-drug targets, they mainly focus on the sequences and functional roles of proteins. Many other properties of proteins have not been fully investigated. Using the DrugBank (version 3.0) database containing nearly 6,816 drug entries including 760 FDA-approved drugs and 1822 of their targets and human UniProt/Swiss-Prot databases, we defined 1578 non-redundant drug target and 17,575 non-drug target proteins. To select these non-redundant protein datasets, we built four datasets (A, B, C, and D) by considering clustering of paralogous proteins. We first reassessed the widely used properties of drug target proteins. We confirmed and extended that drug target proteins (1) are likely to have more hydrophobic, less polar, less PEST sequences, and more signal peptide sequences higher and (2) are more involved in enzyme catalysis, oxidation and reduction in cellular respiration, and operational genes. In this study, we proposed new properties (essentiality, expression pattern, PTMs, and solvent accessibility) for effectively identifying drug target proteins. We found that (1) drug targetability and protein essentiality are decoupled, (2) druggability of proteins has high expression level and tissue specificity, and (3) functional post-translational modification residues are enriched in drug target proteins. In addition, to predict the drug targetability of proteins, we exploited two machine learning methods (Support Vector Machine and Random Forest). When we predicted drug targets by combining previously known protein properties and proposed new properties, an F-score of 0.8307 was obtained. When the newly proposed properties are integrated, the prediction performance is improved and these properties are related to drug targets. We believe that our study will provide a new aspect in inferring drug-target interactions.

Synthetic Zinc Finger Proteins: The Advent of Targeted Gene Regulation and Genome Modification Technologies

PubMed Central

2015-01-01

Conspectus The understanding of gene regulation and the structure and function of the human genome increased dramatically at the end of the 20th century. Yet the technologies for manipulating the genome have been slower to develop. For instance, the field of gene therapy has been focused on correcting genetic diseases and augmenting tissue repair for more than 40 years. However, with the exception of a few very low efficiency approaches, conventional genetic engineering methods have only been able to add auxiliary genes to cells. This has been a substantial obstacle to the clinical success of gene therapies and has also led to severe unintended consequences in several cases. Therefore, technologies that facilitate the precise modification of cellular genomes have diverse and significant implications in many facets of research and are essential for translating the products of the Genomic Revolution into tangible benefits for medicine and biotechnology. To address this need, in the 1990s, we embarked on a mission to develop technologies for engineering protein–DNA interactions with the aim of creating custom tools capable of targeting any DNA sequence. Our goal has been to allow researchers to reach into genomes to specifically regulate, knock out, or replace any gene. To realize these goals, we initially focused on understanding and manipulating zinc finger proteins. In particular, we sought to create a simple and straightforward method that enables unspecialized laboratories to engineer custom DNA-modifying proteins using only defined modular components, a web-based utility, and standard recombinant DNA technology. Two significant challenges we faced were (i) the development of zinc finger domains that target sequences not recognized by naturally occurring zinc finger proteins and (ii) determining how individual zinc finger domains could be tethered together as polydactyl proteins to recognize unique locations within complex genomes. We and others have since used this modular assembly method to engineer artificial proteins and enzymes that activate, repress, or create defined changes to user-specified genes in human cells, plants, and other organisms. We have also engineered novel methods for externally controlling protein activity and delivery, as well as developed new strategies for the directed evolution of protein and enzyme function. This Account summarizes our work in these areas and highlights independent studies that have successfully used the modular assembly approach to create proteins with novel function. We also discuss emerging alternative methods for genomic targeting, including transcription activator-like effectors (TALEs) and CRISPR/Cas systems, and how they complement the synthetic zinc finger protein technology. PMID:24877793

Barcoding heat shock proteins to human diseases: looking beyond the heat shock response.

PubMed

Kakkar, Vaishali; Meister-Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H

2014-04-01

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR)--and thus generally restoring the disturbed protein homeostasis associated with such diseases--has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or 'barcoded' by a different set of HSPs that can rescue specific types of aggregation. Some of these 'non-canonical' HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated--so-called chaperonopathies--which are also discussed in this Review.

Barcoding heat shock proteins to human diseases: looking beyond the heat shock response

PubMed Central

Kakkar, Vaishali; Meister-Broekema, Melanie; Minoia, Melania; Carra, Serena; Kampinga, Harm H.

2014-01-01

There are numerous human diseases that are associated with protein misfolding and the formation of toxic protein aggregates. Activating the heat shock response (HSR) – and thus generally restoring the disturbed protein homeostasis associated with such diseases – has often been suggested as a therapeutic strategy. However, most data on activating the HSR or its downstream targets in mouse models of diseases associated with aggregate formation have been rather disappointing. The human chaperonome consists of many more heat shock proteins (HSPs) that are not regulated by the HSR, however, and researchers are now focusing on these as potential therapeutic targets. In this Review, we summarize the existing literature on a set of aggregation diseases and propose that each of them can be characterized or ‘barcoded’ by a different set of HSPs that can rescue specific types of aggregation. Some of these ‘non-canonical’ HSPs have demonstrated effectiveness in vivo, in mouse models of protein-aggregation disease. Interestingly, several of these HSPs also cause diseases when mutated – so-called chaperonopathies – which are also discussed in this Review. PMID:24719117

New clinical trial uses targeted cancer drug for lymphatic system disease | Center for Cancer Research

Cancer.gov

Nivolumab is a targeted cancer drug that blocks the action of a protein called PD-1 and activates T cells to attack cancer cells without damaging normal cells. Mark Roschewski, M.D., of the Lymphoid Malignancies Branch is leading a study to determine if nivolumab is effective in treating patients with certain diseases of the lymphatic system.

HDAC Inhibitors Target Replication Forks to Take a Stab at Cancer | Center for Cancer Research

Cancer.gov

The stability and function of many proteins within the cell can be altered with the addition or removal of certain chemical groups, including acetyl groups. Therefore, the enzymes that regulate these modifications have an important impact on the cell. One class of such enzymes—histone deacetylases, or HDACs—has been implicated in cancer and has become a target for cancer

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments

PubMed Central

Chung, Lisa M.; Colangelo, Christopher M.; Zhao, Hongyu

2014-01-01

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets. PMID:24905083

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments.

PubMed

Chung, Lisa M; Colangelo, Christopher M; Zhao, Hongyu

2014-06-05

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets.

Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

PubMed

Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

2017-09-01

Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

Targeting G protein coupled receptor-related pathways as emerging molecular therapies

PubMed Central

Ghanemi, Abdelaziz

2013-01-01

G protein coupled receptors (GPCRs) represent the most important targets in modern pharmacology because of the different functions they mediate, especially within brain and peripheral nervous system, and also because of their functional and stereochemical properties. In this paper, we illustrate, via a variety of examples, novel advances about the GPCR-related molecules that have been shown to play diverse roles in GPCR pathways and in pathophysiological phenomena. We have exemplified how those GPCRs’ pathways are, or might constitute, potential targets for different drugs either to stimulate, modify, regulate or inhibit the cellular mechanisms that are hypothesized to govern some pathologic, physiologic, biologic and cellular or molecular aspects both in vivo and in vitro. Therefore, influencing such pathways will, undoubtedly, lead to different therapeutical applications based on the related pharmacological implications. Furthermore, such new properties can be applied in different fields. In addition to offering fruitful directions for future researches, we hope the reviewed data, together with the elements found within the cited references, will inspire clinicians and researchers devoted to the studies on GPCR’s properties. PMID:25972730

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

PubMed Central

Chen, Yingzhi; Zhang, Meng; Jin, Hongyue; Tang, Yisi; Wang, Huiyuan; Xu, Qin; Li, Yaping; Li, Feng; Huang, Yongzhuo

2017-01-01

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation. PMID:27914267

Musashi RNA-Binding Proteins as Cancer Drivers and Novel Therapeutic Targets.

PubMed

Kudinov, Alexander E; Karanicolas, John; Golemis, Erica A; Boumber, Yanis

2017-05-01

Aberrant gene expression that drives human cancer can arise from epigenetic dysregulation. Although much attention has focused on altered activity of transcription factors and chromatin-modulating proteins, proteins that act posttranscriptionally can potently affect expression of oncogenic signaling proteins. The RNA-binding proteins (RBP) Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance. Following identification of Musashi as a regulator of progenitor cell identity in Drosophila , the human Musashi proteins were initially linked to control of maintenance of hematopoietic stem cells, then stem cell compartments for additional cell types. More recently, the Musashi proteins were found to be overexpressed and prognostic of outcome in numerous cancer types, including colorectal, lung, and pancreatic cancers; glioblastoma; and several leukemias. MSI1 and MSI2 bind and regulate the mRNA stability and translation of proteins operating in essential oncogenic signaling pathways, including NUMB/Notch, PTEN/mTOR, TGFβ/SMAD3, MYC, cMET, and others. On the basis of these activities, MSI proteins maintain cancer stem cell populations and regulate cancer invasion, metastasis, and development of more aggressive cancer phenotypes, including drug resistance. Although RBPs are viewed as difficult therapeutic targets, initial efforts to develop MSI-specific inhibitors are promising, and RNA interference-based approaches to inhibiting these proteins have had promising outcomes in preclinical studies. In the interim, understanding the function of these translational regulators may yield insight into the relationship between mRNA expression and protein expression in tumors, guiding tumor-profiling analysis. This review provides a current overview of Musashi as a cancer driver and novel therapeutic target. Clin Cancer Res; 23(9); 2143-53. ©2017 AACR . ©2017 American Association for Cancer Research.

Review and research analysis of computational target methods using BioRuby and in silico screening of herbal lead compounds against pancreatic cancer using R programming.

PubMed

Jayadeepa, R M; Ray, Ankita; Naik, Dhaval; Sanyal, Debendra Nath; Shah, Disha

2014-01-01

Plants and their natural components sophisticated with the cornerstone of traditional conventional medicinal system throughout the globe for many years and extend to furnish mankind with latest remedies. Natural Products act as lead molecules for the synthesis of various potent drugs. In the current research a study is conducted on herbal small molecule and their potential binding chemical affinity to the effect or molecules of major diseases such as pancreatic cancer. Clinical studies demonstrate correlation between Cyclin- Dependent Kinase 4 (CDK4) and malignant progression of Pancreatic Cancer. Using Bioruby Gem's we were able to analyze better characteristics of the target protein. VegaZZ and NAMD were used to minimize the energy of the target protein. Therefore identification of effective, well- tolerated targets was analyzed. Further the target protein was subjected to docking with the anti cancer inhibitors which represents a rational chemo preventive strategy using AutoDock Vina. Later using the dock score top ranked phytochemicals were analyzed for Toxicity Analysis. Using the BioRuby gem we were able to measure the distance between the amino acid. Various R scripting libraries were used to hunt the best leads, as in this case the phytochemicals. Phytochemicals such as Wedelolactones and Catechin were analyzed computationally. This study has presented the various effects of naturally occurring anti pancreatic cancer compounds Catechin, Wedelolactones that inhibits Cyclin Dependent Kinase 4. The study results reveal that compounds use less binding energy to CDK4 and inhibit its activity. Future investigation of other various wet lab studies such as cell line studies will confirm results of these two herbal chemical formulations potential ones for treating Pancreatic Cancer.

The flavivirus capsid protein: Structure, function and perspectives towards drug design.

PubMed

Oliveira, Edson R A; Mohana-Borges, Ronaldo; de Alencastro, Ricardo B; Horta, Bruno A C

2017-01-02

Flaviviruses, such as dengue and zika viruses, are etiologic agents transmitted to humans mainly by arthropods and are of great epidemiological interest. The flavivirus capsid protein is a structural element required for the viral nucleocapsid assembly that presents the classical function of sheltering the viral genome. After decades of research, many reports have shown its different functionalities and influence over cell normal functioning. The subcellular distribution of this protein, which involves accumulation around lipid droplets and nuclear localization, also corroborates with its multi-functional characteristic. As flavivirus diseases are still in need of global control and in view of the possible key functionalities that the capsid protein promotes over flavivirus biology, novel considerations arise towards anti-flavivirus drug research. This review covers the main aspects concerning structural and functional features of the flavivirus C protein, ultimately, highlighting prospects in drug discovery based on this viral target. Copyright © 2016 Elsevier B.V. All rights reserved.

Random Splicing of Several Exons Caused by a Single Base Change in the Target Exon of CRISPR/Cas9 Mediated Gene Knockout.

PubMed

Kapahnke, Marcel; Banning, Antje; Tikkanen, Ritva

2016-12-14

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated sequence 9 (CRISPR/Cas9) system is widely used for genome editing purposes as it facilitates an efficient knockout of a specific gene in, e.g. cultured cells. Targeted double-strand breaks are introduced to the target sequence of the guide RNAs, which activates the cellular DNA repair mechanism for non-homologous-end-joining, resulting in unprecise repair and introduction of small deletions or insertions. Due to this, sequence alterations in the coding region of the target gene frequently cause frame-shift mutations, facilitating degradation of the mRNA. We here show that such CRISPR/Cas9-mediated alterations in the target exon may also result in altered splicing of the respective pre-mRNA, most likely due to mutations of splice-regulatory sequences. Using the human FLOT-1 gene as an example, we demonstrate that such altered splicing products also give rise to aberrant protein products. These may potentially function as dominant-negative proteins and thus interfere with the interpretation of the data generated with these cell lines. Since most researchers only control the consequences of CRISPR knockout at genomic and protein level, our data should encourage to also check the alterations at the mRNA level.

Implementation of a protein profiling platform developed as an academic-pharmaceutical industry collaborative effort.

PubMed

Végvári, Akos; Magnusson, Mattias; Wallman, Lars; Ekström, Simon; Bolmsjö, Gunnar; Nilsson, Johan; Miliotis, Tasso; Ostling, Jörgen; Kjellström, Sven; Ottervald, Jan; Franzén, Bo; Hultberg, Hans; Marko-Varga, György; Laurell, Thomas

2008-06-01

As much attention has devoted to the proteome research during the last few years, biomarker discovery has become an increasingly hot area, potentially enabling the development of new assays for diagnosis and prognosis of severe diseases. This is the field of research interest where efforts originating from both academic and industrial groups should jointly work on solutions. In this paper, we would like to demonstrate the fruitful combination of both research domains where the scientific crossroads sprout fresh ideas from the basic research domain and how these are refined and tethered to industrial standards. We will present an approach that is based on novel microfluidic devices, utilizing their benefits in processing small-volume samples. Our biomarker discovery strategy, built around this platform, involves optimized samples processing (based on SPE and sample enrichment) and fast MALDI-MS readout. The identification of novel biomarkers at low-abundance level has been achieved by the utilization of a miniaturized sample handling platform, which offers clean-up and enrichment of proteins in one step. Complete automation has been realized in the form of a unique robotic instrumentation that is able to extract and transfer 96 samples onto standard MALDI target plates with high throughput. The developed platform was operated with a 60 sample turnaround per hour allowing sensitivities in femtomol regions of medium- and low-abundant target proteins from clinical studies on samples of multiple sclerosis and gastroesophageal reflux disease. Several proteins have been identified as new biomarkers from cerebrospinal fluid and esophagus epithelial cells.

Bioengineering approaches to controlled protein delivery.

PubMed

Kobsa, Serge; Saltzman, W Mark

2008-05-01

Proteins are of crucial importance in all biologic organisms, in terms of both structure and function. Their deficits play central roles in many pathologic states, and their potential as powerful therapeutic agents has been widely recognized. Many issues, however, exist in delivery of biologically active proteins to target tissues and organs. Recent advances in biomedical engineering have lead to development of advanced techniques for controlled delivery of peptides and proteins, paving the way for their efficient use in treating human injury and disease. With a particular emphasis on most recent advances, this review discusses currently available techniques for controlled delivery of proteins and considers future research directions.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

The RAS Problem: Turning Off a Broken Switch

Cancer.gov

The RAS gene is commonly mutated in cancer and researchers are working to better understand how to develop drugs that can target the RAS protein, which for many years has been considered to be “undruggable.”

Complex network theory for the identification and assessment of candidate protein targets.

PubMed

McGarry, Ken; McDonald, Sharon

2018-06-01

In this work we use complex network theory to provide a statistical model of the connectivity patterns of human proteins and their interaction partners. Our intention is to identify important proteins that may be predisposed to be potential candidates as drug targets for therapeutic interventions. Target proteins usually have more interaction partners than non-target proteins, but there are no hard-and-fast rules for defining the actual number of interactions. We devise a statistical measure for identifying hub proteins, we score our target proteins with gene ontology annotations. The important druggable protein targets are likely to have similar biological functions that can be assessed for their potential therapeutic value. Our system provides a statistical analysis of the local and distant neighborhood protein interactions of the potential targets using complex network measures. This approach builds a more accurate model of drug-to-target activity and therefore the likely impact on treating diseases. We integrate high quality protein interaction data from the HINT database and disease associated proteins from the DrugTarget database. Other sources include biological knowledge from Gene Ontology and drug information from DrugBank. The problem is a very challenging one since the data is highly imbalanced between target proteins and the more numerous nontargets. We use undersampling on the training data and build Random Forest classifier models which are used to identify previously unclassified target proteins. We validate and corroborate these findings from the available literature. Copyright © 2018 Elsevier Ltd. All rights reserved.

Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications.

PubMed

Mizukami, Shin; Hori, Yuichiro; Kikuchi, Kazuya

2014-01-21

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

[G protein-coupled receptors in the spot light].

PubMed

Benleulmi-Chaachoua, Abla; Wojciech, Stefanie; Jockers, Ralf

2013-01-01

G protein-coupled receptors (GPCRs), also known as seven transmembrane domain-spanning proteins (7TM), play an important role in tissue homeostasis and cellular and hormonal communication. GPCRs are targeted by a large panel of natural ligands such as photons, ions, metabolites, lipids and proteins but also by numerous drugs. Research efforts in the GPCR field have been rewarded in 2012 by the Nobel Price in Chemistry. The present article briefly summarizes our current knowledge on GPCRs and discusses future challenges in terms of fundamental aspects and therapeutic applications. © Société de Biologie, 2013.

[Protein carbamylation: what it is and why it concerns nephrologists].

PubMed

De Simone, Emanuele; Di Micco, Lucia; La Manna, Gaetano; Di Iorio, Biagio

2018-05-01

Spontaneous urea dissociation in water solution is a prominent source of protein carbamylation in our body. Protein carbamylation is a well-known phenomenon since early seventies. Some years ago, much interest in the diagnostic power of carbamylated protein arouse. Recently the target of the researches focused on its potential cardiovascular pathogenicity. Some authors claimed that this could be a reason for higher cardiovascular mortality in uremic patients. Nutritional therapy, amino acids supplementation and intensive dialysis regimen are some of the therapeutic tools tested to lower the carbamylation burst in this population. Copyright by Società Italiana di Nefrologia SIN, Rome, Italy.

HMPAS: Human Membrane Protein Analysis System

PubMed Central

2013-01-01

Background Membrane proteins perform essential roles in diverse cellular functions and are regarded as major pharmaceutical targets. The significance of membrane proteins has led to the developing dozens of resources related with membrane proteins. However, most of these resources are built for specific well-known membrane protein groups, making it difficult to find common and specific features of various membrane protein groups. Methods We collected human membrane proteins from the dispersed resources and predicted novel membrane protein candidates by using ortholog information and our membrane protein classifiers. The membrane proteins were classified according to the type of interaction with the membrane, subcellular localization, and molecular function. We also made new feature dataset to characterize the membrane proteins in various aspects including membrane protein topology, domain, biological process, disease, and drug. Moreover, protein structure and ICD-10-CM based integrated disease and drug information was newly included. To analyze the comprehensive information of membrane proteins, we implemented analysis tools to identify novel sequence and functional features of the classified membrane protein groups and to extract features from protein sequences. Results We constructed HMPAS with 28,509 collected known membrane proteins and 8,076 newly predicted candidates. This system provides integrated information of human membrane proteins individually and in groups organized by 45 subcellular locations and 1,401 molecular functions. As a case study, we identified associations between the membrane proteins and diseases and present that membrane proteins are promising targets for diseases related with nervous system and circulatory system. A web-based interface of this system was constructed to facilitate researchers not only to retrieve organized information of individual proteins but also to use the tools to analyze the membrane proteins. Conclusions HMPAS provides comprehensive information about human membrane proteins including specific features of certain membrane protein groups. In this system, user can acquire the information of individual proteins and specified groups focused on their conserved sequence features, involved cellular processes, and diseases. HMPAS may contribute as a valuable resource for the inference of novel cellular mechanisms and pharmaceutical targets associated with the human membrane proteins. HMPAS is freely available at http://fcode.kaist.ac.kr/hmpas. PMID:24564858

Structural Genomics and Drug Discovery for Infectious Diseases

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

Anderson, W.F.

The application of structural genomics methods and approaches to proteins from organisms causing infectious diseases is making available the three dimensional structures of many proteins that are potential drug targets and laying the groundwork for structure aided drug discovery efforts. There are a number of structural genomics projects with a focus on pathogens that have been initiated worldwide. The Center for Structural Genomics of Infectious Diseases (CSGID) was recently established to apply state-of-the-art high throughput structural biology technologies to the characterization of proteins from the National Institute for Allergy and Infectious Diseases (NIAID) category A-C pathogens and organisms causing emerging,more » or re-emerging infectious diseases. The target selection process emphasizes potential biomedical benefits. Selected proteins include known drug targets and their homologs, essential enzymes, virulence factors and vaccine candidates. The Center also provides a structure determination service for the infectious disease scientific community. The ultimate goal is to generate a library of structures that are available to the scientific community and can serve as a starting point for further research and structure aided drug discovery for infectious diseases. To achieve this goal, the CSGID will determine protein crystal structures of 400 proteins and protein-ligand complexes using proven, rapid, highly integrated, and cost-effective methods for such determination, primarily by X-ray crystallography. High throughput crystallographic structure determination is greatly aided by frequent, convenient access to high-performance beamlines at third-generation synchrotron X-ray sources.« less

Transient receptor potential canonical 4 and 5 proteins as targets in cancer therapeutics.

PubMed

Gaunt, Hannah J; Vasudev, Naveen S; Beech, David J

2016-10-01

Novel approaches towards cancer therapy are urgently needed. One approach might be to target ion channels mediating Ca 2+ entry because of the critical roles played by Ca 2+ in many cell types, including cancer cells. There are several types of these ion channels, but here we address those formed by assembly of transient receptor potential canonical (TRPC) proteins, particularly those which involve two closely related members of the family: TRPC4 and TRPC5. We focus on these proteins because recent studies point to roles in important aspects of cancer: drug resistance, transmission of drug resistance through extracellular vesicles, tumour vascularisation, and evoked cancer cell death by the TRPC4/5 channel activator (-)-englerin A. We conclude that further research is both justified and necessary before these proteins can be considered as strong targets for anti-cancer cell drug discovery programmes. It is nevertheless already apparent that inhibitors of the channels would be unlikely to cause significant adverse effects, but, rather, have other effects which may be beneficial in the context of cancer and chemotherapy, potentially including suppression of innate fear, visceral pain and pathological cardiac remodelling.

A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

PubMed

Agrotis, Alexander; Ketteler, Robin

2015-01-01

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

Biomolecular engineering of intracellular switches in eukaryotes

PubMed Central

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

2010-01-01

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

Of monkeys and men: immunomic profiling of sera from humans and non-human primates resistant to schistosomiasis reveals novel potential vaccine candidates.

PubMed

Pearson, Mark S; Becker, Luke; Driguez, Patrick; Young, Neil D; Gaze, Soraya; Mendes, Tiago; Li, Xiao-Hong; Doolan, Denise L; Midzi, Nicholas; Mduluza, Takafira; McManus, Donald P; Wilson, R Alan; Bethony, Jeffrey M; Nausch, Norman; Mutapi, Francisca; Felgner, Philip L; Loukas, Alex

2015-01-01

Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR) to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80), tetraspanins, glutathione-S-transferases, and glucose transporters (SGTP1), as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to S. japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognized by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens.

[Programmed necrosis mediated by receptor-interacting protein 3: a new target for liver disease research].

PubMed

Zhang, J; Jing, Y; Li, Y N; Zhou, L; Wang, B M

2016-09-20

Hepatocyte death mainly includes apoptosis and necrosis and is a critical process in the pathophysiological mechanism of liver injury caused by various reasons. Recent studies have shown that key regulatory molecules in the inhibition of apoptosis such as caspase cannot be used as targets for inhibiting disease progression in clinical practice. In recent years, programmed necrosis mediated by receptor-interacting protein 3(RIP3)becomes a new hot research topic. It not only plays an important role in inducing inflammatory response, but also is closely regulated by intracellular signal factors, and it is a type of active cell death which can be interfered with. Compared with apoptosis, programmed necrosis is accompanied by the release of various inflammatory factors, which significantly affects local immune microenvironment. RIP3-mediated programmed necrosis has been taken seriously in many diseases. Although its mechanism of action in liver disease remains unclear, the results of recent studies confirmed its important role in the development of liver disease. This article reviews the research advances in the role of RIP3-mediated programmed necrosis signaling pathway in liver disease of various causes and investigates the possibility of RIP3-mediated programmed necrosis as a new target in the treatment of liver disease.

The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

PubMed Central

Kunze, Markus; Berger, Johannes

2015-01-01

The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s) encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes. PMID:26441678

Computational design of protein interactions: designing proteins that neutralize influenza by inhibiting its hemagglutinin surface protein

NASA Astrophysics Data System (ADS)

Fleishman, Sarel

2012-02-01

Molecular recognition underlies all life processes. Design of interactions not seen in nature is a test of our understanding of molecular recognition and could unlock the vast potential of subtle control over molecular interaction networks, allowing the design of novel diagnostics and therapeutics for basic and applied research. We developed the first general method for designing protein interactions. The method starts by computing a region of high affinity interactions between dismembered amino acid residues and the target surface and then identifying proteins that can harbor these residues. Designs are tested experimentally for binding the target surface and successful ones are affinity matured using yeast cell surface display. Applied to the conserved stem region of influenza hemagglutinin we designed two unrelated proteins that, following affinity maturation, bound hemagglutinin at subnanomolar dissociation constants. Co-crystal structures of hemagglutinin bound to the two designed binders were within 1Angstrom RMSd of their models, validating the accuracy of the design strategy. One of the designed proteins inhibits the conformational changes that underlie hemagglutinin's cell-invasion functions and blocks virus infectivity in cell culture, suggesting that such proteins may in future serve as diagnostics and antivirals against a wide range of pathogenic influenza strains. We have used this method to obtain experimentally validated binders of several other target proteins, demonstrating the generality of the approach. We discuss the combination of modeling and high-throughput characterization of design variants which has been key to the success of this approach, as well as how we have used the data obtained in this project to enhance our understanding of molecular recognition. References: Science 332:816 JMB, in press Protein Sci 20:753

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

PubMed

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

2014-12-28

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

PharmMapper 2017 update: a web server for potential drug target identification with a comprehensive target pharmacophore database.

PubMed

Wang, Xia; Shen, Yihang; Wang, Shiwei; Li, Shiliang; Zhang, Weilin; Liu, Xiaofeng; Lai, Luhua; Pei, Jianfeng; Li, Honglin

2017-07-03

The PharmMapper online tool is a web server for potential drug target identification by reversed pharmacophore matching the query compound against an in-house pharmacophore model database. The original version of PharmMapper includes more than 7000 target pharmacophores derived from complex crystal structures with corresponding protein target annotations. In this article, we present a new version of the PharmMapper web server, of which the backend pharmacophore database is six times larger than the earlier one, with a total of 23 236 proteins covering 16 159 druggable pharmacophore models and 51 431 ligandable pharmacophore models. The expanded target data cover 450 indications and 4800 molecular functions compared to 110 indications and 349 molecular functions in our last update. In addition, the new web server is united with the statistically meaningful ranking of the identified drug targets, which is achieved through the use of standard scores. It also features an improved user interface. The proposed web server is freely available at http://lilab.ecust.edu.cn/pharmmapper/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory

PubMed Central

Morris, Chris; Pajon, Anne; Griffiths, Susanne L.; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M.; Wilter da Silva, Alan; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S.; Stuart, David I.; Henrick, Kim; Esnouf, Robert M.

2011-01-01

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service. PMID:21460443

Recent Progress and Development of Crystal Structure Analysis of Enzymes and Other Proteins

NASA Astrophysics Data System (ADS)

Tanokura, Masaru; Nagata, Koji; Miyazono, Ken-Ichi; Miyakawa, Takuya; Okai, Masahiko

Structural biology has made tremendous progress in this decade. Here we briefly introduce the Target Proteins Research Program, a national project promoted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. The program aims to reveal the structure and function of proteins that are of great importance in both academic research and industrial application. We also summarize the results of structure-function analyses of (i) transcriptional regulatory proteins useful for the breading of drought and heat stress tolerant crops, (ii) useful enzymes for the production of chiral compounds, and (iii) useful enzymes for the degradation of environmental pollution substances. These results can be utilized in various areas of industries, to enhance food production, to improve the efficiency of pharmaceutical compound production, and to promote the bioremediation of contaminated soil and water.

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.

PubMed

Morris, Chris; Pajon, Anne; Griffiths, Susanne L; Daniel, Ed; Savitsky, Marc; Lin, Bill; Diprose, Jonathan M; da Silva, Alan Wilter; Pilicheva, Katya; Troshin, Peter; van Niekerk, Johannes; Isaacs, Neil; Naismith, James; Nave, Colin; Blake, Richard; Wilson, Keith S; Stuart, David I; Henrick, Kim; Esnouf, Robert M

2011-04-01

The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.

Open reading frames associated with cancer in the dark matter of the human genome.

PubMed

Delgado, Ana Paula; Brandao, Pamela; Chapado, Maria Julia; Hamid, Sheilin; Narayanan, Ramaswamy

2014-01-01

The uncharacterized proteins (open reading frames, ORFs) in the human genome offer an opportunity to discover novel targets for cancer. A systematic analysis of the dark matter of the human proteome for druggability and biomarker discovery is crucial to mining the genome. Numerous data mining tools are available to mine these ORFs to develop a comprehensive knowledge base for future target discovery and validation. Using the Genetic Association Database, the ORFs of the human dark matter proteome were screened for evidence of association with neoplasms. The Phenome-Genome Integrator tool was used to establish phenotypic association with disease traits including cancer. Batch analysis of the tools for protein expression analysis, gene ontology and motifs and domains was used to characterize the ORFs. Sixty-two ORFs were identified for neoplasm association. The expression Quantitative Trait Loci (eQTL) analysis identified thirteen ORFs related to cancer traits. Protein expression, motifs and domain analysis and genome-wide association studies verified the relevance of these OncoORFs in diverse tumors. The OncoORFs are also associated with a wide variety of human diseases and disorders. Our results link the OncoORFs to diverse diseases and disorders. This suggests a complex landscape of the uncharacterized proteome in human diseases. These results open the dark matter of the proteome to novel cancer target research. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

NASA Astrophysics Data System (ADS)

Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

2016-03-01

Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2

PubMed Central

Ma, Yufei; Wang, Haiming; Yan, Dan; Wei, Yanquan; Cao, Yuhua; Yi, Peiwei; Zhang, Hailu; Deng, Zongwu; Dai, Jianwu; Liu, Xiangtao; Luo, Jianxun; Zhang, Zhijun; Sun, Shiqi; Guo, Huichen

2016-01-01

Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research. PMID:26996514

Signaling networks in TMPRSS2-ERG positive prostate cancers: Do we need a Pied Piper or sharpshooter to deal with "at large" fused oncoprotein.

PubMed

Lin, X; Qureshi, M Z; Romero, M A; Yaylim, I; Arif, S; Ucak, I; Fayyaz, S; Farooqi, A A; Mansoor, Q; Ismail, M

2017-02-28

Overwhelmingly increasing scientific evidence has provided near complete resolution of prostate cancer landscape and it is now more understandable that wide ranging factors underlies its development and progression. Increasingly it is being realized that genetic/epigenetic factors, Intra-tumoral and inter-tumoral heterogeneity, loss of apoptosis, dysregulations of spatio-temporally controlled signaling cascades, Darwinian evolution in response to therapeutic pressures play instrumental role in prostate carcinogenesis. Moreover, multi-directional patterns of spread between primary tumors and metastatic sites have also been studied extensively in prostate cancer. Research over the years has gradually and systematically revealed closer association between tumor phenotype and type of gene fusion. Latest developments in deep sequencing technologies have shown that gene fusions originate in a non-random, cell type dependent manner and are much more frequent than previously surmised. These findings enabled sub-classification and categorization of seemingly identical diseases. Furthermore, research methodologies have shown that many gene fusions inform us about risk stratification and many chimeric proteins encoded by the fused genes are being studied as drug target/s. We partition this multi-component review into the molecular basis of formation of fusion transcripts, how protein network is regulated in fusion positive prostate cancer cells and therapeutic strategies which are currently being investigated to efficiently target fusion transcript and its protein product.

Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

PubMed

Mielecki, Marcin; Lesyng, Bogdan

2016-01-01

Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

Properties of Protein Drug Target Classes

PubMed Central

Bull, Simon C.; Doig, Andrew J.

2015-01-01

Accurate identification of drug targets is a crucial part of any drug development program. We mined the human proteome to discover properties of proteins that may be important in determining their suitability for pharmaceutical modulation. Data was gathered concerning each protein’s sequence, post-translational modifications, secondary structure, germline variants, expression profile and drug target status. The data was then analysed to determine features for which the target and non-target proteins had significantly different values. This analysis was repeated for subsets of the proteome consisting of all G-protein coupled receptors, ion channels, kinases and proteases, as well as proteins that are implicated in cancer. Machine learning was used to quantify the proteins in each dataset in terms of their potential to serve as a drug target. This was accomplished by first inducing a random forest that could distinguish between its targets and non-targets, and then using the random forest to quantify the drug target likeness of the non-targets. The properties that can best differentiate targets from non-targets were primarily those that are directly related to a protein’s sequence (e.g. secondary structure). Germline variants, expression levels and interactions between proteins had minimal discriminative power. Overall, the best indicators of drug target likeness were found to be the proteins’ hydrophobicities, in vivo half-lives, propensity for being membrane bound and the fraction of non-polar amino acids in their sequences. In terms of predicting potential targets, datasets of proteases, ion channels and cancer proteins were able to induce random forests that were highly capable of distinguishing between targets and non-targets. The non-target proteins predicted to be targets by these random forests comprise the set of the most suitable potential future drug targets, and should therefore be prioritised when building a drug development programme. PMID:25822509

Proteolytic activity during senescence of plants

NASA Technical Reports Server (NTRS)

Huffaker, R. C.

1990-01-01

Although information has rapidly developed concerning the intracellular localization of plant proteins, relatively few reports concern the intracellular location of endo- and exo-proteolytic activities. Relatively few proteases have been purified, characterized, and associated with a specific cellular location. With the exception of the processing proteases involved in transport of proteins across membranes, little progress has yet been made concerning determination of in vivo products of specific proteases. Information on the turnover of individual proteins and the assessment of rate-limiting steps in pathways as proteins are turned over is steadily appearing. Since chloroplasts are the major site of both protein synthesis and, during senescence, degradation, it was important to show unambiguously that chloroplasts can degrade their own constituents. Another important contribution was to obtain evidence that the chloroplasts contain proteases capable of degrading their constituents. This work has been more tenuous because of the low activities found and the possibility of contamination by vacuolar enzymes during the isolation of organelles. The possible targeting of cytoplasmic proteins for degradation by facilitating their transport into vacuoles is a field which hopefully will develop more rapidly in the future. Information on targeting of proteins for degradation via the ubiquitin (Ub) degradation pathway is developing rapidly. Future research must determine how much unity exists across the different eukaryotic systems. At present, it has important implications for protein turnover in plants, since apparently Ub is involved in the degradation of phytochrome. Little information has been developed regarding what triggers increased proteolysis with the onset of senescence, although it appears to involve protein synthesis. Thus far, the evidence indicates that the complement of proteases prior to senescence is sufficient to carry out the observed protein degradation. This field of study has great practical implications, e.g. maintaining photosynthesis during seed-fill in order to obtain greater crop yields. The current use of stay green' variants in the populations of several crop plants to produce increased yields shows the potential for future development. The near future should see exciting discoveries in these areas of research that will have far reaching effects on the construction of transgenic plants for future research accomplishments and agricultural use.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

PubMed

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A strategy to find novel candidate anti-Alzheimer's disease drugs by constructing interaction networks between drug targets and natural compounds in medical plants.

PubMed

Chen, Bi-Wen; Li, Wen-Xing; Wang, Guang-Hui; Li, Gong-Hua; Liu, Jia-Qian; Zheng, Jun-Juan; Wang, Qian; Li, Hui-Juan; Dai, Shao-Xing; Huang, Jing-Fei

2018-01-01

Alzheimer' disease (AD) is an ultimately fatal degenerative brain disorder that has an increasingly large burden on health and social care systems. There are only five drugs for AD on the market, and no new effective medicines have been discovered for many years. Chinese medicinal plants have been used to treat diseases for thousands of years, and screening herbal remedies is a way to develop new drugs. We used molecular docking to screen 30,438 compounds from Traditional Chinese Medicine (TCM) against a comprehensive list of AD target proteins. TCM compounds in the top 0.5% of binding affinity scores for each target protein were selected as our research objects. Structural similarities between existing drugs from DrugBank database and selected TCM compounds as well as the druggability of our candidate compounds were studied. Finally, we searched the CNKI database to obtain studies on anti-AD Chinese plants from 2007 to 2017, and only clinical studies were included. A total of 1,476 compounds (top 0.5%) were selected as drug candidates. Most of these compounds are abundantly found in plants used for treating AD in China, especially the plants from two genera Panax and Morus. We classified the compounds by single target and multiple targets and analyzed the interactions between target proteins and compounds. Analysis of structural similarity revealed that 17 candidate anti-AD compounds were structurally identical to 14 existing approved drugs. Most of them have been reported to have a positive effect in AD. After filtering for compound druggability, we identified 11 anti-AD compounds with favorable properties, seven of which are found in anti-AD Chinese plants. Of 11 anti-AD compounds, four compounds 5,862, 5,863, 5,868, 5,869 have anti-inflammatory activity. The compound 28,814 mainly has immunoregulatory activity. The other six compounds have not yet been reported for any biology activity at present. Natural compounds from TCM provide a broad prospect for the screening of anti-AD drugs. In this work, we established networks to systematically study the connections among natural compounds, approved drugs, TCM plants and AD target proteins with the goal of identifying promising drug candidates. We hope that our study will facilitate in-depth research for the treatment of AD in Chinese medicine.

Molecular Tools for Facilitative Carbohydrate Transporters (Gluts).

PubMed

Tanasova, Marina; Fedie, Joseph R

2017-09-19

Facilitative carbohydrate transporters-Gluts-have received wide attention over decades due to their essential role in nutrient uptake and links with various metabolic disorders, including diabetes, obesity, and cancer. Endeavors directed towards understanding the mechanisms of Glut-mediated nutrient uptake have resulted in a multidisciplinary research field spanning protein chemistry, chemical biology, organic synthesis, crystallography, and biomolecular modeling. Gluts became attractive targets for cancer research and medicinal chemistry, leading to the development of new approaches to cancer diagnostics and providing avenues for cancer-targeting therapeutics. In this review, the current state of knowledge of the molecular interactions behind Glut-mediated sugar uptake, Glut-targeting probes, therapeutics, and inhibitors are discussed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics in cardiovascular research: global and targeted strategies

PubMed Central

Shen, Xiaomeng; Young, Rebeccah; Canty, John M.; Qu, Jun

2014-01-01

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation. PMID:24920501

Mind the GAP: A Novel Tumor-Promoting Mechanism | Center for Cancer Research

Cancer.gov

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

Drug-Target Interactions: Prediction Methods and Applications.

PubMed

Anusuya, Shanmugam; Kesherwani, Manish; Priya, K Vishnu; Vimala, Antonydhason; Shanmugam, Gnanendra; Velmurugan, Devadasan; Gromiha, M Michael

2018-01-01

Identifying the interactions between drugs and target proteins is a key step in drug discovery. This not only aids to understand the disease mechanism, but also helps to identify unexpected therapeutic activity or adverse side effects of drugs. Hence, drug-target interaction prediction becomes an essential tool in the field of drug repurposing. The availability of heterogeneous biological data on known drug-target interactions enabled many researchers to develop various computational methods to decipher unknown drug-target interactions. This review provides an overview on these computational methods for predicting drug-target interactions along with available webservers and databases for drug-target interactions. Further, the applicability of drug-target interactions in various diseases for identifying lead compounds has been outlined. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target.

PubMed

Imbert, Isabelle; Botto, Jean-Marie; Farra, Claude D; Domloge, Nouha

2012-06-01

Telomere shortening is considered as one of the main characteristics of cellular aging by limiting cellular division. Besides the fundamental advances through the discoveries of telomere and telomerase, which were recognized by a Nobel Prize, telomere protection remains an essential area of research. Recently, it was evidenced that studying the cross-talks between the proteins associated with telomere should provide a better understanding of the mechanistic basis for telomere-associated aging phenotypes. In this review, we discuss the current knowledge on telomere shortening, telomerase activity, and the essential role of telomere binding proteins in telomere stabilization and telomere-end protection. This review highlights the capacity of telomere binding proteins to limit cellular senescence and to maintain skin tissue homeostasis, which is of key importance to reduce accelerated tissue aging. Future studies addressing telomere protection and limitation of DNA damage response in human skin should include investigations on telomere binding proteins. As little is known about the expression of telomere binding proteins in human skin and modulation of their expression with aging, it remains an interesting field of skin research and a key area for future skin protection and anti-aging developments. © 2012 Wiley Periodicals, Inc.

The Protein Structure Initiative Structural Biology Knowledgebase Technology Portal: a structural biology web resource.

PubMed

Gifford, Lida K; Carter, Lester G; Gabanyi, Margaret J; Berman, Helen M; Adams, Paul D

2012-06-01

The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/ ) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB ( http://sbkb.org ), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a functional sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.

Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications.

PubMed

Jimenez, Connie R; Verheul, Henk M W

2014-01-01

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

Perspectives on pathway perturbation: Focused research to enhance 3R objectives

EPA Science Inventory

In vitro high-throughput screening (HTS) and in silico technologies are emerging as 21st century tools for hazard identification. Computational methods that strategically examine cross-species conservation of protein sequence/structural information for chemical molecular targets ...

Molecular insights into cancer therapeutic effects of the dietary medicinal phytochemical withaferin A.

PubMed

Chirumamilla, Chandra Sekhar; Pérez-Novo, Claudina; Van Ostade, Xaveer; Vanden Berghe, Wim

2017-05-01

Despite the worldwide research efforts to combat cancer, it remains a leading cause of death. Although various specific kinase inhibitors already have been approved for clinical cancer treatment, occurrence of intrinsic or acquired resistance and intermittent response over longer periods limits long-term success of single kinase-targeted therapies. In this respect, there is a renewed interest in polypharmaceutical natural compounds, which simultaneously target various hyperactivated kinases involved in tumour-inflammation, angiogenesis, cell survival, proliferation, metastasis and angiogenesis. The dietary medicinal phytochemical withaferin A (WA), isolated from Withaferin somnifera (popular Indian name Ashwagandha), holds promise as a novel anti-cancer agent, which targets multiple cell survival kinase pathways, including IκB kinase/NF-κB, PI3 kinase/protein kinase B/mammalian target of rapamycin and mitogen-activated protein kinase/extracellular signal-regulated kinase amongst others. In this review, we propose a novel mechanism of WA-dependent kinase inhibition via electrophilic covalent targeting of cysteine residues in conserved kinase activation domains (kinase cysteinome), which could underlie its pleiotropic therapeutic effects in cancer signalling.

Computational Studies of Snake Venom Toxins

PubMed Central

Ojeda, Paola G.; Caballero, Julio; Kaas, Quentin; González, Wendy

2017-01-01

Most snake venom toxins are proteins, and participate to envenomation through a diverse array of bioactivities, such as bleeding, inflammation, and pain, cytotoxic, cardiotoxic or neurotoxic effects. The venom of a single snake species contains hundreds of toxins, and the venoms of the 725 species of venomous snakes represent a large pool of potentially bioactive proteins. Despite considerable discovery efforts, most of the snake venom toxins are still uncharacterized. Modern bioinformatics tools have been recently developed to mine snake venoms, helping focus experimental research on the most potentially interesting toxins. Some computational techniques predict toxin molecular targets, and the binding mode to these targets. This review gives an overview of current knowledge on the ~2200 sequences, and more than 400 three-dimensional structures of snake toxins deposited in public repositories, as well as of molecular modeling studies of the interaction between these toxins and their molecular targets. We also describe how modern bioinformatics have been used to study the snake venom protein phospholipase A2, the small basic myotoxin Crotamine, and the three-finger peptide Mambalgin. PMID:29271884

Peptide-based antibody alternatives for biological sensing in austere environments

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

2017-02-01

The most critical component of a biosensor, the biorecognition element, must exhibit high selectivity and strong affinity for a target of interest in operational sensing. Monoclonal antibodies are the current standard reagents for such devices, but their adaptability, manufacturability, and stability greatly limit their effectiveness in fieldable sensors. Peptides have emerged as potential antibody replacements in such applications due to their similar binding performance, extreme chemical and thermal stabilities, and on-demand scalability. In conjunction with modeling capabilities, work at the Army Research Lab focuses on protein catalyzed capture (PCC) agent technology and bacterial display for the discovery of these novel peptide binding reagents. The synthetic, bottom-up PCC agent technology uses an iterative, in situ "click chemistry" approach to produce high performing peptides against specific epitopes translatable to the protein target. Bacterial display allows rapid reagent discovery due to the combination of fast bacterial growth and effective peptide sequence enrichment through multiple rounds of biopanning. Recent advances in both methods are highlighted in regards to the discovery of reagents against Army high priority protein targets for soldier safety, performance, and diagnostics.

A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells.

PubMed

Cao, Jicong; Arha, Manish; Sudrik, Chaitanya; Mukherjee, Abhirup; Wu, Xia; Kane, Ravi S

2015-04-30

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein-RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5' untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A quick reality check for microRNA target prediction.

PubMed

Kast, Juergen

2011-04-01

The regulation of protein abundance by microRNA (miRNA)-mediated repression of mRNA translation is a rapidly growing area of interest in biochemical research. In animal cells, the miRNA seed sequence does not perfectly match that of the mRNA it targets, resulting in a large number of possible miRNA targets and varied extents of repression. Several software tools are available for the prediction of miRNA targets, yet the overlap between them is limited. Jovanovic et al. have developed and applied a targeted, quantitative approach to validate predicted miRNA target proteins. Using a proteome database, they have set up and tested selected reaction monitoring assays for approximately 20% of more than 800 predicted let-7 targets, as well as control genes in Caenorhabditis elegans. Their results demonstrate that such assays can be developed quickly and with relative ease, and applied in a high-throughput setup to verify known and identify novel miRNA targets. They also show, however, that the choice of the biological system and material has a noticeable influence on the frequency, extent and direction of the observed changes. Nonetheless, selected reaction monitoring assays, such as those developed by Jovanovic et al., represent an attractive new tool in the study of miRNA function at the organism level.

RFDT: A Rotation Forest-based Predictor for Predicting Drug-Target Interactions Using Drug Structure and Protein Sequence Information.

PubMed

Wang, Lei; You, Zhu-Hong; Chen, Xing; Yan, Xin; Liu, Gang; Zhang, Wei

2018-01-01

Identification of interaction between drugs and target proteins plays an important role in discovering new drug candidates. However, through the experimental method to identify the drug-target interactions remain to be extremely time-consuming, expensive and challenging even nowadays. Therefore, it is urgent to develop new computational methods to predict potential drugtarget interactions (DTI). In this article, a novel computational model is developed for predicting potential drug-target interactions under the theory that each drug-target interaction pair can be represented by the structural properties from drugs and evolutionary information derived from proteins. Specifically, the protein sequences are encoded as Position-Specific Scoring Matrix (PSSM) descriptor which contains information of biological evolutionary and the drug molecules are encoded as fingerprint feature vector which represents the existence of certain functional groups or fragments. Four benchmark datasets involving enzymes, ion channels, GPCRs and nuclear receptors, are independently used for establishing predictive models with Rotation Forest (RF) model. The proposed method achieved the prediction accuracy of 91.3%, 89.1%, 84.1% and 71.1% for four datasets respectively. In order to make our method more persuasive, we compared our classifier with the state-of-theart Support Vector Machine (SVM) classifier. We also compared the proposed method with other excellent methods. Experimental results demonstrate that the proposed method is effective in the prediction of DTI, and can provide assistance for new drug research and development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

PubMed Central

Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

2014-01-01

The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success. PMID:25341935

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

PubMed Central

Crowther, Gregory J.; Hillesland, Heidi K.; Keyloun, Katelyn R.; Reid, Molly C.; Lafuente-Monasterio, Maria Jose; Ghidelli-Disse, Sonja; Leonard, Stephen E.; He, Panqing; Jones, Jackson C.; Krahn, Mallory M.; Mo, Jack S.; Dasari, Kartheek S.; Fox, Anna M. W.; Boesche, Markus; El Bakkouri, Majida; Rivas, Kasey L.; Leroy, Didier; Hui, Raymond; Drewes, Gerard; Maly, Dustin J.; Van Voorhis, Wesley C.; Ojo, Kayode K.

2016-01-01

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible. PMID:26934697

Toward Omics-Based, Systems Biomedicine, and Path and Drug Discovery Methodologies for Depression-Inflammation Research.

PubMed

Maes, Michael; Nowak, Gabriel; Caso, Javier R; Leza, Juan Carlos; Song, Cai; Kubera, Marta; Klein, Hans; Galecki, Piotr; Noto, Cristiano; Glaab, Enrico; Balling, Rudi; Berk, Michael

2016-07-01

Meta-analyses confirm that depression is accompanied by signs of inflammation including increased levels of acute phase proteins, e.g., C-reactive protein, and pro-inflammatory cytokines, e.g., interleukin-6. Supporting the translational significance of this, a meta-analysis showed that anti-inflammatory drugs may have antidepressant effects. Here, we argue that inflammation and depression research needs to get onto a new track. Firstly, the choice of inflammatory biomarkers in depression research was often too selective and did not consider the broader pathways. Secondly, although mild inflammatory responses are present in depression, other immune-related pathways cannot be disregarded as new drug targets, e.g., activation of cell-mediated immunity, oxidative and nitrosative stress (O&NS) pathways, autoimmune responses, bacterial translocation, and activation of the toll-like receptor and neuroprogressive pathways. Thirdly, anti-inflammatory treatments are sometimes used without full understanding of their effects on the broader pathways underpinning depression. Since many of the activated immune-inflammatory pathways in depression actually confer protection against an overzealous inflammatory response, targeting these pathways may result in unpredictable and unwanted results. Furthermore, this paper discusses the required improvements in research strategy, i.e., path and drug discovery processes, omics-based techniques, and systems biomedicine methodologies. Firstly, novel methods should be employed to examine the intracellular networks that control and modulate the immune, O&NS and neuroprogressive pathways using omics-based assays, including genomics, transcriptomics, proteomics, metabolomics, epigenomics, immunoproteomics and metagenomics. Secondly, systems biomedicine analyses are essential to unravel the complex interactions between these cellular networks, pathways, and the multifactorial trigger factors and to delineate new drug targets in the cellular networks or pathways. Drug discovery processes should delineate new drugs targeting the intracellular networks and immune-related pathways.

Protein degradation machinery is present broadly during early development in the sea urchin.

PubMed

Zazueta-Novoa, Vanesa; Wessel, Gary M

2014-07-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Protein degradation machinery is present broadly during early development in the sea urchin

PubMed Central

Zazueta-Novoa, Vanesa; Wessel, Gary M.

2014-01-01

Ubiquitin-dependent proteosome-mediated proteolysis is an important pathway of degradation that controls the timed destruction of cellular proteins in all tissues. All intracellular proteins and many extracellular proteins are continually being hydrolyzed to their constituent amino acids as a result of their recognition by E3 ligases for specific targeting of ubiquitination. Gustavus is a member of an ECS-type E3 ligase which interacts with Vasa, a DEAD-box RNA helicase, to regulate its localization during sea urchin embryonic development, and Gustavus mRNA accumulation is highly localized and dynamic during development. We tested if the core complex for Gustavus function was present in the embryo and if other SOCS box proteins also had restricted expression profiles that would inform future research. Expression patterns of the key members of the proteasomal function, such as the E3 core complex which interacts with Gustavus, and other E3-SOCS box proteins, are widely spread and dynamic in early development of the embryo suggesting broad core complex availability in the proteasome degradation pathway and temporal/spatial enrichments of various E3 ligase dependent targeting mechanisms. PMID:24963879

Protein Tyrosine Nitration during Development and Abiotic Stress Response in Plants

PubMed Central

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

2016-01-01

In recent years, the study of nitric oxide (NO) in plant systems has attracted the attention of many researchers. A growing number of investigations have shown the significance of NO as a signal molecule or as a molecule involved in the response against (a)biotic processes. NO can be responsible of the post-translational modifications (NO-PTM) of target proteins by mechanisms such as the nitration of tyrosine residues. The study of protein tyrosine nitration during development and under biotic and adverse environmental conditions has increased in the last decade; nevertheless, there is also an endogenous nitration which seems to have regulatory functions. Moreover, the advance in proteome techniques has enabled the identification of new nitrated proteins, showing the high variability among plant organs, development stage and species. Finally, it may be important to discern between a widespread protein nitration because of greater RNS content, and the specific nitration of key targets which could affect cell-signaling processes. In view of the above point, we present a mini-review that offers an update about the endogenous protein tyrosine nitration, during plant development and under several abiotic stress conditions. PMID:27895655

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

Chemokine Signaling in Allergic Contact Dermatitis: Toward Targeted Therapies.

PubMed

Smith, Jeffrey S; Rajagopal, Sudarshan; Atwater, Amber Reck

2018-06-22

Allergic contact dermatitis (ACD) is a common skin disease that results in significant cost and morbidity. Despite its high prevalence, therapeutic options are limited. Allergic contact dermatitis is regulated primarily by T cells within the adaptive immune system, but also by natural killer and innate lymphoid cells within the innate immune system. The chemokine receptor system, consisting of chemokine peptides and chemokine G protein-coupled receptors, is a critical regulator of inflammatory processes such as ACD. Specific chemokine signaling pathways are selectively up-regulated in ACD, most prominently CXCR3 and its endogenous chemokines CXCL9, CXCL10, and CXCL11. Recent research demonstrates that these 3 chemokines are not redundant and indeed activate distinct intracellular signaling profiles such as those activated by heterotrimeric G proteins and β-arrestin adapter proteins. Such differential signaling provides an attractive therapeutic target for novel ACD therapies and other inflammatory diseases.

The power and promise of "rewiring" the mitogen-activated protein kinase network in prostate cancer therapeutics.

PubMed

Papatsoris, Athanasios G; Karamouzis, Michalis V; Papavassiliou, Athanasios G

2007-03-01

Prostate cancer is the most frequently diagnosed cancer among men and the second leading cause of male cancer deaths. Initially, tumor growth is androgen dependent and thus responsive to pharmacologic androgen deprivation, but there is a high rate of treatment failure because the disease evolves in an androgen-independent state. Growing evidence suggests that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade represents a pivotal molecular circuitry participating directly or indirectly in prostate cancer evolution. The crucial role of the protein elements comprising this complex signal transduction network makes them potential targets for pharmacologic interference. Here, we will delineate the current knowledge regarding the involvement of the Ras/MAPK pathway in prostate carcinogenesis, spotlight ongoing research concerning the development of novel targeted agents such as the Ras/MAPK inhibitors in prostate cancer, and discuss the future perspectives of their therapeutic efficacy.

[GPCRs heterodimerization: a new way towards the discovery of function for the orphan receptors?].

PubMed

Levoye, Angélique; Jockers, Ralf

2007-01-01

G protein-coupled receptors (GPCRs), also called seven transmembrane domain (7TM) proteins, represent the largest family of cell surface receptors. GPCRs control a variety of physiological processes, are involved in multiple diseases and are major drug targets. Despite a vast effort of academic and industrial research, more than one hundred receptors remain orphans. These orphan GPCRs offer a great potential for drug discovery, as almost 60% of currently prescribed drugs target GPCRs. Deorphenization strategies have concentrated mainly on the identification of the natural ligands of these proteins. Recent advances have shown that orphan GPCRs, similar to orphan nuclear receptors, can regulate the function of non-orphan receptors by heterodimerization. These findings not only help to better understand the extraordinary diversity of GPCRs, but also open new perspectives for the identification of the function of these orphan receptors that hold great therapeutic potential.

GPCRs and EGFR - Cross-talk of membrane receptors in cancer.

PubMed

Köse, Meryem

2017-08-15

G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) are two important classes of cell surface receptors proven to be highly tractable as drug targets. Both receptor classes are involved in various complex (patho-) physiological processes in the human body including cellular growth and differentiation. More recently, accumulating data suggest that GPCR-induced activation of EGFR, the prototyp of RTKs represents a major mechanism in various cancers. The present review will focus on this cross-talk with particular emphasis on intracellular scaffold proteins regulating EGFR transactivation. It will give an overview about the current status of the research and future directions, highlight recent trends in the field, and discuss the potential of therapeutic strategies combining GPCR and EGFR targeting on the one hand and specific targeting of the cross-talk on the other hand in cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

PubMed Central

Tycko, Josh; Myer, Vic E.; Hsu, Patrick D.

2016-01-01

Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

Non-canonical modulators of nuclear receptors.

PubMed

Tice, Colin M; Zheng, Ya-Jun

2016-09-01

Like G protein-coupled receptors (GPCRs) and protein kinases, nuclear receptors (NRs) are a rich source of pharmaceutical targets. Over 80 NR-targeting drugs have been approved for 18 NRs. The focus of drug discovery in NRs has hitherto been on identifying ligands that bind to the canonical ligand binding pockets of the C-terminal ligand binding domains (LBDs). Due to the development of drug resistance and selectivity concerns, there has been considerable interest in exploring other, non-canonical ligand binding sites. Unfortunately, the potencies of compounds binding at other sites have generally not been sufficient for clinical development. However, the situation has changed dramatically over the last 3years, as compounds with sufficient potency have been reported for several NR targets. Here we review recent developments in this area from a medicinal chemistry point of view in the hope of stimulating further interest in this area of research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity.

PubMed

Chen, Lianguo; Au, Doris W T; Hu, Chenyan; Peterson, Drew R; Zhou, Bingsheng; Qian, Pei-Yuan

2017-02-07

Environmental pollutants are capable of concomitantly inducing diverse toxic effects. However, it is largely unknown which effects are directly induced and which effects are secondary, thus calling for definitive identification of the initiating molecular event for a pollutant to elucidate the mechanism of toxicity. In the present study, affinity pull-down assays were used to identify target proteins for 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT), a costal pollutant of emerging concern, in various tissues (e.g., brain, liver, plasma, and gonad) from marine medaka (Oryzias melastigma) and zebrafish (Danio rerio). Pull-down results showed that, in male and female brains from medaka and zebrafish, DCOIT had a consistently high affinity for G protein alpha subunits (Gα), suggesting the targeted effects of DCOIT on signaling transduction from G protein-coupled receptors (GPCRs) and an extrapolatable mode of action in teleost brains. Validation using recombinant proteins and molecular docking analysis confirmed that binding of DCOIT to Gα protein competitively inhibited its activation by substrate. Considering the involvement of GPCRs in the regulation of myriad biological processes, including the hypothalamus-pituitary-gonadal-liver axis, binding of DCOIT to upstream Gα proteins in the brain may provide a plausible explanation for the diversity of toxic effects resulting from DCOIT challenge, especially abnormal hormonal production through the mitogen-activated protein kinase pathway. A new mechanism of action based on GPCR signaling is thus hypothesized for endocrine disrupting chemicals and warrants further research to clearly elucidate the link between GPCR signaling and endocrine disruption.

Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress

PubMed Central

2015-01-01

Protein structure underlies essential biological processes and provides a blueprint for molecular mimicry that drives drug discovery. Although small molecules represent the lion’s share of agents that target proteins for therapeutic benefit, there remains no substitute for the natural properties of proteins and their peptide subunits in the majority of biological contexts. The peptide α-helix represents a common structural motif that mediates communication between signaling proteins. Because peptides can lose their shape when taken out of context, developing chemical interventions to stabilize their bioactive structure remains an active area of research. The all-hydrocarbon staple has emerged as one such solution, conferring α-helical structure, protease resistance, cellular penetrance, and biological activity upon successful incorporation of a series of design and application principles. Here, we describe our more than decade-long experience in developing stapled peptides as biomedical research tools and prototype therapeutics, highlighting lessons learned, pitfalls to avoid, and keys to success. PMID:24601557

Gene targeting in embryonic stem cells, II: conditional technologies

USDA-ARS?s Scientific Manuscript database

Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

PubMed

Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

2016-02-19

Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

PubMed

Kroj, Thomas; Chanclud, Emilie; Michel-Romiti, Corinne; Grand, Xavier; Morel, Jean-Benoit

2016-04-01

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

PubMed Central

2013-01-01

Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their protein expression levels were within a range of 0.637-101 fmol/μg protein. The largest difference in the levels between the three strains was 2.2-fold for 13 molecules, although bcrp and mct1 displayed statistically significant differences between C57BL/6J and the other strain(s). Highly sensitive simultaneous absolute quantification achieved by QTAP will increase the usefulness of proteomics in biological sciences and is expected to advance the new research field of pharmacoproteomics (PPx). PMID:23758935

Targets of drugs are generally, and targets of drugs having side effects are specifically good spreaders of human interactome perturbations.

PubMed

Perez-Lopez, Áron R; Szalay, Kristóf Z; Türei, Dénes; Módos, Dezső; Lenti, Katalin; Korcsmáros, Tamás; Csermely, Peter

2015-05-11

Network-based methods are playing an increasingly important role in drug design. Our main question in this paper was whether the efficiency of drug target proteins to spread perturbations in the human interactome is larger if the binding drugs have side effects, as compared to those which have no reported side effects. Our results showed that in general, drug targets were better spreaders of perturbations than non-target proteins, and in particular, targets of drugs with side effects were also better spreaders of perturbations than targets of drugs having no reported side effects in human protein-protein interaction networks. Colorectal cancer-related proteins were good spreaders and had a high centrality, while type 2 diabetes-related proteins showed an average spreading efficiency and had an average centrality in the human interactome. Moreover, the interactome-distance between drug targets and disease-related proteins was higher in diabetes than in colorectal cancer. Our results may help a better understanding of the network position and dynamics of drug targets and disease-related proteins, and may contribute to develop additional, network-based tests to increase the potential safety of drug candidates.

Targets of drugs are generally, and targets of drugs having side effects are specifically good spreaders of human interactome perturbations

NASA Astrophysics Data System (ADS)

Perez-Lopez, Áron R.; Szalay, Kristóf Z.; Türei, Dénes; Módos, Dezső; Lenti, Katalin; Korcsmáros, Tamás; Csermely, Peter

2015-05-01

Network-based methods are playing an increasingly important role in drug design. Our main question in this paper was whether the efficiency of drug target proteins to spread perturbations in the human interactome is larger if the binding drugs have side effects, as compared to those which have no reported side effects. Our results showed that in general, drug targets were better spreaders of perturbations than non-target proteins, and in particular, targets of drugs with side effects were also better spreaders of perturbations than targets of drugs having no reported side effects in human protein-protein interaction networks. Colorectal cancer-related proteins were good spreaders and had a high centrality, while type 2 diabetes-related proteins showed an average spreading efficiency and had an average centrality in the human interactome. Moreover, the interactome-distance between drug targets and disease-related proteins was higher in diabetes than in colorectal cancer. Our results may help a better understanding of the network position and dynamics of drug targets and disease-related proteins, and may contribute to develop additional, network-based tests to increase the potential safety of drug candidates.

Targets of drugs are generally, and targets of drugs having side effects are specifically good spreaders of human interactome perturbations

PubMed Central

Perez-Lopez, Áron R.; Szalay, Kristóf Z.; Türei, Dénes; Módos, Dezső; Lenti, Katalin; Korcsmáros, Tamás; Csermely, Peter

2015-01-01

Network-based methods are playing an increasingly important role in drug design. Our main question in this paper was whether the efficiency of drug target proteins to spread perturbations in the human interactome is larger if the binding drugs have side effects, as compared to those which have no reported side effects. Our results showed that in general, drug targets were better spreaders of perturbations than non-target proteins, and in particular, targets of drugs with side effects were also better spreaders of perturbations than targets of drugs having no reported side effects in human protein-protein interaction networks. Colorectal cancer-related proteins were good spreaders and had a high centrality, while type 2 diabetes-related proteins showed an average spreading efficiency and had an average centrality in the human interactome. Moreover, the interactome-distance between drug targets and disease-related proteins was higher in diabetes than in colorectal cancer. Our results may help a better understanding of the network position and dynamics of drug targets and disease-related proteins, and may contribute to develop additional, network-based tests to increase the potential safety of drug candidates. PMID:25960144

Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms.

PubMed Central

Maeda, Kenji; Finnie, Christine; Svensson, Birte

2004-01-01

Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds. PMID:14636158

Computer applications making rapid advances in high throughput microbial proteomics (HTMP).

PubMed

Anandkumar, Balakrishna; Haga, Steve W; Wu, Hui-Fen

2014-02-01

The last few decades have seen the rise of widely-available proteomics tools. From new data acquisition devices, such as MALDI-MS and 2DE to new database searching softwares, these new products have paved the way for high throughput microbial proteomics (HTMP). These tools are enabling researchers to gain new insights into microbial metabolism, and are opening up new areas of study, such as protein-protein interactions (interactomics) discovery. Computer software is a key part of these emerging fields. This current review considers: 1) software tools for identifying the proteome, such as MASCOT or PDQuest, 2) online databases of proteomes, such as SWISS-PROT, Proteome Web, or the Proteomics Facility of the Pathogen Functional Genomics Resource Center, and 3) software tools for applying proteomic data, such as PSI-BLAST or VESPA. These tools allow for research in network biology, protein identification, functional annotation, target identification/validation, protein expression, protein structural analysis, metabolic pathway engineering and drug discovery.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

PubMed Central

Schnepf, E.; Crickmore, N.; Van Rie, J.; Lereclus, D.; Baum, J.; Feitelson, J.; Zeigler, D. R.; Dean, D. H.

1998-01-01

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit. PMID:9729609

Precision medicine driven by cancer systems biology.

PubMed

Filipp, Fabian V

2017-03-01

Molecular insights from genome and systems biology are influencing how cancer is diagnosed and treated. We critically evaluate big data challenges in precision medicine. The melanoma research community has identified distinct subtypes involving chronic sun-induced damage and the mitogen-activated protein kinase driver pathway. In addition, despite low mutation burden, non-genomic mitogen-activated protein kinase melanoma drivers are found in membrane receptors, metabolism, or epigenetic signaling with the ability to bypass central mitogen-activated protein kinase molecules and activating a similar program of mitogenic effectors. Mutation hotspots, structural modeling, UV signature, and genomic as well as non-genomic mechanisms of disease initiation and progression are taken into consideration to identify resistance mutations and novel drug targets. A comprehensive precision medicine profile of a malignant melanoma patient illustrates future rational drug targeting strategies. Network analysis emphasizes an important role of epigenetic and metabolic master regulators in oncogenesis. Co-occurrence of driver mutations in signaling, metabolic, and epigenetic factors highlights how cumulative alterations of our genomes and epigenomes progressively lead to uncontrolled cell proliferation. Precision insights have the ability to identify independent molecular pathways suitable for drug targeting. Synergistic treatment combinations of orthogonal modalities including immunotherapy, mitogen-activated protein kinase inhibitors, epigenetic inhibitors, and metabolic inhibitors have the potential to overcome immune evasion, side effects, and drug resistance.

Of Monkeys and Men: Immunomic Profiling of Sera from Humans and Non-Human Primates Resistant to Schistosomiasis Reveals Novel Potential Vaccine Candidates

PubMed Central

Pearson, Mark S.; Becker, Luke; Driguez, Patrick; Young, Neil D.; Gaze, Soraya; Mendes, Tiago; Li, Xiao-Hong; Doolan, Denise L.; Midzi, Nicholas; Mduluza, Takafira; McManus, Donald P.; Wilson, R. Alan; Bethony, Jeffrey M.; Nausch, Norman; Mutapi, Francisca; Felgner, Philip L.; Loukas, Alex

2015-01-01

Schistosoma haematobium affects more than 100 million people throughout Africa and is the causative agent of urogenital schistosomiasis. The parasite is strongly associated with urothelial cancer in infected individuals and as such is designated a group I carcinogen by the International Agency for Research on Cancer. Using a protein microarray containing schistosome proteins, we sought to identify antigens that were the targets of protective IgG1 immune responses in S. haematobium-exposed individuals that acquire drug-induced resistance (DIR) to schistosomiasis after praziquantel treatment. Numerous antigens with known vaccine potential were identified, including calpain (Smp80), tetraspanins, glutathione-S-transferases, and glucose transporters (SGTP1), as well as previously uncharacterized proteins. Reactive IgG1 responses were not elevated in exposed individuals who did not acquire DIR. To complement our human subjects study, we screened for antigen targets of rhesus macaques rendered resistant to S. japonicum by experimental infection followed by self-cure, and discovered a number of new and known vaccine targets, including major targets recognized by our human subjects. This study has further validated the immunomics-based approach to schistosomiasis vaccine antigen discovery and identified numerous novel potential vaccine antigens. PMID:25999951

Potential of apoptotic pathway-targeted cancer therapeutic research: Where do we stand?

PubMed Central

Baig, S; Seevasant, I; Mohamad, J; Mukheem, A; Huri, H Z; Kamarul, T

2016-01-01

Underneath the intricacy of every cancer lies mysterious events that impel the tumour cell and its posterity into abnormal growth and tissue invasion. Oncogenic mutations disturb the regulatory circuits responsible for the governance of versatile cellular functions, permitting tumour cells to endure deregulated proliferation, resist to proapoptotic insults, invade and erode normal tissues and above all escape apoptosis. This disruption of apoptosis has been highly implicated in various malignancies and has been exploited as an anticancer strategy. Owing to the fact that apoptosis causes minimal inflammation and damage to the tissue, apoptotic cell death-based therapy has been the centre of attraction for the development of anticancer drugs. Increased understanding of the molecular pathways underlying apoptosis has enabled scientists to establish unique approaches targeting apoptosis pathways in cancer therapeutics. In this review, we reconnoitre the two major pathways (intrinsic and extrinsic) targeted cancer therapeutics, steering toward chief modulators of these pathways, such as B-cell lymphoma 2 protein family members (pro- and antiapoptotic), inhibitor of apoptosis proteins, and the foremost thespian of extrinsic pathway regulator, tumour necrosis factor-related apoptosis-inducing agent. Together, we also will have a look from clinical perspective to address the agents (drugs) and therapeutic strategies adopted to target these specific proteins/pathways that have entered clinical trials. PMID:26775709

An integrated structure- and system-based framework to identify new targets of metabolites and known drugs

PubMed Central

Naveed, Hammad; Hameed, Umar S.; Harrus, Deborah; Bourguet, William; Arold, Stefan T.; Gao, Xin

2015-01-01

Motivation: The inherent promiscuity of small molecules towards protein targets impedes our understanding of healthy versus diseased metabolism. This promiscuity also poses a challenge for the pharmaceutical industry as identifying all protein targets is important to assess (side) effects and repositioning opportunities for a drug. Results: Here, we present a novel integrated structure- and system-based approach of drug-target prediction (iDTP) to enable the large-scale discovery of new targets for small molecules, such as pharmaceutical drugs, co-factors and metabolites (collectively called ‘drugs’). For a given drug, our method uses sequence order–independent structure alignment, hierarchical clustering and probabilistic sequence similarity to construct a probabilistic pocket ensemble (PPE) that captures promiscuous structural features of different binding sites on known targets. A drug’s PPE is combined with an approximation of its delivery profile to reduce false positives. In our cross-validation study, we use iDTP to predict the known targets of 11 drugs, with 63% sensitivity and 81% specificity. We then predicted novel targets for these drugs—two that are of high pharmacological interest, the peroxisome proliferator-activated receptor gamma and the oncogene B-cell lymphoma 2, were successfully validated through in vitro binding experiments. Our method is broadly applicable for the prediction of protein-small molecule interactions with several novel applications to biological research and drug development. Availability and implementation: The program, datasets and results are freely available to academic users at http://sfb.kaust.edu.sa/Pages/Software.aspx. Contact: xin.gao@kaust.edu.sa and stefan.arold@kaust.edu.sa Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26286808

Structure of the Get3 targeting factor in complex with its membrane protein cargo

DOE PAGES

Mateja, Agnieszka; Paduch, Marcin; Chang, Hsin-Yang; ...

2015-03-06

Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins (GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. In this paper, we reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans themore » Get3 homodimer. Finally, our data elucidate the mechanism of TA protein recognition and shielding by Get3 and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors.« less

Comprehensive predictions of target proteins based on protein-chemical interaction using virtual screening and experimental verifications.

PubMed

Kobayashi, Hiroki; Harada, Hiroko; Nakamura, Masaomi; Futamura, Yushi; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-Ichiro; Shin-Ya, Kazuo; Doi, Takayuki; Takahashi, Takashi; Natsume, Tohru; Imoto, Masaya; Sakakibara, Yasubumi

2012-04-05

Identification of the target proteins of bioactive compounds is critical for elucidating the mode of action; however, target identification has been difficult in general, mostly due to the low sensitivity of detection using affinity chromatography followed by CBB staining and MS/MS analysis. We applied our protocol of predicting target proteins combining in silico screening and experimental verification for incednine, which inhibits the anti-apoptotic function of Bcl-xL by an unknown mechanism. One hundred eighty-two target protein candidates were computationally predicted to bind to incednine by the statistical prediction method, and the predictions were verified by in vitro binding of incednine to seven proteins, whose expression can be confirmed in our cell system.As a result, 40% accuracy of the computational predictions was achieved successfully, and we newly found 3 incednine-binding proteins. This study revealed that our proposed protocol of predicting target protein combining in silico screening and experimental verification is useful, and provides new insight into a strategy for identifying target proteins of small molecules.

Inter-kingdom prediction certainty evaluation of protein subcellular localization tools: microbial pathogenesis approach for deciphering host microbe interaction.

PubMed

Khan, Abdul Arif; Khan, Zakir; Kalam, Mohd Abul; Khan, Azmat Ali

2018-01-01

Microbial pathogenesis involves several aspects of host-pathogen interactions, including microbial proteins targeting host subcellular compartments and subsequent effects on host physiology. Such studies are supported by experimental data, but recent detection of bacterial proteins localization through computational eukaryotic subcellular protein targeting prediction tools has also come into practice. We evaluated inter-kingdom prediction certainty of these tools. The bacterial proteins experimentally known to target host subcellular compartments were predicted with eukaryotic subcellular targeting prediction tools, and prediction certainty was assessed. The results indicate that these tools alone are not sufficient for inter-kingdom protein targeting prediction. The correct prediction of pathogen's protein subcellular targeting depends on several factors, including presence of localization signal, transmembrane domain and molecular weight, etc., in addition to approach for subcellular targeting prediction. The detection of protein targeting in endomembrane system is comparatively difficult, as the proteins in this location are channelized to different compartments. In addition, the high specificity of training data set also creates low inter-kingdom prediction accuracy. Current data can help to suggest strategy for correct prediction of bacterial protein's subcellular localization in host cell. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

Butterfield, D Allan; Perluigi, Marzia

2017-03-01

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

Quantification of non-coding RNA target localization diversity and its application in cancers.

PubMed

Cheng, Lixin; Leung, Kwong-Sak

2018-04-01

Subcellular localization is pivotal for RNAs and proteins to implement biological functions. The localization diversity of protein interactions has been studied as a crucial feature of proteins, considering that the protein-protein interactions take place in various subcellular locations. Nevertheless, the localization diversity of non-coding RNA (ncRNA) target proteins has not been systematically studied, especially its characteristics in cancers. In this study, we provide a new algorithm, non-coding RNA target localization coefficient (ncTALENT), to quantify the target localization diversity of ncRNAs based on the ncRNA-protein interaction and protein subcellular localization data. ncTALENT can be used to calculate the target localization coefficient of ncRNAs and measure how diversely their targets are distributed among the subcellular locations in various scenarios. We focus our study on long non-coding RNAs (lncRNAs), and our observations reveal that the target localization diversity is a primary characteristic of lncRNAs in different biotypes. Moreover, we found that lncRNAs in multiple cancers, differentially expressed cancer lncRNAs, and lncRNAs with multiple cancer target proteins are prone to have high target localization diversity. Furthermore, the analysis of gastric cancer helps us to obtain a better understanding that the target localization diversity of lncRNAs is an important feature closely related to clinical prognosis. Overall, we systematically studied the target localization diversity of the lncRNAs and uncovered its association with cancer.

Protein turnover measurement using selected reaction monitoring-mass spectrometry (SRM-MS)

PubMed Central

Holman, Stephen W.; Hammond, Dean E.; Simpson, Deborah M.; Waters, John; Hurst, Jane L.

2016-01-01

Protein turnover represents an important mechanism in the functioning of cells, with deregulated synthesis and degradation of proteins implicated in many diseased states. Therefore, proteomics strategies to measure turnover rates with high confidence are of vital importance to understanding many biological processes. In this study, the more widely used approach of non-targeted precursor ion signal intensity (MS1) quantification is compared with selected reaction monitoring (SRM), a data acquisition strategy that records data for specific peptides, to determine if improved quantitative data would be obtained using a targeted quantification approach. Using mouse liver as a model system, turnover measurement of four tricarboxylic acid cycle proteins was performed using both MS1 and SRM quantification strategies. SRM outperformed MS1 in terms of sensitivity and selectivity of measurement, allowing more confident determination of protein turnover rates. SRM data are acquired using cheaper and more widely available tandem quadrupole mass spectrometers, making the approach accessible to a larger number of researchers than MS1 quantification, which is best performed on high mass resolution instruments. SRM acquisition is ideally suited to focused studies where the turnover of tens of proteins is measured, making it applicable in determining the dynamics of proteins complexes and complete metabolic pathways. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644981

Facile Method for the Site-Specific, Covalent Attachment of full-length IgG onto Nanoparticles

PubMed Central

Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T.

2014-01-01

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzycyclooctyne-modified nanoparticles, via copper-free click chemistry. PMID:24729432

Facile method for the site-specific, covalent attachment of full-length IgG onto nanoparticles.

PubMed

Hui, James Zhe; Al Zaki, Ajlan; Cheng, Zhiliang; Popik, Vladimir; Zhang, Hongtao; Luning Prak, Eline T; Tsourkas, Andrew

2014-08-27

Antibodies, most commonly IgGs, have been widely used as targeting ligands in research and therapeutic applications due to their wide array of targets, high specificity and proven efficacy. Many of these applications require antibodies to be conjugated onto surfaces (e.g. nanoparticles and microplates); however, most conventional bioconjugation techniques exhibit low crosslinking efficiencies, reduced functionality due to non-site-specific labeling and random surface orientation, and/or require protein engineering (e.g. cysteine handles), which can be technically challenging. To overcome these limitations, we have recombinantly expressed Protein Z, which binds the Fc region of IgG, with an UV active non-natural amino acid benzoylphenyalanine (BPA) within its binding domain. Upon exposure to long wavelength UV light, the BPA is activated and forms a covalent link between the Protein Z and the bound Fc region of IgG. This technology was combined with expressed protein ligation (EPL), which allowed for the introduction of a fluorophore and click chemistry-compatible azide group onto the C-terminus of Protein Z during the recombinant protein purification step. This enabled the crosslinked-Protein Z-IgG complexes to be efficiently and site-specifically attached to aza-dibenzocyclooctyne-modified nanoparticles, via copper-free click chemistry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The nonstructural proteins of Pneumoviruses are remarkably distinct in substrate diversity and specificity.

PubMed

Ribaudo, Michael; Barik, Sailen

2017-11-06

Interferon (IFN) inhibits viruses by inducing several hundred cellular genes, aptly named 'interferon (IFN)-stimulated genes' (ISGs). The only two RNA viruses of the Pneumovirus genus of the Paramyxoviridae family, namely Respiratory Syncytial Virus (RSV) and Pneumonia Virus of Mice (PVM), each encode two nonstructural (NS) proteins that share no sequence similarity but yet suppress IFN. Since suppression of IFN underlies the ability of these viruses to replicate in the host cells, the mechanism of such suppression has become an important area of research. This Short Report is an important extension of our previous efforts in defining this mechanism. We show that, like their PVM counterparts, the RSV NS proteins also target multiple members of the ISG family. While significantly extending the substrate repertoire of the RSV NS proteins, these results, unexpectedly, also reveal that the target preferences of the NS proteins of the two viruses are entirely different. This is surprising since the two Pneumoviruses are phylogenetically close with similar genome organization and gene function, and the NS proteins of both also serve as suppressors of host IFN response. The finding that the NS proteins of the two highly similar viruses suppress entirely different members of the ISG family raises intriguing questions of pneumoviral NS evolution and mechanism of action.

Tier-1 assays for assessing the toxicity of insecticidal proteins produced by genetically engineered plants to non-target arthropods.

PubMed

Li, Yun-He; Romeis, Jörg; Wu, Kong-Ming; Peng, Yu-Fa

2014-04-01

In assessing an insect-resistant genetically engineered (IRGE) crop before its commercialization, researchers normally use so-called "Tier-1 assays" as the initial step to determine the effects of the crop on non-target organisms. In these tests, the insecticidal proteins (IPs) produced by the IRGEs are added to the diets of test organisms in the laboratory. Test organisms in such assays can be directly exposed to much higher concentrations of the test IPs than they would encounter in the field. The results of Tier-1 assays are thus more conservative than those generated in studies in which the organisms are exposed to the IPs by feeding on IRGE plant tissue or in the case of predators or parasites, by feeding on invertebrate prey or hosts that have fed on IRGE plant tissue. In this report, we consider three important factors that must be considered in Tier-1 assays: (i) methods for delivery of the IP to the test organisms; (ii) the need for and selection of compounds used as positive controls; and (iii) methods for monitoring the concentration, stability and bioactivity of the IP during the assay. We also analyze the existing data from Tier-1 assays regarding the toxicity of Bt Cry proteins to non-target arthropod species. The data indicate that the widely used Bt proteins have no direct toxicity to non-target organisms. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron‐specific antigens

PubMed Central

Vojdani, Aristo

2016-01-01

Abstract Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co‐occurrence of anti‐MBP and anti‐MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA‐human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. PMID:27610592

VaProS: a database-integration approach for protein/genome information retrieval.

PubMed

Gojobori, Takashi; Ikeo, Kazuho; Katayama, Yukie; Kawabata, Takeshi; Kinjo, Akira R; Kinoshita, Kengo; Kwon, Yeondae; Migita, Ohsuke; Mizutani, Hisashi; Muraoka, Masafumi; Nagata, Koji; Omori, Satoshi; Sugawara, Hideaki; Yamada, Daichi; Yura, Kei

2016-12-01

Life science research now heavily relies on all sorts of databases for genome sequences, transcription, protein three-dimensional (3D) structures, protein-protein interactions, phenotypes and so forth. The knowledge accumulated by all the omics research is so vast that a computer-aided search of data is now a prerequisite for starting a new study. In addition, a combinatory search throughout these databases has a chance to extract new ideas and new hypotheses that can be examined by wet-lab experiments. By virtually integrating the related databases on the Internet, we have built a new web application that facilitates life science researchers for retrieving experts' knowledge stored in the databases and for building a new hypothesis of the research target. This web application, named VaProS, puts stress on the interconnection between the functional information of genome sequences and protein 3D structures, such as structural effect of the gene mutation. In this manuscript, we present the notion of VaProS, the databases and tools that can be accessed without any knowledge of database locations and data formats, and the power of search exemplified in quest of the molecular mechanisms of lysosomal storage disease. VaProS can be freely accessed at http://p4d-info.nig.ac.jp/vapros/ .

From Bioengineering to CRISPR/Cas9 – A Personal Retrospective of 20 Years of Research in Programmable Genome Targeting

PubMed Central

Jeltsch, Albert

2018-01-01

Genome targeting of restriction enzymes and DNA methyltransferases has many important applications including genome and epigenome editing. 15–20 years ago, my group was involved in the development of approaches for programmable genome targeting, aiming to connect enzymes with an oligodeoxynucleotide (ODN), which could form a sequence-specific triple helix at the genomic target site. Importantly, the target site of such enzyme-ODN conjugate could be varied simply by altering the ODN sequence promising great applicative values. However, this approach was facing many problems including the preparation and purification of the enzyme-ODN conjugates, their efficient delivery into cells, slow kinetics of triple helix formation and the requirement of a poly-purine target site sequence. Hence, for several years genome and epigenome editing approaches mainly were based on Zinc fingers and TAL proteins as targeting devices. More recently, CRISPR/Cas systems were discovered, which use a bound RNA for genome targeting that forms an RNA/DNA duplex with one DNA strand of the target site. These systems combine all potential advantages of the once imagined enzyme-ODN conjugates and avoid all main disadvantageous. Consequently, the application of CRISPR/Cas in genome and epigenome editing has exploded in recent years. We can draw two important conclusions from this example of research history. First, evolution still is the better bioengineer than humans and, whenever tested in parallel, natural solutions outcompete engineered ones. Second, CRISPR/Cas system were discovered in pure, curiosity driven, basic research, highlighting that it is basic, bottom-up research paving the way for fundamental innovation. PMID:29434619

Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da; Zhou, Feifan; Wang, Huiying; Tang, Yonghong

2009-03-01

The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin αvβ3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin αvβ3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin αvβ3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

Targeting activator protein 1 signaling pathway by bioactive natural agents: Possible therapeutic strategy for cancer prevention and intervention.

PubMed

Tewari, Devesh; Nabavi, Seyed Fazel; Nabavi, Seyed Mohammad; Sureda, Antoni; Farooqi, Ammad Ahmad; Atanasov, Atanas G; Vacca, Rosa Anna; Sethi, Gautam; Bishayee, Anupam

2018-02-01

Activator protein 1 (AP-1) is a key transcription factor in the control of several cellular processes responsible for cell survival proliferation and differentiation. Dysfunctional AP-1 expression and activity are involved in several severe diseases, especially inflammatory disorders and cancer. Therefore, targeting AP-1 has recently emerged as an attractive therapeutic strategy for cancer prevention and therapy. This review summarizes our current understanding of AP-1 biology and function as well as explores and discusses several natural bioactive compounds modulating AP-1-associated signaling pathways for cancer prevention and intervention. Current limitations, challenges, and future directions of research are also critically discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering Proteins for Thermostability with iRDP Web Server

PubMed Central

Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C. G.

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements. PMID:26436543

Engineering Proteins for Thermostability with iRDP Web Server.

PubMed

Panigrahi, Priyabrata; Sule, Manas; Ghanate, Avinash; Ramasamy, Sureshkumar; Suresh, C G

2015-01-01

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

T Cells that Recognize HPV Protein Can Target Virus-Infected Cells | Center for Cancer Research

Cancer.gov

Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

PubMed Central

Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Histone Deacetylase Inhibitors as a Novel Targeted Therapy Against Non-small Cell Lung Cancer: Where Are We Now and What Should We Expect?

PubMed

Damaskos, Christos; Tomos, Ioannis; Garmpis, Nikolaos; Karakatsani, Anna; Dimitroulis, Dimitrios; Garmpi, Anna; Spartalis, Eleftherios; Kampolis, Christos F; Tsagkari, Eleni; Loukeri, Angeliki A; Margonis, Georgios-Antonios; Spartalis, Michael; Andreatos, Nikolaos; Schizas, Dimitrios; Kokkineli, Stefania; Antoniou, Efstathios A; Nonni, Afroditi; Tsourouflis, Gerasimos; Markatos, Konstantinos; Kontzoglou, Konstantinos; Kostakis, Alkiviadis; Tomos, Periklis

2018-01-01

Non-small cell lung cancer constitutes the most common type of lung cancer, accounting for 85-90% of lung cancer, and is a leading cause of cancer-related death. Despite the progress during the past years, poor prognosis remains a challenge and requires further research and development of novel antitumor treatment. Recently, the role of histone deacetylases in gene expression has emerged showing their regulation of the acetylation of histone proteins and other non-histone protein targets and their role in chromatin organization, while their inhibitors, the histone deacetylase inhibitors, have been proposed to have a potential therapeutic role in diverse malignancies, including non-small cell lung cancer. This review article focuses on the role of histone deacetylase inhibitors in the treatment of non-small cell lung cancer and the major molecular mechanisms underlying their antitumor activity recognized so far. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

PubMed Central

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-01-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. PMID:24792163

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

PubMed

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

2016-03-18

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Approaches to achieve high-level heterologous protein production in plants.

PubMed

Streatfield, Stephen J

2007-01-01

Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies.

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

PubMed

Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

2018-01-16

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of bioinformatics tools to their full potential to analyse protein-protein interactions as a comprehensive network. Copyright © 2017 Elsevier B.V. All rights reserved.

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

PubMed Central

Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

2015-01-01

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

PubMed

Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

2014-07-16

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.

Protein-Polymer Conjugates: Synthetic Approaches by Controlled Radical Polymerizations & Interesting Applications

PubMed Central

Grover, Gregory N.; Maynard, Heather D.

2011-01-01

Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, a key aspect in therapeutic design, anchoring conjugates to surfaces, and utilizing these materials for supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to make protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted. PMID:21071260

UniDrug-target: a computational tool to identify unique drug targets in pathogenic bacteria.

PubMed

Chanumolu, Sree Krishna; Rout, Chittaranjan; Chauhan, Rajinder S

2012-01-01

Targeting conserved proteins of bacteria through antibacterial medications has resulted in both the development of resistant strains and changes to human health by destroying beneficial microbes which eventually become breeding grounds for the evolution of resistances. Despite the availability of more than 800 genomes sequences, 430 pathways, 4743 enzymes, 9257 metabolic reactions and protein (three-dimensional) 3D structures in bacteria, no pathogen-specific computational drug target identification tool has been developed. A web server, UniDrug-Target, which combines bacterial biological information and computational methods to stringently identify pathogen-specific proteins as drug targets, has been designed. Besides predicting pathogen-specific proteins essentiality, chokepoint property, etc., three new algorithms were developed and implemented by using protein sequences, domains, structures, and metabolic reactions for construction of partial metabolic networks (PMNs), determination of conservation in critical residues, and variation analysis of residues forming similar cavities in proteins sequences. First, PMNs are constructed to determine the extent of disturbances in metabolite production by targeting a protein as drug target. Conservation of pathogen-specific protein's critical residues involved in cavity formation and biological function determined at domain-level with low-matching sequences. Last, variation analysis of residues forming similar cavities in proteins sequences from pathogenic versus non-pathogenic bacteria and humans is performed. The server is capable of predicting drug targets for any sequenced pathogenic bacteria having fasta sequences and annotated information. The utility of UniDrug-Target server was demonstrated for Mycobacterium tuberculosis (H37Rv). The UniDrug-Target identified 265 mycobacteria pathogen-specific proteins, including 17 essential proteins which can be potential drug targets. UniDrug-Target is expected to accelerate pathogen-specific drug targets identification which will increase their success and durability as drugs developed against them have less chance to develop resistances and adverse impact on environment. The server is freely available at http://117.211.115.67/UDT/main.html. The standalone application (source codes) is available at http://www.bioinformatics.org/ftp/pub/bioinfojuit/UDT.rar.

Computer-based prediction of mitochondria-targeting peptides.

PubMed

Martelli, Pier Luigi; Savojardo, Castrense; Fariselli, Piero; Tasco, Gianluca; Casadio, Rita

2015-01-01

Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. Subcellular localization is a feature necessary for understanding the protein role and the compartment where the mature protein is active and very difficult to characterize experimentally. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is possible to recognize a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with experimentally characterized targeting peptides. Furthermore, we illustrate our newly implemented web server and its usage on the whole human proteome in order to infer mitochondrial targeting peptides, their cleavage sites, and whether the targeting peptide regions contain or not arginine-rich recurrent motifs. By this, we add some other 2,800 human proteins to the 124 ones already experimentally annotated with a mitochondrial targeting peptide.

Characterization of the targeting signal in mitochondrial β-barrel proteins

PubMed Central

Jores, Tobias; Klinger, Anna; Groß, Lucia E.; Kawano, Shin; Flinner, Nadine; Duchardt-Ferner, Elke; Wöhnert, Jens; Kalbacher, Hubert; Endo, Toshiya; Schleiff, Enrico; Rapaport, Doron

2016-01-01

Mitochondrial β-barrel proteins are synthesized on cytosolic ribosomes and must be specifically targeted to the organelle before their integration into the mitochondrial outer membrane. The signal that assures such precise targeting and its recognition by the organelle remained obscure. In the present study we show that a specialized β-hairpin motif is this long searched for signal. We demonstrate that a synthetic β-hairpin peptide competes with the import of mitochondrial β-barrel proteins and that proteins harbouring a β-hairpin peptide fused to passenger domains are targeted to mitochondria. Furthermore, a β-hairpin motif from mitochondrial proteins targets chloroplast β-barrel proteins to mitochondria. The mitochondrial targeting depends on the hydrophobicity of the β-hairpin motif. Finally, this motif interacts with the mitochondrial import receptor Tom20. Collectively, we reveal that β-barrel proteins are targeted to mitochondria by a dedicated β-hairpin element, and this motif is recognized at the organelle surface by the outer membrane translocase. PMID:27345737

Detection of alternative splice variants at the proteome level in Aspergillus flavus.

PubMed

Chang, Kung-Yen; Georgianna, D Ryan; Heber, Steffen; Payne, Gary A; Muddiman, David C

2010-03-05

Identification of proteins from proteolytic peptides or intact proteins plays an essential role in proteomics. Researchers use search engines to match the acquired peptide sequences to the target proteins. However, search engines depend on protein databases to provide candidates for consideration. Alternative splicing (AS), the mechanism where the exon of pre-mRNAs can be spliced and rearranged to generate distinct mRNA and therefore protein variants, enable higher eukaryotic organisms, with only a limited number of genes, to have the requisite complexity and diversity at the proteome level. Multiple alternative isoforms from one gene often share common segments of sequences. However, many protein databases only include a limited number of isoforms to keep minimal redundancy. As a result, the database search might not identify a target protein even with high quality tandem MS data and accurate intact precursor ion mass. We computationally predicted an exhaustive list of putative isoforms of Aspergillus flavus proteins from 20 371 expressed sequence tags to investigate whether an alternative splicing protein database can assign a greater proportion of mass spectrometry data. The newly constructed AS database provided 9807 new alternatively spliced variants in addition to 12 832 previously annotated proteins. The searches of the existing tandem MS spectra data set using the AS database identified 29 new proteins encoded by 26 genes. Nine fungal genes appeared to have multiple protein isoforms. In addition to the discovery of splice variants, AS database also showed potential to improve genome annotation. In summary, the introduction of an alternative splicing database helps identify more proteins and unveils more information about a proteome.

Development of proteome-wide binding reagents for research and diagnostics.

PubMed

Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda

2013-12-01

Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Template-Based Protein Structure Reconstruction Method Using Deep Autoencoder Learning.

PubMed

Li, Haiou; Lyu, Qiang; Cheng, Jianlin

2016-12-01

Protein structure prediction is an important problem in computational biology, and is widely applied to various biomedical problems such as protein function study, protein design, and drug design. In this work, we developed a novel deep learning approach based on a deeply stacked denoising autoencoder for protein structure reconstruction. We applied our approach to a template-based protein structure prediction using only the 3D structural coordinates of homologous template proteins as input. The templates were identified for a target protein by a PSI-BLAST search. 3DRobot (a program that automatically generates diverse and well-packed protein structure decoys) was used to generate initial decoy models for the target from the templates. A stacked denoising autoencoder was trained on the decoys to obtain a deep learning model for the target protein. The trained deep model was then used to reconstruct the final structural model for the target sequence. With target proteins that have highly similar template proteins as benchmarks, the GDT-TS score of the predicted structures is greater than 0.7, suggesting that the deep autoencoder is a promising method for protein structure reconstruction.

Peptide and protein delivery using new drug delivery systems.

PubMed

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

2013-01-01

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

Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein

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

Su, Hua-Poo; Golden, Joseph W.; Gittis, Apostolos G.

2007-11-25

Medical countermeasures to prevent or treat smallpox are needed due to the potential use of poxviruses as biological weapons. Safety concerns with the currently available smallpox vaccine indicate a need for research on alternative poxvirus vaccine strategies. Molecular vaccines involving the use of proteins and/or genes and recombinant antibodies are among the strategies under current investigation. The poxvirus L1 protein, encoded by the L1R open reading frame, is the target of neutralizing antibodies and has been successfully used as a component of both protein subunit and DNA vaccines. L1-specific monoclonal antibodies (e.g., mouse monoclonal antibody mAb-7D11, mAb-10F5) with potent neutralizingmore » activity bind L1 in a conformation-specific manner. This suggests that proper folding of the L1 protein used in molecular vaccines will affect the production of neutralizing antibodies and protection. Here, we co-crystallized the Fab fragment of mAb-7D11 with the L1 protein. The crystal structure of the complex between Fab-7D11 and L1 reveals the basis for the conformation-specific binding as recognition of a discontinuous epitope containing two loops that are held together by a disulfide bond. The structure of this important conformational epitope of L1 will contribute to the development of molecular poxvirus vaccines and also provides a novel target for anti-poxvirus drugs. In addition, the sequence and structure of Fab-7D11 will contribute to the development of L1-targeted immunotherapeutics.« less

A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

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

Zhou, X.; Sun, Q; Kini, R

2008-01-01

The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstratemore » a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.« less

Ligand cluster-based protein network and ePlatton, a multi-target ligand finder.

PubMed

Du, Yu; Shi, Tieliu

2016-01-01

Small molecules are information carriers that make cells aware of external changes and couple internal metabolic and signalling pathway systems with each other. In some specific physiological status, natural or artificial molecules are used to interact with selective biological targets to activate or inhibit their functions to achieve expected biological and physiological output. Millions of years of evolution have optimized biological processes and pathways and now the endocrine and immune system cannot work properly without some key small molecules. In the past thousands of years, the human race has managed to find many medicines against diseases by trail-and-error experience. In the recent decades, with the deepening understanding of life and the progress of molecular biology, researchers spare no effort to design molecules targeting one or two key enzymes and receptors related to corresponding diseases. But recent studies in pharmacogenomics have shown that polypharmacology may be necessary for the effects of drugs, which challenge the paradigm, 'one drug, one target, one disease'. Nowadays, cheminformatics and structural biology can help us reasonably take advantage of the polypharmacology to design next-generation promiscuous drugs and drug combination therapies. 234,591 protein-ligand interactions were extracted from ChEMBL. By the 2D structure similarity, 13,769 ligand emerged from 156,151 distinct ligands which were recognized by 1477 proteins. Ligand cluster- and sequence-based protein networks (LCBN, SBN) were constructed, compared and analysed. For assisting compound designing, exploring polypharmacology and finding possible drug combination, we integrated the pathway, disease, drug adverse reaction and the relationship of targets and ligand clusters into the web platform, ePlatton, which is available at http://www.megabionet.org/eplatton. Although there were some disagreements between the LCBN and SBN, communities in both networks were largely the same with normalized mutual information at 0.9. The study of target and ligand cluster promiscuity underlying the LCBN showed that light ligand clusters were more promiscuous than the heavy one and that highly connected nodes tended to be protein kinases and involved in phosphorylation. ePlatton considerably reduced the redundancy of the ligand set of targets and made it easy to deduce the possible relationship between compounds and targets, pathways and side effects. ePlatton behaved reliably in validation experiments and also fast in virtual screening and information retrieval.Graphical abstractCluster exemplars and ePlatton's mechanism.

Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance.

PubMed

Lai, Shu-Jung; Tu, I-Fan; Wu, Wan-Ling; Yang, Jhih-Tian; Luk, Louis Y P; Lai, Mei-Chin; Tsai, Yu-Hsuan; Wu, Shih-Hsiung

2017-05-25

Phosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions. Here we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori. In summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level.

How bacteria hack the matrix and dodge the bullets of immunity.

PubMed

Paulsson, Magnus; Riesbeck, Kristian

2018-06-30

Haemophilus influenzae , Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential. Copyright ©ERS 2018.

Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

PubMed Central

Delfosse, Kathleen; Wozny, Michael R.; Jaipargas, Erica-Ashley; Barton, Kiah A.; Anderson, Cole; Mathur, Jaideep

2016-01-01

Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes. PMID:26834765

Prediction of hot spots in protein interfaces using a random forest model with hybrid features.

PubMed

Wang, Lin; Liu, Zhi-Ping; Zhang, Xiang-Sun; Chen, Luonan

2012-03-01

Prediction of hot spots in protein interfaces provides crucial information for the research on protein-protein interaction and drug design. Existing machine learning methods generally judge whether a given residue is likely to be a hot spot by extracting features only from the target residue. However, hot spots usually form a small cluster of residues which are tightly packed together at the center of protein interface. With this in mind, we present a novel method to extract hybrid features which incorporate a wide range of information of the target residue and its spatially neighboring residues, i.e. the nearest contact residue in the other face (mirror-contact residue) and the nearest contact residue in the same face (intra-contact residue). We provide a novel random forest (RF) model to effectively integrate these hybrid features for predicting hot spots in protein interfaces. Our method can achieve accuracy (ACC) of 82.4% and Matthew's correlation coefficient (MCC) of 0.482 in Alanine Scanning Energetics Database, and ACC of 77.6% and MCC of 0.429 in Binding Interface Database. In a comparison study, performance of our RF model exceeds other existing methods, such as Robetta, FOLDEF, KFC, KFC2, MINERVA and HotPoint. Of our hybrid features, three physicochemical features of target residues (mass, polarizability and isoelectric point), the relative side-chain accessible surface area and the average depth index of mirror-contact residues are found to be the main discriminative features in hot spots prediction. We also confirm that hot spots tend to form large contact surface areas between two interacting proteins. Source data and code are available at: http://www.aporc.org/doc/wiki/HotSpot.

Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX.

PubMed

Xu, Jiehua; Teng, I-Ting; Zhang, Liqin; Delgado, Stefanie; Champanhac, Carole; Cansiz, Sena; Wu, Cuichen; Shan, Hong; Tan, Weihong

2015-01-01

Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

PubMed

Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

2017-08-18

Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

Comprehensive quantitative analysis of central carbon and amino-acid metabolism in Saccharomyces cerevisiae under multiple conditions by targeted proteomics

PubMed Central

Costenoble, Roeland; Picotti, Paola; Reiter, Lukas; Stallmach, Robert; Heinemann, Matthias; Sauer, Uwe; Aebersold, Ruedi

2011-01-01

Decades of biochemical research have identified most of the enzymes that catalyze metabolic reactions in the yeast Saccharomyces cerevisiae. The adaptation of metabolism to changing nutritional conditions, in contrast, is much less well understood. As an important stepping stone toward such understanding, we exploit the power of proteomics assays based on selected reaction monitoring (SRM) mass spectrometry to quantify abundance changes of the 228 proteins that constitute the central carbon and amino-acid metabolic network in the yeast Saccharomyces cerevisiae, at five different metabolic steady states. Overall, 90% of the targeted proteins, including families of isoenzymes, were consistently detected and quantified in each sample, generating a proteomic data set that represents a nutritionally perturbed biological system at high reproducibility. The data set is near comprehensive because we detect 95–99% of all proteins that are required under a given condition. Interpreted through flux balance modeling, the data indicate that S. cerevisiae retains proteins not necessarily used in a particular environment. Further, the data suggest differential functionality for several metabolic isoenzymes. PMID:21283140

Targeting pH regulating proteins for cancer therapy-Progress and limitations.

PubMed

Parks, Scott K; Pouysségur, Jacques

2017-04-01

Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pH i ) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pH i regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pH i in the continued presence of external acidification (pH e ). Considerable experimentation has revealed targets that successfully disrupt tumour pH i regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na + /H + exchangers (NHEs), carbonic anhydrases (CAs), Na + /HCO 3 - co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pH i when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

Therapeutic antibodies: market considerations, disease targets and bioprocessing.

PubMed

Elvin, John G; Couston, Ruairidh G; van der Walle, Christopher F

2013-01-02

Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Genomics and integrated systems biology in Plasmodium falciparum: a path to malaria control and eradication.

PubMed

Le Roch, K G; Chung, D-W D; Ponts, N

2012-01-01

The first draft of the human malaria parasite's genome was released in 2002. Since then, the malaria scientific community has witnessed a steady embrace of new and powerful functional genomic studies. Over the years, these approaches have slowly revolutionized malaria research and enabled the comprehensive, unbiased investigation of various aspects of the parasite's biology. These genome-wide analyses delivered a refined annotation of the parasite's genome, delivered a better knowledge of its RNA, proteins and metabolite derivatives, and fostered the discovery of new vaccine and drug targets. Despite the positive impacts of these genomic studies, most research and investment still focus on protein targets, drugs and vaccine candidates that were known before the publication of the parasite genome sequence. However, recent access to next-generation sequencing technologies, along with an increased number of genome-wide applications, is expanding the impact of the parasite genome on biomedical research, contributing to a paradigm shift in research activities that may possibly lead to new optimized diagnosis and treatments. This review provides an update of Plasmodium falciparum genome sequences and an overview of the rapid development of genomics and system biology applications that have an immense potential of creating powerful tools for a successful malaria eradication campaign. © 2011 Blackwell Publishing Ltd.

Comparative Analysis of Predicted Plastid-Targeted Proteomes of Sequenced Higher Plant Genomes

PubMed Central

Schaeffer, Scott; Harper, Artemus; Raja, Rajani; Jaiswal, Pankaj; Dhingra, Amit

2014-01-01

Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding plastid biology, preferentially based on model systems is deficient. New plant genomes are expected to enable the identification of potentially new plastid-targeted proteins that will aid in studying novel roles of plastids. PMID:25393533

A Roadmap for Tick-Borne Flavivirus Research in the "Omics" Era.

PubMed

Grabowski, Jeffrey M; Hill, Catherine A

2017-01-01

Tick-borne flaviviruses (TBFs) affect human health globally. Human vaccines provide protection against some TBFs, and antivirals are available, yet TBF-specific control strategies are limited. Advances in genomics offer hope to understand the viral complement transmitted by ticks, and to develop disruptive, data-driven technologies for virus detection, treatment, and control. The genome assemblies of Ixodes scapularis , the North American tick vector of the TBF, Powassan virus, and other tick vectors, are providing insights into tick biology and pathogen transmission and serve as nucleation points for expanded genomic research. Systems biology has yielded insights to the response of tick cells to viral infection at the transcript and protein level, and new protein targets for vaccines to limit virus transmission. Reverse vaccinology approaches have moved candidate tick antigenic epitopes into vaccine development pipelines. Traditional drug and in silico screening have identified candidate antivirals, and target-based approaches have been developed to identify novel acaricides. Yet, additional genomic resources are required to expand TBF research. Priorities include genome assemblies for tick vectors, "omic" studies involving high consequence pathogens and vectors, and emphasizing viral metagenomics, tick-virus metabolomics, and structural genomics of TBF and tick proteins. Also required are resources for forward genetics, including the development of tick strains with quantifiable traits, genetic markers and linkage maps. Here we review the current state of genomic research on ticks and tick-borne viruses with an emphasis on TBFs. We outline an ambitious 10-year roadmap for research in the "omics era," and explore key milestones needed to accomplish the goal of delivering three new vaccines, antivirals and acaricides for TBF control by 2030.

A Roadmap for Tick-Borne Flavivirus Research in the “Omics” Era

PubMed Central

Grabowski, Jeffrey M.; Hill, Catherine A.

2017-01-01

Tick-borne flaviviruses (TBFs) affect human health globally. Human vaccines provide protection against some TBFs, and antivirals are available, yet TBF-specific control strategies are limited. Advances in genomics offer hope to understand the viral complement transmitted by ticks, and to develop disruptive, data-driven technologies for virus detection, treatment, and control. The genome assemblies of Ixodes scapularis, the North American tick vector of the TBF, Powassan virus, and other tick vectors, are providing insights into tick biology and pathogen transmission and serve as nucleation points for expanded genomic research. Systems biology has yielded insights to the response of tick cells to viral infection at the transcript and protein level, and new protein targets for vaccines to limit virus transmission. Reverse vaccinology approaches have moved candidate tick antigenic epitopes into vaccine development pipelines. Traditional drug and in silico screening have identified candidate antivirals, and target-based approaches have been developed to identify novel acaricides. Yet, additional genomic resources are required to expand TBF research. Priorities include genome assemblies for tick vectors, “omic” studies involving high consequence pathogens and vectors, and emphasizing viral metagenomics, tick-virus metabolomics, and structural genomics of TBF and tick proteins. Also required are resources for forward genetics, including the development of tick strains with quantifiable traits, genetic markers and linkage maps. Here we review the current state of genomic research on ticks and tick-borne viruses with an emphasis on TBFs. We outline an ambitious 10-year roadmap for research in the “omics era,” and explore key milestones needed to accomplish the goal of delivering three new vaccines, antivirals and acaricides for TBF control by 2030. PMID:29312896

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

PubMed

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Computational multiscale modeling in protein--ligand docking.

PubMed

Taufer, Michela; Armen, Roger; Chen, Jianhan; Teller, Patricia; Brooks, Charles

2009-01-01

In biological systems, the binding of small molecule ligands to proteins is a crucial process for almost every aspect of biochemistry and molecular biology. Enzymes are proteins that function by catalyzing specific biochemical reactions that convert reactants into products. Complex organisms are typically composed of cells in which thousands of enzymes participate in complex and interconnected biochemical pathways. Some enzymes serve as sequential steps in specific pathways (such as energy metabolism), while others function to regulate entire pathways and cellular functions [1]. Small molecule ligands can be designed to bind to a specific enzyme and inhibit the biochemical reaction. Inhibiting the activity of key enzymes may result in the entire biochemical pathways being turned on or off [2], [3]. Many small molecule drugs marketed today function in this generic way as enzyme inhibitors. If research identifies a specific enzyme as being crucial to the progress of disease, then this enzyme may be targeted with an inhibitor, which may slow down or reverse the progress of disease. In this way, enzymes are targeted from specific pathogens (e.g., virus, bacteria, fungi) for infectious diseases [4], [5], and human enzymes are targeted for noninfectious diseases such as cardiovascular disease, cancer, diabetes, and neurodegenerative diseases [6].

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

PubMed

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.

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

PubMed Central

Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

2015-01-01

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

PubMed

Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

2015-01-21

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

Pac-Man for biotechnology: co-opting degrons for targeted protein degradation to control and alter cell function.

PubMed

Yu, Geng; Rosenberg, Julian N; Betenbaugh, Michael J; Oyler, George A

2015-12-01

Protein degradation in normal living cells is precisely regulated to match the cells' physiological requirements. The selectivity of protein degradation is determined by an elaborate degron-tagging system. Degron refers to an amino acid sequence that encodes a protein degradation signal, which is oftentimes a poly-ubiquitin chain that can be transferred to other proteins. Current understanding of ubiquitination dependent and independent protein degradation processes has expanded the application of degrons for targeted protein degradation and novel cell engineering strategies. Recent findings suggest that small molecules inducing protein association can be exploited to create degrons that target proteins for degradation. Here, recent applications of degron-based targeted protein degradation in eukaryotic organisms are reviewed. The degron mediated protein degradation represents a rapidly tunable methodology to control protein abundance, which has broad application in therapeutics and cellular function control and monitoring. Copyright © 2015. Published by Elsevier Ltd.

ASD: a comprehensive database of allosteric proteins and modulators

PubMed Central

Huang, Zhimin; Zhu, Liang; Cao, Yan; Wu, Geng; Liu, Xinyi; Chen, Yingyi; Wang, Qi; Shi, Ting; Zhao, Yaxue; Wang, Yuefei; Li, Weihua; Li, Yixue; Chen, Haifeng; Chen, Guoqiang; Zhang, Jian

2011-01-01

Allostery is the most direct, rapid and efficient way of regulating protein function, ranging from the control of metabolic mechanisms to signal-transduction pathways. However, an enormous amount of unsystematic allostery information has deterred scientists who could benefit from this field. Here, we present the AlloSteric Database (ASD), the first online database that provides a central resource for the display, search and analysis of structure, function and related annotation for allosteric molecules. Currently, ASD contains 336 allosteric proteins from 101 species and 8095 modulators in three categories (activators, inhibitors and regulators). Proteins are annotated with a detailed description of allostery, biological process and related diseases, and modulators with binding affinity, physicochemical properties and therapeutic area. Integrating the information of allosteric proteins in ASD should allow for the identification of specific allosteric sites of a given subtype among proteins of the same family that can potentially serve as ideal targets for experimental validation. In addition, modulators curated in ASD can be used to investigate potent allosteric targets for the query compound, and also help chemists to implement structure modifications for novel allosteric drug design. Therefore, ASD could be a platform and a starting point for biologists and medicinal chemists for furthering allosteric research. ASD is freely available at http://mdl.shsmu.edu.cn/ASD/. PMID:21051350

Applying chaperones to protein-misfolding disorders: molecular chaperones against α-synuclein in Parkinson's disease.

PubMed

Chaari, Ali; Hoarau-Véchot, Jessica; Ladjimi, Moncef

2013-09-01

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of a protein called α-synuclein (α-syn) into inclusions known as lewy bodies (LB) within neurons. This accumulation is also due to insufficient formation and activity of dopamine produced in certain neurons within the substantia nigra. Lewy bodies are the pathological hallmark of the idiopathic disorder and the cascade that allows α-synuclein to misfold, aggregate and form these inclusions has been the subject of intensive research. Targeting these early steps of oligomerization is one of the main therapeutic approaches in order to develop neurodegenerative-modifying agents. Because the folding and refolding of alpha synuclein is the key point of this cascade, we are interested in this review to summarize the role of some molecular chaperones proteins such as Hsp70, Hsp90 and small heat shock proteins (sHsp) and Hsp 104. Hsp70 and its co-chaperone, Hsp70 and small heat shock proteins can prevent neurodegeneration by preventing α-syn misfolding, oligomerization and aggregation in vitro and in Parkinson disease animal models. Hsp104 is able to resolve disordered protein aggregates and cross beta amyloid conformers. Together, these chaperones have a complementary effect and can be a target for therapeutic intervention in PD. Published by Elsevier B.V.

Endoplasmic reticulum localized PerA is required for cell wall integrity, azole drug resistance, and virulence in Aspergillus fumigatus

PubMed Central

Chung, Dawoon; Thammahong, Arsa; Shepardson, Kelly M.; Blosser, Sara J.; Cramer, Robert A.

2014-01-01

Summary GPI-anchoring is a universal and critical post-translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI-anchored, and disruption of GPI-anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI-anchored protein functions, our current knowledge of GPI lipid remodeling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodeling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β-glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow derived macrophages relative to wild type. Given the structural specificity of fungal GPI-anchors, which is different from humans, understanding GPI lipid remodeling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target. PMID:24779420

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

Cancer.gov

The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

Implementation of a solid target production facility

NASA Astrophysics Data System (ADS)

Tochon-Danguy, H. J.; Poniger, S. S.; Sachinidis, J. I.; Panopoulos, H. P.; Scott, A. M.

2012-12-01

The desire to utilize long-lived PET isotopes in Australia has significantly increased over the years and several research projects for labelling of peptides, proteins and biomolecules, including labelling of recombinant antibodies has been restricted due to the limited availability of suitable isotopes. This need has led to the recent installation and commissioning of a new facility dedicated to fully automated solid target isotope production, including 24I, 64Cu, 89Zr and 86Y at the Austin Health Centre for PET.

HIV and Drug Resistance: Hitting a Moving Target | Center for Cancer Research

Cancer.gov

Prior research revealed how HIV-1 makes its destructive entry into the target cell by fusing together the cholesterol-rich lipid bilayer of the viral envelope—made with key glycoproteins gp120 and gp41—and the host cell’s plasma membrane. Cell-viral interactions begin with the binding of gp120 to the CD4 receptor molecule on the target cell, followed by gp120 binding to coreceptors. These coreceptors likely reside in structures called lipid rafts—areas in the cell plasma membrane that are rich in cholesterol, saturated fatty acids, and certain proteins that facilitate the entry of viruses into host cells. Finally, sequences in gp41 trigger the fusion of the viral and cellular lipid bilayers. The lipid rafts are then involved in the production of new viral particles.

Identification and Herc5-mediated ISGylation of novel target proteins.

PubMed

Takeuchi, Tomoharu; Inoue, Satoshi; Yokosawa, Hideyoshi

2006-09-22

ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: the E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L- and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

Improving compound-protein interaction prediction by building up highly credible negative samples.

PubMed

Liu, Hui; Sun, Jianjiang; Guan, Jihong; Zheng, Jie; Zhou, Shuigeng

2015-06-15

Computational prediction of compound-protein interactions (CPIs) is of great importance for drug design and development, as genome-scale experimental validation of CPIs is not only time-consuming but also prohibitively expensive. With the availability of an increasing number of validated interactions, the performance of computational prediction approaches is severely impended by the lack of reliable negative CPI samples. A systematic method of screening reliable negative sample becomes critical to improving the performance of in silico prediction methods. This article aims at building up a set of highly credible negative samples of CPIs via an in silico screening method. As most existing computational models assume that similar compounds are likely to interact with similar target proteins and achieve remarkable performance, it is rational to identify potential negative samples based on the converse negative proposition that the proteins dissimilar to every known/predicted target of a compound are not much likely to be targeted by the compound and vice versa. We integrated various resources, including chemical structures, chemical expression profiles and side effects of compounds, amino acid sequences, protein-protein interaction network and functional annotations of proteins, into a systematic screening framework. We first tested the screened negative samples on six classical classifiers, and all these classifiers achieved remarkably higher performance on our negative samples than on randomly generated negative samples for both human and Caenorhabditis elegans. We then verified the negative samples on three existing prediction models, including bipartite local model, Gaussian kernel profile and Bayesian matrix factorization, and found that the performances of these models are also significantly improved on the screened negative samples. Moreover, we validated the screened negative samples on a drug bioactivity dataset. Finally, we derived two sets of new interactions by training an support vector machine classifier on the positive interactions annotated in DrugBank and our screened negative interactions. The screened negative samples and the predicted interactions provide the research community with a useful resource for identifying new drug targets and a helpful supplement to the current curated compound-protein databases. Supplementary files are available at: http://admis.fudan.edu.cn/negative-cpi/. © The Author 2015. Published by Oxford University Press.

Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks

DTIC Science & Technology

2012-09-21

Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks Paulo Shakarian1*, J. Kenneth Wickiser2 1 Paulo Shakarian...significantly attacked. Citation: Shakarian P, Wickiser JK (2012) Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks...to 00-00-2012 4. TITLE AND SUBTITLE Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks 5a. CONTRACT NUMBER 5b

New Partnership Could Help Identify Drugs to Target Cancers Driven by KRAS Mutations | Frederick National Laboratory for Cancer Research

Cancer.gov

More than 100,000 newly diagnosed cases of cancer each year in the United States are subsequently linked to mutations in the KRAS protein. In response to this urgent problem, a new partnership agreement involving the Frederick National Laboratory for

New study shows normally helpful natural bacteria may also trigger lupus | Center for Cancer Research

Cancer.gov

CCR scientists have discovered that a protein produced by bacteria that naturally inhabit our bodies may trigger the autoimmune disease lupus. The results of the study could unveil an entirely new set of drug targets for treating lupus and other autoimmune diseases. Read more…

A universal entropy-driven mechanism for thioredoxin–target recognition

PubMed Central

Palde, Prakash B.; Carroll, Kate S.

2015-01-01

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx–protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx–target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein–protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx–target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition. PMID:26080424

Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains.

PubMed

Privat, Nicolas; Levavasseur, Etienne; Yildirim, Serfildan; Hannaoui, Samia; Brandel, Jean-Philippe; Laplanche, Jean-Louis; Béringue, Vincent; Seilhean, Danielle; Haïk, Stéphane

2017-10-06

Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrP Sc ). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrP Sc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the Theileria parva sporozoite proteome.

PubMed

Nyagwange, James; Tijhaar, Edwin; Ternette, Nicola; Mobegi, Fredrick; Tretina, Kyle; Silva, Joana C; Pelle, Roger; Nene, Vishvanath

2018-03-01

East Coast fever is a lymphoproliferative disease caused by the tick-borne protozoan parasite Theileria parva. The sporozoite stage of this parasite, harboured and released from the salivary glands of the tick Rhipicephalus appendiculatus during feeding, invades and establishes infection in bovine lymphocytes. Blocking this initial stage of invasion presents a promising vaccine strategy for control of East Coast fever and can in part be achieved by targeting the major sporozoite surface protein p67. To support research on the biology of T. parva and the identification of additional candidate vaccine antigens, we report on the sporozoite proteome as defined by LC-MS/MS analysis. In total, 4780 proteins were identified in an enriched preparation of sporozoites. Of these, 2007 were identified as T. parva proteins, representing close to 50% of the total predicted parasite proteome. The remaining 2773 proteins were derived from the tick vector. The identified sporozoite proteins include a set of known T. parva antigens targeted by antibodies and cytotoxic T cells from cattle that are immune to East Coast fever. We also identified proteins predicted to be orthologs of Plasmodium falciparum sporozoite surface molecules and invasion organelle proteins, and proteins that may contribute to the phenomenon of bovine lymphocyte transformation. Overall, these data establish a protein expression profile of T. parva sporozoites as an important starting point for further study of a parasitic species which has considerable agricultural impact. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Inferring protein domains associated with drug side effects based on drug-target interaction network.

PubMed

Iwata, Hiroaki; Mizutani, Sayaka; Tabei, Yasuo; Kotera, Masaaki; Goto, Susumu; Yamanishi, Yoshihiro

2013-01-01

Most phenotypic effects of drugs are involved in the interactions between drugs and their target proteins, however, our knowledge about the molecular mechanism of the drug-target interactions is very limited. One of challenging issues in recent pharmaceutical science is to identify the underlying molecular features which govern drug-target interactions. In this paper, we make a systematic analysis of the correlation between drug side effects and protein domains, which we call "pharmacogenomic features," based on the drug-target interaction network. We detect drug side effects and protein domains that appear jointly in known drug-target interactions, which is made possible by using classifiers with sparse models. It is shown that the inferred pharmacogenomic features can be used for predicting potential drug-target interactions. We also discuss advantages and limitations of the pharmacogenomic features, compared with the chemogenomic features that are the associations between drug chemical substructures and protein domains. The inferred side effect-domain association network is expected to be useful for estimating common drug side effects for different protein families and characteristic drug side effects for specific protein domains.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

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

Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a smallmore » change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.« less

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis.

PubMed

Wilson, Kris; Webster, Scott P; Iredale, John P; Zheng, Xiaozhong; Homer, Natalie Z; Pham, Nhan T; Auer, Manfred; Mole, Damian J

2017-12-15

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

NASA Astrophysics Data System (ADS)

Wilson, Kris; Webster, Scott P.; Iredale, John P.; Zheng, Xiaozhong; Homer, Natalie Z.; Pham, Nhan T.; Auer, Manfred; Mole, Damian J.

2018-01-01

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

PubMed Central

Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

2018-01-01

ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection. PMID:29437971

Prioritization of Biomarker Targets in Human Umbilical Cord Blood: Identification of Proteins in Infant Blood Serving as Validated Biomarkers in Adults

PubMed Central

Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R.; Witter, Frank R.

2012-01-01

Background: Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped. Objectives: Our goal was to accelerate biomarker research on UCB by exploring its detectable protein content and providing a priority list of potential biomarkers based on known proteins involved in disease pathways. Methods: We explored cord blood serum proteins by profiling a UCB pool of 12 neonates with different backgrounds using a combination of isoelectric focusing and liquid chromatography coupled with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) and by comparing results with information contained in metabolic and disease databases available for adult blood. Results: A total of 1,210 UCB proteins were identified with a protein-level false discovery rate of ~ 5% as estimated by naïve target-decoy and MAYU approaches, signifying a 6-fold increase in the number of UCB proteins described to date. Identified proteins correspond to 138 different metabolic and disease pathways and provide a platform of mechanistically linked biomarker candidates for tracking disruptions in cellular processes. Moreover, among the identified proteins, 38 were found to be approved biomarkers for adult blood. Conclusions: The results of this study advance current knowledge of the human cord blood serum proteome. They showcase the potential of UCB as a diagnostic medium for assessing infant health by detection and identification of candidate biomarkers for known disease pathways using a global, nontargeted approach. These biomarkers may inform about mechanisms of exposure–disease relationships. Furthermore, biomarkers approved by the U.S. Food and Drug Administration for screening in adult blood were detected in UCB and represent high-priority targets for immediate validation. PMID:22538116

Prioritization of biomarker targets in human umbilical cord blood: identification of proteins in infant blood serving as validated biomarkers in adults.

PubMed

Hansmeier, Nicole; Chao, Tzu-Chiao; Goldman, Lynn R; Witter, Frank R; Halden, Rolf U

2012-05-01

Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped. Our goal was to accelerate biomarker research on UCB by exploring its detectable protein content and providing a priority list of potential biomarkers based on known proteins involved in disease pathways. We explored cord blood serum proteins by profiling a UCB pool of 12 neonates with different backgrounds using a combination of isoelectric focusing and liquid chromatography coupled with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) and by comparing results with information contained in metabolic and disease databases available for adult blood. A total of 1,210 UCB proteins were identified with a protein-level false discovery rate of ~ 5% as estimated by naïve target-decoy and MAYU approaches, signifying a 6-fold increase in the number of UCB proteins described to date. Identified proteins correspond to 138 different metabolic and disease pathways and provide a platform of mechanistically linked biomarker candidates for tracking disruptions in cellular processes. Moreover, among the identified proteins, 38 were found to be approved biomarkers for adult blood. The results of this study advance current knowledge of the human cord blood serum proteome. They showcase the potential of UCB as a diagnostic medium for assessing infant health by detection and identification of candidate biomarkers for known disease pathways using a global, nontargeted approach. These biomarkers may inform about mechanisms of exposure-disease relationships. Furthermore, biomarkers approved by the U.S. Food and Drug Administration for screening in adult blood were detected in UCB and represent high-priority targets for immediate validation.

Engineering and Application of Zinc Finger Proteins and TALEs for Biomedical Research.

PubMed

Kim, Moon-Soo; Kini, Anu Ganesh

2017-08-01

Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.

Deubiquitylation of deubiquitylases.

PubMed

Haq, Saba; Ramakrishna, Suresh

2017-06-01

Deubiquitylating enzymes (DUBs) reverse the ubiquitylation of target proteins, thereby regulating diverse cellular functions. In contrast to the plethora of research being conducted on the ability of DUBs to counter the degradation of cellular proteins or auto-ubiquitylated E3 ligases, very little is known about the mechanisms of DUB regulation. In this review paper, we summarize a novel possible mechanism of DUB deubiquitylation by other DUBs. The available data suggest the need for further experiments to validate and characterize this notion of 'Dubbing DUBs'. The current studies indicate that the idea of deubiquitylation of DUBs by other DUBs is still in its infancy. Nevertheless, future research holds the promise of validation of this concept. © 2017 The Authors.

Deubiquitylation of deubiquitylases

PubMed Central

Haq, Saba

2017-01-01

Deubiquitylating enzymes (DUBs) reverse the ubiquitylation of target proteins, thereby regulating diverse cellular functions. In contrast to the plethora of research being conducted on the ability of DUBs to counter the degradation of cellular proteins or auto-ubiquitylated E3 ligases, very little is known about the mechanisms of DUB regulation. In this review paper, we summarize a novel possible mechanism of DUB deubiquitylation by other DUBs. The available data suggest the need for further experiments to validate and characterize this notion of ‘Dubbing DUBs’. The current studies indicate that the idea of deubiquitylation of DUBs by other DUBs is still in its infancy. Nevertheless, future research holds the promise of validation of this concept. PMID:28659380

Recording information on protein complexes in an information management system.

PubMed

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

2011-08-01

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

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

PubMed Central

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A.; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells. PMID:25360666

Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

PubMed

Autelitano, François; Loyaux, Denis; Roudières, Sébastien; Déon, Catherine; Guette, Frédérique; Fabre, Philippe; Ping, Qinggong; Wang, Su; Auvergne, Romane; Badarinarayana, Vasudeo; Smith, Michael; Guillemot, Jean-Claude; Goldman, Steven A; Natesan, Sridaran; Ferrara, Pascual; August, Paul

2014-01-01

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

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

PubMed

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.

Staying Alive: Cancer Cells Expressing Mutant KRas Depend on ERH for Survival | Center for Cancer Research

Cancer.gov

The small G-protein KRas acts like a molecular switch, turning on and off pro-growth signaling pathways within cells when appropriate. In a large number of cancers, KRas is permanently turned on by a variety of mutations and drives the constant growth of these tumor cells. KRas itself has proved to be a poor drug target so researchers in the laboratory of Ji Luo, Ph.D., in CCR’s Medical Oncology Branch decided to look for other pathways that are essential for the growth of cells expressing mutant KRas. These pathways could present new drug targets, and blocking their activities might selectively affect cells that express mutant KRas.

Identifying relationships between unrelated pharmaceutical target proteins on the basis of shared active compounds.

PubMed

Miljković, Filip; Kunimoto, Ryo; Bajorath, Jürgen

2017-08-01

Computational exploration of small-molecule-based relationships between target proteins from different families. Target annotations of drugs and other bioactive compounds were systematically analyzed on the basis of high-confidence activity data. A total of 286 novel chemical links were established between distantly related or unrelated target proteins. These relationships involved a total of 1859 bioactive compounds including 147 drugs and 141 targets. Computational analysis of large amounts of compounds and activity data has revealed unexpected relationships between diverse target proteins on the basis of compounds they share. These relationships are relevant for drug discovery efforts. Target pairs that we have identified and associated compound information are made freely available.

Autotaxin: A protein with two faces

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

Tania, Mousumi; Khan, Md. Asaduzzaman; Zhang, Huaiyuan

Research highlights: {yields} Autotaxin (ATX) has lysophospholipase D activity. {yields} ATX catalyzes the formation of lysophosphatidic acid (LPA). {yields} LPA is a mitogen, and thus is responsible for cancer. {yields} ATX also catalyzes the formation of anti-cancerous cyclic phosphatidic acid. {yields} Autotaxin is a novel target of cancer therapy research. -- Abstract: Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses.more » The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.« less

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability.

PubMed

Yoshida, Keisuke; Hisabori, Toru

2016-07-05

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

PubMed Central

Yoshida, Keisuke; Hisabori, Toru

2016-01-01

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

Efficient genome editing by FACS enrichment of paired D10A Cas9 nickases coupled with fluorescent proteins.

PubMed

Gopalappa, Ramu; Song, Myungjae; Chandrasekaran, Arun Pandian; Das, Soumyadip; Haq, Saba; Koh, Hyun Chul; Ramakrishna, Suresh

2018-05-31

Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of double-nicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.

Exo-Dye-based assay for rapid, inexpensive, and sensitive detection of DNA-binding proteins.

PubMed

Chen, Zaozao; Ji, Meiju; Hou, Peng; Lu, Zuhong

2006-07-07

We reported herein a rapid, inexpensive, and sensitive technique for detecting sequence-specific DNA-binding proteins. In this technique, the common exonuclease III (ExoIII) footprinting assay is coupled with simple SYBR Green I staining for monitoring the activities of DNA-binding proteins. We named this technique as ExoIII-Dye-based assay. In this assay, a duplex probe was designed to detect DNA-binding protein. One side of the probe contains one protein-binding site, and another side of it contains five protruding bases at 3' end for protection from ExoIII digestion. If a target protein is present, it will bind to binding sites of probe and produce a physical hindrance to ExoIII, which protects the duplex probe from digestion of ExoIII. SYBR Green I will bind to probe, which results in high fluorescence intensity. On the contrary, in the absence of the target protein, the naked duplex probe will be degraded by ExoIII. SYBR Green I will be released, which results in a low fluorescence intensity. In this study, we employed this technique to successfully detect transcription factor NF-kappaB in crude cell extracts. Moreover, it could also be used to evaluate the binding affinity of NF-kappaB. This technique has therefore wide potential application in research, medical diagnosis, and drug discovery.

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

PubMed

Kumeta, Masahiro; Panina, Yulia; Yamazaki, Hiroya; Takeyasu, Kunio; Yoshimura, Shige H

2018-06-13

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

The prediction of candidate genes for cervix related cancer through gene ontology and graph theoretical approach.

PubMed

Hindumathi, V; Kranthi, T; Rao, S B; Manimaran, P

2014-06-01

With rapidly changing technology, prediction of candidate genes has become an indispensable task in recent years mainly in the field of biological research. The empirical methods for candidate gene prioritization that succors to explore the potential pathway between genetic determinants and complex diseases are highly cumbersome and labor intensive. In such a scenario predicting potential targets for a disease state through in silico approaches are of researcher's interest. The prodigious availability of protein interaction data coupled with gene annotation renders an ease in the accurate determination of disease specific candidate genes. In our work we have prioritized the cervix related cancer candidate genes by employing Csaba Ortutay and his co-workers approach of identifying the candidate genes through graph theoretical centrality measures and gene ontology. With the advantage of the human protein interaction data, cervical cancer gene sets and the ontological terms, we were able to predict 15 novel candidates for cervical carcinogenesis. The disease relevance of the anticipated candidate genes was corroborated through a literature survey. Also the presence of the drugs for these candidates was detected through Therapeutic Target Database (TTD) and DrugMap Central (DMC) which affirms that they may be endowed as potential drug targets for cervical cancer.

A designed repeat protein as an affinity capture reagent

PubMed Central

Speltz, Elizabeth B.; Brown, Rebecca S.H.; Hajare, Holly S.; Schlieker, Christian; Regan, Lynne

2017-01-01

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class - tetratricopeptide repeat (TPR) proteins. In previous work we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena 2011; Main 2003]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena 2009; Cortajarena 2008; Jackrel 2009; Kajander 2007]. Here we focus on the development of one such TPR-peptide interaction for a practical application – affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications. PMID:26517897

Characterization of single chain antibody targets through yeast two hybrid

PubMed Central

2010-01-01

Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine. PMID:20727208

[Mathematical modeling: an essential tool for the study of therapeutic targeting in solid tumors].

PubMed

Saidak, Zuzana; Giacobbi, Anne-Sophie; Morisse, Mony Chenda; Mammeri, Youcef; Galmiche, Antoine

2017-12-01

Recent progress in biology has made the study of the medical treatment of cancer more effective, but it has also revealed the large complexity of carcinogenesis and cell signaling. For many types of cancer, several therapeutic targets are known and in some cases drugs against these targets exist. Unfortunately, the target proteins often work in networks, resulting in functional adaptation and the development of resilience/resistance to medical treatment. The use of mathematical modeling makes it possible to carry out system-level analyses for improved study of therapeutic targeting in solid tumours. We present the main types of mathematical models used in cancer research and we provide examples illustrating the relevance of these approaches in molecular oncobiology. © 2017 médecine/sciences – Inserm.

Alarmins firing arthritis: Helpful diagnostic tools and promising therapeutic targets.

PubMed

Lavric, Miha; Miranda-García, María Auxiliadora; Holzinger, Dirk; Foell, Dirk; Wittkowski, Helmut

2017-07-01

Alarmins are endogenous molecules with homeostatic roles that have reached the focus of research in inflammatory arthritis in the last two decades, mostly due to their ability to indicate tissue related damage after active or passive release from injured cells. From HMGB1, S100A8/A9 and S100A12 proteins, over heat-shock proteins (HSPs) and purine metabolites (e.g. uric acid, ATP) to altered matrix proteins and interleukin-33 (IL-33), a number of alarmins have been determined until now as having a role in rheumatoid arthritis, psoriatic and juvenile idiopathic arthritis, as well as spondyloarthritis and gout. Although formerly being linked to initiation and chronification of inflammatory arthritis, driving auto- and paracrine inflammatory loops, more recent research has also unraveled the alarmins' role in the crosstalk between innate and adaptive immunity and in resolution of inflammation. Providing a state-of-the-art overview of known alarmins, this review lists the known modes of action and pathologic contribution of alarmins to inflammatory arthritis, as well as biomarker potential of alarmins in the clinical setting for tracking disease severity. Based upon research on animal experimental models (CIA, AIA) and clinical trials, a look is made into potentially viable strategies for modifying alarmin secretion and their target receptor (e.g. TLR, RAGE) interaction with the purpose of attenuating arthritic disease. Copyright © 2016 Société française de rhumatologie. Published by Elsevier SAS. All rights reserved.

Future therapeutic targets for the treatment and prevention of cholesterol gallstones.

PubMed

Castro-Torres, Ibrahim Guillermo; de Jesús Cárdenas-Vázquez, René; Velázquez-González, Claudia; Ventura-Martínez, Rosa; De la O-Arciniega, Minarda; Naranjo-Rodríguez, Elia Brosla; Martínez-Vázquez, Mariano

2015-10-15

The formation of cholesterol gallstones involves very complex imbalances, such as alterations in the secretion of biliary lipids (which involves the ABCG5, ABCG8, ABCB4 and ABCB11 transporters), biochemical and immunological reactions in the gallbladder that produce biliary sludge (mucins), physicochemical changes in the structure of cholesterol (crystallization), alterations in gallbladder motility, changes in the intestinal absorption of cholesterol (ABCG5/8 transporters and Niemann-Pick C1L1 protein) and alterations in small intestine motility. Some of these proteins have been studied at the clinical and experimental levels, but more research is required. In this review, we discuss the results of studies on some molecules involved in the pathophysiology of gallstones that may be future therapeutic targets to prevent the development of this disease, and possible sites for treatment based mainly on the absorption of intestinal cholesterol (Niemann-Pick C1L1 and ABCG5/8 proteins). Copyright © 2015. Published by Elsevier B.V.

Epigenetic pathway targets for the treatment of disease: accelerating progress in the development of pharmacological tools: IUPHAR Review 11

PubMed Central

Tough, David F; Lewis, Huw D; Rioja, Inmaculada; Lindon, Matthew J; Prinjha, Rab K

2014-01-01

The properties of a cell are determined both genetically by the DNA sequence of its genes and epigenetically through processes that regulate the pattern, timing and magnitude of expression of its genes. While the genetic basis of disease has been a topic of intense study for decades, recent years have seen a dramatic increase in the understanding of epigenetic regulatory mechanisms and a growing appreciation that epigenetic misregulation makes a significant contribution to human disease. Several large protein families have been identified that act in different ways to control the expression of genes through epigenetic mechanisms. Many of these protein families are finally proving tractable for the development of small molecules that modulate their function and represent new target classes for drug discovery. Here, we provide an overview of some of the key epigenetic regulatory proteins and discuss progress towards the development of pharmacological tools for use in research and therapy. PMID:25060293

[Activity of NTDs Drug-discovery Research Consortium].

PubMed

Namatame, Ichiji

2016-01-01

Neglected tropical diseases (NTDs) are an extremely important issue facing global health care. To improve "access to health" where people are unable to access adequate medical care due to poverty and weak healthcare systems, we have established two consortiums: the NTD drug discovery research consortium, and the pediatric praziquantel consortium. The NTD drug discovery research consortium, which involves six institutions from industry, government, and academia, as well as an international non-profit organization, is committed to developing anti-protozoan active compounds for three NTDs (Leishmaniasis, Chagas disease, and African sleeping sickness). Each participating institute will contribute their efforts to accomplish the following: selection of drug targets based on information technology, and drug discovery by three different approaches (in silico drug discovery, "fragment evolution" which is a unique drug designing method of Astellas Pharma, and phenotypic screening with Astellas' compound library). The consortium has established a brand new database (Integrated Neglected Tropical Disease Database; iNTRODB), and has selected target proteins for the in silico and fragment evolution drug discovery approaches. Thus far, we have identified a number of promising compounds that inhibit the target protein, and we are currently trying to improve the anti-protozoan activity of these compounds. The pediatric praziquantel consortium was founded in July 2012 to develop and register a new praziquantel pediatric formulation for the treatment of schistosomiasis. Astellas Pharma has been a core member in this consortium since its establishment, and has provided expertise and technology in the area of pediatric formulation development and clinical development.

Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

PubMed Central

2012-01-01

Background The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. Results Our findings demonstrate that a miRNA’s functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. Conclusions We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment. PMID:22929553

New Molecular Targets of Anticancer Therapy - Current Status and Perspectives.

PubMed

Zajac, Marianna; Muszalska, Izabela; Jelinska, Anna

2016-01-01

Molecularly targeted anticancer therapy involves the use of drugs or other substances affecting specific molecular targets that play a part in the development, progression and spread of a given neoplasm. By contrast, the majority of classical chemotherapeutics act on all rapidly proliferating cells, both healthy and cancerous ones. Target anticancer drugs are designed to achieve a particular aim and they usually act cytostatically, not cytotoxically like classical chemotherapeutics. At present, more than 300 biological molecular targets have been identified. The proteins involved in cellular metabolism include (among others) receptor proteins, signal transduction proteins, mRNA thread matrix synthesis proteins participating in neoplastic transformation, cell cycle control proteins, functional and structural proteins. The receptor proteins that are targeted by currently used anticancer drugs comprise the epithelial growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor(VEGFR). Target anticancer drugs may affect extracellular receptor domains (antibodies) or intracellular receptor domains (tyrosine kinase inhibitors). The blocking of the mRNA thread containing information about the structure of oncogenes (signal transduction proteins) is another molecular target of anticancer drugs. That type of treatment, referred to as antisense therapy, is in clinical trials. When the synthesis of genetic material is disturbed, in most cases the passage to the next cycle phase is blocked. The key proteins responsible for the blockage are cyclines and cycline- dependent kinases (CDK). Clinical trials are focused on natural and synthetic substances capable of blocking various CDKs. The paper discusses the molecular targets and chemical structure of target anticancer drugs that have been approved for and currently applied in antineoplastic therapy together with indications and contraindications for their application.

Strategic incorporation of fluorine in the drug discovery of new-generation antitubercular agents targeting bacterial cell division protein FtsZ⋆

PubMed Central

Ojima, Iwao; Awasthi, Divya; Wei, Longfei; Haranahalli, Krupanandan

2016-01-01

This article presents an account of our research on the discovery and development of new-generation fluorine-containing antibacterial agents against drug-resistant tuberculosis, targeting FtsZ. FtsZ is an essential protein for bacterial cell division and a highly promising therapeutic target for antibacterial drug discovery. Through design, synthesis and semi-HTP screening of libraries of novel benzimidazoles, followed by SAR studies, we identified highly potent lead compounds. However, these lead compounds were found to lack sufficient metabolic and plasma stabilities. Accordingly, we have performed extensive study on the strategic incorporation of fluorine into lead compounds to improve pharmacological properties. This study has led to the development of highly efficacious fluorine-containing benzimidazoles as potential drug candidates. We have also performed computational docking analysis of these novel FtsZ inhibitors to identify their putative binding site. Based on the structural data and docking analysis, a plausible mode-of-action for this novel class of FtsZ inhibitors is proposed. PMID:28555087

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

PubMed Central

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development. PMID:28638238

Differential Impact of Plasma Proteins on the Adhesion Efficiency of Vascular-Targeted Carriers (VTCs) in Blood of Common Laboratory Animals.

PubMed

Namdee, Katawut; Sobczynski, Daniel J; Onyskiw, Peter J; Eniola-Adefeso, Omolola

2015-12-16

Vascular-targeted carrier (VTC) interaction with human plasma is known to reduce targeted adhesion efficiency in vitro. However, the role of plasma proteins on the adhesion efficiency of VTCs in laboratory animals remains unknown. Here, in vitro blood flow assays are used to explore the effects of plasma from mouse, rabbit, and porcine on VTC adhesion. Porcine blood exhibited a strong negative plasma effect on VTC adhesion while no significant plasma effect was found with rabbit and mouse blood. A brush density poly(ethylene glycol) (PEG) on VTCs was effective at improving adhesion of microsized, but not nanosized, VTCs in porcine blood. Overall, the results suggest that porcine models, as opposed to mouse, can serve as better models in preclinical research for predicting the in vivo functionality of VTCs for use in humans. These considerations hold great importance for the design of various pharmaceutical products and development of reliable drug delivery systems.

Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A.

PubMed

Watanabe, Mariko; Iwakawa, Hiro-Oki; Tadakuma, Hisashi; Tomari, Yukihide

2017-10-13

Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major.

PubMed

Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

2017-01-01

We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens , only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens . Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development.

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Graphene/graphene oxide and their derivatives in the separation/isolation and preconcentration of protein species: A review.

PubMed

Chen, Xuwei; Hai, Xin; Wang, Jianhua

2016-05-30

The distinctive/unique electrical, chemical and optical properties make graphene/graphene oxide-based materials popular in the field of analytical chemistry. Its large surface offers excellent capacity to anchor target analyte, making it an powerful sorbent in the adsorption and preconcentration of trace level analyte of interest in the field of sample preparation. The large delocalized π-electron system of graphene framework provides strong affinity to species containing aromatic rings, such as proteins, and the abundant active sites on its surface offers the chance to modulate adsorption tendency towards specific protein via functional modification/decoration. This review provides an overview of the current research on graphene/graphene oxide-based materials as attractive and powerful adsorption media in the separation/isolation and preconcentration of protein species from biological sample matrixes. These practices are aiming at providing protein sample of high purity for further investigations and applications, or to achieve certain extent of enrichment prior to quantitative assay. In addition, the challenges and future perspectives in the related research fields have been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets.

PubMed

Vasaikar, Suhas; Bhatia, Pooja; Bhatia, Partap G; Chu Yaiw, Koon

2016-11-21

In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

PubMed Central

Navarre, William Wiley; Schneewind, Olaf

1999-01-01

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

Watching the action unfold: New cryo-EM images capture CRISPR’s interaction with target DNA | Center for Cancer Research

Cancer.gov

Using the Nobel-prize winning technique of cryo-EM, researchers led by CCR Senior Investigator Sriram Subramaniam, Ph.D., have captured a series of highly detailed images of a protein complex belonging to the CRISPR system that can be used by bacteria to recognize and destroy foreign DNA. The images reveal the molecule’s form before and after its interaction with DNA and help

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

PubMed

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

2017-11-14

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

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology

PubMed Central

Pawar, Sumit; Ungricht, Rosemarie; Tiefenboeck, Peter; Leroux, Jean-Christophe

2017-01-01

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER. PMID:28826471

The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study.

PubMed

Burslem, George M; Smith, Blake E; Lai, Ashton C; Jaime-Figueroa, Saul; McQuaid, Daniel C; Bondeson, Daniel P; Toure, Momar; Dong, Hanqing; Qian, Yimin; Wang, Jing; Crew, Andrew P; Hines, John; Crews, Craig M

2018-01-18

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP)

PubMed Central

Wang, Jigang; Zhang, Chong-Jing; Zhang, Jianbin; He, Yingke; Lee, Yew Mun; Chen, Songbi; Lim, Teck Kwang; Ng, Shukie; Shen, Han-Ming; Lin, Qingsong

2015-01-01

Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs. PMID:25600173

Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

PubMed

Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

2017-11-17

With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

THE DELICATE BALANCE BETWEEN SECRETED PROTEIN FOLDING AND ENDOPLASMIC RETICULUM-ASSOCIATED DEGRADATION IN HUMAN PHYSIOLOGY

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.

2014-01-01

Protein folding is a complex, error-prone process that often results in an irreparable protein by-product. These by-products can be recognized by cellular quality control machineries and targeted for proteasome-dependent degradation. The folding of proteins in the secretory pathway adds another layer to the protein folding “problem,” as the endoplasmic reticulum maintains a unique chemical environment within the cell. In fact, a growing number of diseases are attributed to defects in secretory protein folding, and many of these by-products are targeted for a process known as endoplasmic reticulum-associated degradation (ERAD). Since its discovery, research on the mechanisms underlying the ERAD pathway has provided new insights into how ERAD contributes to human health during both normal and diseases states. Links between ERAD and disease are evidenced from the loss of protein function as a result of degradation, chronic cellular stress when ERAD fails to keep up with misfolded protein production, and the ability of some pathogens to coopt the ERAD pathway. The growing number of ERAD substrates has also illuminated the differences in the machineries used to recognize and degrade a vast array of potential clients for this pathway. Despite all that is known about ERAD, many questions remain, and new paradigms will likely emerge. Clearly, the key to successful disease treatment lies within defining the molecular details of the ERAD pathway and in understanding how this conserved pathway selects and degrades an innumerable cast of substrates. PMID:22535891

Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

PubMed Central

Andreassen, Paul R.; Ren, Keqin

2016-01-01

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

Target discovery focused approaches to overcome bottlenecks in the exploitation of antimycobacterial natural products.

PubMed

Baptista, Rafael; Bhowmick, Sumana; Nash, Robert J; Baillie, Les; Mur, Luis Aj

2018-04-01

Tuberculosis is a major global health hazard. The search for new antimycobacterials has focused on such as screening combinational chemistry libraries or designing chemicals to target predefined pockets of essential bacterial proteins. The relative ineffectiveness of these has led to a reappraisal of natural products for new antimycobacterial drug leads. However, progress has been limited, we suggest through a failure in many cases to define the drug target and optimize the hits using this information. We highlight methods of target discovery needed to develop a drug into a candidate for clinical trials. We incorporate these into suggested analysis pipelines which could inform the research strategies to accelerate the development of new drug leads from natural products.

Bronchoprotection and bronchorelaxation in asthma: New targets, and new ways to target the old ones.

PubMed

Pera, Tonio; Penn, Raymond B

2016-08-01

Despite over 50years of inhaled beta-agonists and corticosteroids as the default management or rescue drugs for asthma, recent research suggests that new therapeutic options are likely to emerge. This belief stems from both an improved understanding of what causes and regulates airway smooth muscle (ASM) contraction, and the identification of new targets whose inhibition or activation can relax ASM. In this review we discuss the recent findings that provide new insight into ASM contractile regulation, a revolution in pharmacology that identifies new ways to "tune" G protein-coupled receptors to improve therapeutic efficacy, and the discovery of several novel targets/approaches capable of effecting bronchoprotection or bronchodilation. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeting the proteasome pathway.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2009-05-01

The ubiquitin-proteasome pathway functions as a main pathway in intracellular protein degradation and plays a vital role in almost all cellular events. Various inhibitors of this pathway have been developed for research purposes. The recent approval of bortezomib (PS-341, Velcade, a proteasome inhibitor, for the treatment of multiple myeloma has opened the way to the discovery of drugs targeting the proteasome and other components of the ubiquitin-proteasome pathway. We review the current understanding of the ubiquitin-proteasome pathway and inhibitors targeting this pathway, including proteasome inhibitors, as candidate drugs for chemical therapy. Preclinical and clinical data for inhibitors of the proteasome and the ubiquitin-proteasome pathway are discussed. The proteasome and other members in the ubiquitin-proteasome pathway have emerged as novel therapeutic targets.

Tumor-Intrinsic and Tumor-Extrinsic Factors Impacting Hsp90-Targeted Therapy

PubMed Central

Alarcon, S. V.; Mollapour, M.; Lee, M.-J.; Tsutsumi, S.; Lee, S.; Kim, Y. S.; Prince, T.; Apolo, A.; Giaccone, G.; Xu, W.; Neckers, L. M.; Trepel, J. B.

2012-01-01

In 1994 the first heat shock protein 90 (Hsp90) inhibitor was identified and Hsp90 was reported to be a target for anticancer therapeutics. In the past 18 years there have been 17 distinct Hsp90 inhibitors entered into clinical trial, and the small molecule Hsp90 inhibitors have been highly valuable as probes of the role of Hsp90 and its client proteins in cancer. Although no Hsp90 inhibitor has achieved regulatory approval, recently there has been significant progress in Hsp90 inhibitor clinical development, and in the past year RECIST responses have been documented in HER2-positive breast cancer and EML4-ALK-positive non-small cell lung cancer. All of the clinical Hsp90 inhibitors studied to date are specific in their target, i.e. they bind exclusively to Hsp90 and two related heat shock proteins. However, Hsp90 inhibitors are markedly pleiotropic, causing degradation of over 200 client proteins and impacting critical multiprotein complexes. Furthermore, it has only recently been appreciated that Hsp90 inhibitors can, paradoxically, cause transient activation of the protein kinase clients they are chaperoning, resulting in initiation of signal transduction and significant physiological events in both tumor and tumor microenvironment. An additional area of recent progress in Hsp90 research is in studies of the posttranslational modifications of Hsp90 itself and Hsp90 co-chaperone proteins. Together, a picture is emerging in which the impact of Hsp90 inhibitors is shaped by the tumor intracellular and extracellular milieu, and in which Hsp90 inhibitors impact tumor and host on a microenvironmental and systems level. Here we review the tumor intrinsic and extrinsic factors that impact the efficacy of small molecules engaging the Hsp90 chaperone machine. PMID:22804236

Human Spermatozoa Contain Multiple Targets for Protein S-Nitrosylation: An Alternative Mechanism of the Modulation of Sperm Function by Nitric Oxide?

PubMed Central

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

2009-01-01

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

Is Phosphoproteomics Ready for Clinical Research?

PubMed Central

Iliuk, Anton B.; Tao, W. Andy

2012-01-01

Background For many diseases such as cancer where phosphorylation-dependent signaling is the foundation of disease onset and progression, single-gene testing and genomic profiling alone are not sufficient in providing most critical information. The reason for this is that in these activated pathways the signaling changes and drug resistance are often not directly correlated with changes in protein expression levels. In order to obtain the essential information needed to evaluate pathway activation or the effects of certain drugs and therapies on the molecular level, the analysis of changes in protein phosphorylation is critical. Methods Existing approaches do not differentiate clinical disease subtypes on the protein and signaling pathway level, and therefore hamper the predictive management of the disease and the selection of therapeutic targets. Conclusions The mini-review examines the impact of emerging systems biology tools and the possibility of applying phosphoproteomics to clinical research. PMID:23159844

Inferring protein domains associated with drug side effects based on drug-target interaction network

PubMed Central

2013-01-01

Background Most phenotypic effects of drugs are involved in the interactions between drugs and their target proteins, however, our knowledge about the molecular mechanism of the drug-target interactions is very limited. One of challenging issues in recent pharmaceutical science is to identify the underlying molecular features which govern drug-target interactions. Results In this paper, we make a systematic analysis of the correlation between drug side effects and protein domains, which we call "pharmacogenomic features," based on the drug-target interaction network. We detect drug side effects and protein domains that appear jointly in known drug-target interactions, which is made possible by using classifiers with sparse models. It is shown that the inferred pharmacogenomic features can be used for predicting potential drug-target interactions. We also discuss advantages and limitations of the pharmacogenomic features, compared with the chemogenomic features that are the associations between drug chemical substructures and protein domains. Conclusion The inferred side effect-domain association network is expected to be useful for estimating common drug side effects for different protein families and characteristic drug side effects for specific protein domains. PMID:24565527

The fate and transport of the Cry1Ab protein in an agricultural field and laboratory aquatic microcosms.

PubMed

Strain, Katherine E; Lydy, Michael J

2015-08-01

Genetically engineered crops expressing insecticidal crystalline proteins derived from Bacillus thuringiensis (Bt), were commercialized almost two decades ago as a means to manage agricultural pests. The Bt proteins are highly specific and only lethal upon ingestion, limiting the scope of toxicity to target insects. However, concern of exposure to non-target organisms and negative public perceptions regarding Bt crops has caused controversy surrounding their use. The objective of this research was to monitor the fate and transport of a Bt protein, Cry1Ab, in a large-scale agricultural field containing maize expressing the Cry1Ab protein and a non-Bt near isoline, and in aquatic microcosms. The highest environmental concentrations of the Cry1Ab protein were found in runoff water and sediment, up to 130ngL(-1) and 143ngg(-1) dry weight, respectively, with the Cry1Ab protein detected in both Bt and non-Bt maize fields. As surface runoff and residual crop debris can transport Bt proteins to waterways adjacent to agricultural fields, a series of laboratory experiments were conducted to determine the potential fate of the Cry1Ab protein under different conditions. The results showed that sediment type and temperature can influence the degradation of the Cry1Ab protein in an aquatic system and that the Cry1Ab protein can persist for up to two months. Although Cry1Ab protein concentrations measured in the field soil indicate little exposure to terrestrial organisms, the consistent input of Bt-contaminated runoff and crop debris into agricultural waterways is relevant to understanding potential consequences to aquatic species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification of Proteins Fused to Maltose-Binding Protein.

PubMed

Lebendiker, Mario; Danieli, Tsafi

2017-01-01

Maltose-Binding Protein (MBP) is one of the most popular fusion partners being used for producing recombinant proteins in bacterial cells. MBP allows the use of a simple capture affinity step on Amylose-Agarose or Dextrin-Sepharose columns, resulting in a protein that is often 70-90 % pure in a single step. In addition to protein isolation applications, MBP provides a high degree of translation, and facilitates the proper folding and solubility of the target protein. This paper describes efficient procedures for isolating highly purified MBP target proteins. Special attention is given to considerations for downstream applications such as structural determination studies, protein activity assays, and assessing the chemical characteristics of the target protein.

Biosafety research for non-target organism risk assessment of RNAi-based GE plants

PubMed Central

Roberts, Andrew F.; Devos, Yann; Lemgo, Godwin N. Y.; Zhou, Xuguo

2015-01-01

RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of a symposium session at the 13th ISBGMO in Cape Town, South Africa. This paper continues that discussion by proposing research areas that may be beneficial for future environmental risk assessments of RNAi-based genetically modified plants, with a particular focus on non-target organism assessment. PMID:26594220

Kinase inhibitors for CNS diseases: an analysis of the recent patent literature.

PubMed

Amigoni, Federica; Legnaghi, Elena; Pevarello, Paolo

2012-05-01

Protein kinases (PKs), as members of an important target class in current pharmaceutical research, have been mostly exploited so far in therapeutic areas such as oncology and inflammation. However, basic research on some PKs as key components of molecular mechanisms underlying neurodegeneration and neuroprotection may translate into new medicines for CNS diseases in the next few years. This review is an account of recent patents dealing with kinase inhibitors primarily designed for CNS indications. CNS-directed patents on kinase modulators published after 2008 were surveyed using SciFinder(®) and public patent search engines. Some PK targets, such as GSK-3β, CDK5, ROCK and p38α MAPK, continue to attract interest even though a clinical proof-of-concept is yet to be attained in a CNS setting. Less established PKs such as LRRK2, MLK, PAK and DAPK-1 hold promise as valuable targets of the future.

Identification of a Novel Cancer Biomarker | Center for Cancer Research

Cancer.gov

During cancer development, cells accumulate a variety of mutations which alter their normal components and activities. One potential change is in the carbohydrate or sugar polymers which decorate proteins predominately found on the cell surface. The accessibility of these residues makes them ideal targets for the development of diagnostics or therapeutics.

High-Fat Diet Linked to Prostate Cancer Metastasis

Cancer.gov

A new study in mice has revealed a molecular link between a high-fat diet and the growth and spread of prostate cancer. As this Cancer Currents post explains, researchers also showed that an anti-obesity drug that targets a protein that controls fat synthesis could potentially be used to treat metastatic prostate cance

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

Zgurskaya, Helen; Smith, Jeremy

ORNL leveraged powerful supercomputing to support research led by University of Oklahoma scientists to identify chemicals that seek out and disrupt bacterial proteins called efflux pumps, known to be a major cause of antibiotic resistance. By running simulations on Titan, the team selected molecules most likely to target and potentially disable the assembly of efflux pumps found in E. coli bacteria cells.

New Drug Self-Assembles and Delivers Itself to Its Target | Center for Cancer Research

Cancer.gov

Viruses self-assemble with high precision and disassemble upon fusion with the cell membrane and deliver this way their cargo to the interior of the cells. Using this talent, viruses are able to inject whole proteins and large nucleic acid molecules into certain types of cells.

Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

NASA Astrophysics Data System (ADS)

Shah, Dhiral Ashwin

Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. The same concept of naturally occurring protein-protein interactions can also be implemented to selectively bring intracellular protein targets in close proximity to proteasomal degradation machinery in cells and effect their depletion from the cellular compartments. This approach will be able to not only target entire pool of proteins to ubiquitination-mediated degradation, but also to specific sub-pools of posttranslationally modified proteins in the cell, provided peptides having distinct binding affinities are identified for posttranslational modifications. This system can then be tested for intracellular protein delivery using nanoparticle carriers to identify roles of different posttranslational modifications on the protein's activity. In future work, we propose to develop a cellular detection system, based on GFP complementation, which can be used to evaluate the efficiency of different protein delivery carriers to internalize proteins into the cell cytosol. We envision the application of nanoscale materials as intracellular protein delivery vehicles to target diverse cell signaling pathways at the posttranslational level, and subsequent metabolic manipulation, which may have interesting therapeutic properties and can potentially target stem cell fate.

Interplay between cancer cell cycle and metabolism: Challenges, targets and therapeutic opportunities.

PubMed

Roy, Debmalya; Sheng, Gao Ying; Herve, Semukunzi; Carvalho, Evandro; Mahanty, Arpan; Yuan, Shengtao; Sun, Li

2017-05-01

A growing interest has emerged in the field of studying the cross-talk between cancer cell cycle and metabolism. In this review, we aimed to present how metabolism and cell cycle are correlated and how cancer cells get energy to drive cell cycle. Cell proliferation and cell death largely depend on the metabolic activity of the cell. Cell cycle proteins, e.g. cyclin D, cyclin dependent kinase (CDK), some pro-apoptotic and anti-apoptotic proteins, and P53 have been shown to be regulated by metabolic crosstalk. Dysregulation of this cross-talk between metabolism and cell cycle leads to degenerative disorder(s) and cancer. It is not fully understood the actual reason of aberration between metabolism and cell cycle, but it is a hallmark of cancer research. Herein, we discussed the role of some regulatory molecules relative of cell cycle and metabolism and highlight how they control the function of each other. We also pointed out, current therapeutic opportunities and some additional crucial therapeutic targets on these fields that could be a breakthrough in cancer research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation.

PubMed

Zhao, Likun; Zhang, Jiangyan; Xu, Huiming; Geng, Hao; Cheng, Yongqiang

2016-09-07

Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity.

Chemical biology based on target-selective degradation of proteins and carbohydrates using light-activatable organic molecules.

PubMed

Toshima, Kazunobu

2013-05-01

Proteins and carbohydrates play crucial roles in a wide range of biological processes, including serious diseases. The development of novel and innovative methods for selective control of specific proteins and carbohydrates functions has attracted much attention in the field of chemical biology. In this account article, the development of novel chemical tools, which can degrade target proteins and carbohydrates by irradiation with a specific wavelength of light under mild conditions without any additives, is introduced. This novel class of photochemical agents promise bright prospects for finding not only molecular-targeted bioprobes for understanding of the structure-activity relationships of proteins and carbohydrates but also novel therapeutic drugs targeting proteins and carbohydrates.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

PubMed

Hetényi, Anasztázia; Hegedűs, Zsófia; Fajka-Boja, Roberta; Monostori, Éva; Kövér, Katalin E; Martinek, Tamás A

2016-12-01

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1 H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15 N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15 N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Protein Knockdown Technology: Application of Ubiquitin Ligase to Cancer Therapy.

PubMed

Ohoka, Nobumichi; Shibata, Norihito; Hattori, Takayuki; Naito, Mikihiko

2016-01-01

Selective degradation of pathogenic proteins by small molecules in cells is a novel approach for development of therapeutic agents against various diseases, including cancer. We and others have developed a protein knockdown technology with a series of hybrid small compounds, called SNIPERs (Specific and Nongenetic IAP-dependent Protein ERasers); and peptidic chimeric molecules, called PROTACs (proteolysis-targeting chimeric molecules), which induce selective degradation of target proteins via the ubiquitin-proteasome pathway. These compounds include two different ligands connected by a linker; one is a ligand for a ubiquitin ligase and the other is a ligand for the target protein, which are expected to crosslink these proteins in cells. Theoretically, any cytosolic protein can be targeted for degradation by this technology. To date, several SNIPERs and PROTACs against various oncogenic proteins have been developed, which specifically induce polyubiquitylation and proteasomal degradation of the oncogenic proteins, resulting in cell death, growth arrest, or impaired migration of cancer cells. Thus, this protein knockdown technology has a great potential for cancer therapy.

Target Identification of Grape Seed Extract in Colorectal Cancer using Drug Affinity Responsive Target Stability (DARTS) Technique: Role of Endoplasmic Reticulum Stress Response Proteins

PubMed Central

Derry, Molly M.; Somasagara, Ranganatha; Raina, Komal; Kumar, Sushil; Gomez, Joe; Patel, Manisha; Agarwal, Rajesh; Agarwal, Chapla

2014-01-01

Various natural agents, including grape seed extract (GSE), have shown considerable chemopreventive and anti-cancer efficacy against different cancers in pre-clinical studies; however, their specific protein targets are largely unknown and thus, their clinical usefulness is marred by limited scientific evidences about their direct cellular targets. Accordingly, herein, employing, for the first time, the recently developed drug affinity responsive target stability (DARTS) technique, we aimed to profile the potential protein targets of GSE in human colorectal cancer (CRC) cells. Unlike other methods, which can cause chemical alteration of the drug components to allow for detection, this approach relies on the fact that a drug bound protein may become less susceptible to proteolysis and hence the enriched proteins can be detected by Mass Spectroscopy methods. Our results, utilizing the DARTS technique followed by examination of the spectral output by LC/MS and the MASCOT data, revealed that GSE targets endoplasmic reticulum (ER) stress response proteins resulting in overall down regulation of proteins involved in translation and that GSE also causes oxidative protein modifications, specifically on methionine amino acids residues on its protein targets. Corroborating these findings, mechanistic studies revealed that GSE indeed caused ER stress and strongly inhibited PI3k-Akt–mTOR pathway for its biological effects in CRC cells. Furthermore, bioenergetics studies indicated that GSE also interferes with glycolysis and mitochondrial metabolism in CRC cells. Together, the present study identifying GSE molecular targets in CRC cells, combined with its efficacy in vast pre-clinical CRC models, further supports its usefulness for CRC prevention and treatment. PMID:24724981

Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.

PubMed

Scott, Daniel J; Kummer, Lutz; Egloff, Pascal; Bathgate, Ross A D; Plückthun, Andreas

2014-11-01

The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Apoptin towards safe and efficient anticancer therapies.

PubMed

Backendorf, Claude; Noteborn, Mathieu H M

2014-01-01

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent.Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

PubMed

Jagdeo, Julienne M; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M; Jan, Eric

2018-04-15

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed t erminal a mine i sotopic l abeling of s ubstrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3C pro s) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3C pro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3C pro -targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3C pro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3C pro substrates in vivo , we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection. Copyright © 2018 Jagdeo et al.

Large-scale identification of target proteins of a glycosyltransferase isozyme by Lectin-IGOT-LC/MS, an LC/MS-based glycoproteomic approach

PubMed Central

Sugahara, Daisuke; Kaji, Hiroyuki; Sugihara, Kazushi; Asano, Masahide; Narimatsu, Hisashi

2012-01-01

Model organisms containing deletion or mutation in a glycosyltransferase-gene exhibit various physiological abnormalities, suggesting that specific glycan motifs on certain proteins play important roles in vivo. Identification of the target proteins of glycosyltransferase isozymes is the key to understand the roles of glycans. Here, we demonstrated the proteome-scale identification of the target proteins specific for a glycosyltransferase isozyme, β1,4-galactosyltransferase-I (β4GalT-I). Although β4GalT-I is the most characterized glycosyltransferase, its distinctive contribution to β1,4-galactosylation has been hardly described so far. We identified a large number of candidates for the target proteins specific to β4GalT-I by comparative analysis of β4GalT-I-deleted and wild-type mice using the LC/MS-based technique with the isotope-coded glycosylation site-specific tagging (IGOT) of lectin-captured N-glycopeptides. Our approach to identify the target proteins in a proteome-scale offers common features and trends in the target proteins, which facilitate understanding of the mechanism that controls assembly of a particular glycan motif on specific proteins. PMID:23002422

A comparative proteomic strategy for subcellular proteome research: ICAT approach coupled with bioinformatics prediction to ascertain rat liver mitochondrial proteins and indication of mitochondrial localization for catalase.

PubMed

Jiang, Xiao-Sheng; Dai, Jie; Sheng, Quan-Hu; Zhang, Lei; Xia, Qi-Chang; Wu, Jia-Rui; Zeng, Rong

2005-01-01

Subcellular proteomics, as an important step to functional proteomics, has been a focus in proteomic research. However, the co-purification of "contaminating" proteins has been the major problem in all the subcellular proteomic research including all kinds of mitochondrial proteome research. It is often difficult to conclude whether these "contaminants" represent true endogenous partners or artificial associations induced by cell disruption or incomplete purification. To solve such a problem, we applied a high-throughput comparative proteome experimental strategy, ICAT approach performed with two-dimensional LC-MS/MS analysis, coupled with combinational usage of different bioinformatics tools, to study the proteome of rat liver mitochondria prepared with traditional centrifugation (CM) or further purified with a Nycodenz gradient (PM). A total of 169 proteins were identified and quantified convincingly in the ICAT analysis, in which 90 proteins have an ICAT ratio of PM:CM>1.0, while another 79 proteins have an ICAT ratio of PM:CM<1.0. Almost all the proteins annotated as mitochondrial according to Swiss-Prot annotation, bioinformatics prediction, and literature reports have a ratio of PM:CM>1.0, while proteins annotated as extracellular or secreted, cytoplasmic, endoplasmic reticulum, ribosomal, and so on have a ratio of PM:CM<1.0. Catalase and AP endonuclease 1, which have been known as peroxisomal and nuclear, respectively, have shown a ratio of PM:CM>1.0, confirming the reports about their mitochondrial location. Moreover, the 125 proteins with subcellular location annotation have been used as a testing dataset to evaluate the efficiency for ascertaining mitochondrial proteins by ICAT analysis and the bioinformatics tools such as PSORT, TargetP, SubLoc, MitoProt, and Predotar. The results indicated that ICAT analysis coupled with combinational usage of different bioinformatics tools could effectively ascertain mitochondrial proteins and distinguish contaminant proteins and even multilocation proteins. Using such a strategy, many novel proteins, known proteins without subcellular location annotation, and even known proteins that have been annotated as other locations have been strongly indicated for their mitochondrial location.

Targeting Neuroblastoma Cell Surface Proteins: Recommendations for Homology Modeling of hNET, ALK, and TrkB.

PubMed

Haddad, Yazan; Heger, Zbyněk; Adam, Vojtech

2017-01-01

Targeted therapy is a promising approach for treatment of neuroblastoma as evident from the large number of targeting agents employed in clinical practice today. In the absence of known crystal structures, researchers rely on homology modeling to construct template-based theoretical structures for drug design and testing. Here, we discuss three candidate cell surface proteins that are suitable for homology modeling: human norepinephrine transporter (hNET), anaplastic lymphoma kinase (ALK), and neurotrophic tyrosine kinase receptor 2 (NTRK2 or TrkB). When choosing templates, both sequence identity and structure quality are important for homology modeling and pose the first of many challenges in the modeling process. Homology modeling of hNET can be improved using template models of dopamine and serotonin transporters instead of the leucine transporter (LeuT). The extracellular domains of ALK and TrkB are yet to be exploited by homology modeling. There are several idiosyncrasies that require direct attention throughout the process of model construction, evaluation and refinement. Shifts/gaps in the alignment between the template and target, backbone outliers and side-chain rotamer outliers are among the main sources of physical errors in the structures. Low-conserved regions can be refined with loop modeling method. Residue hydrophobicity, accessibility to bound metals or glycosylation can aid in model refinement. We recommend resolving these idiosyncrasies as part of "good modeling practice" to obtain highest quality model. Decreasing physical errors in protein structures plays major role in the development of targeting agents and understanding of chemical interactions at the molecular level.

Epigenetic Drug Repositioning for Alzheimer's Disease Based on Epigenetic Targets in Human Interactome.

PubMed

Chatterjee, Paulami; Roy, Debjani; Rathi, Nitin

2018-01-01

Epigenetics has emerged as an important field in drug discovery. Alzheimer's disease (AD), the leading neurodegenerative disorder throughout the world, is shown to have an epigenetic basis. Currently, there are very few effective epigenetic drugs available for AD. In this work, for the first time we have proposed 14 AD repositioning epigenetic drugs and identified their targets from extensive human interactome. Interacting partners of the AD epigenetic proteins were identified from the extensive human interactome to construct Epigenetic Protein-Protein Interaction Network (EP-PPIN). Epigenetic Drug-Target Network (EP-DTN) was constructed with the drugs associated with the proteins of EP-PPIN. Regulation of non-coding RNAs associated with the target proteins of these drugs was also studied. AD related target proteins, epigenetic targets, enriched pathways, and functional categories of the proposed repositioning drugs were also studied. The proposed 14 AD epigenetic repositioning drugs have overlapping targets and miRs with known AD epigenetic targets and miRs. Furthermore, several shared functional categories and enriched pathways were obtained for these drugs with FDA approved epigenetic drugs and known AD drugs. The findings of our work might provide insight into future AD epigenetic-therapeutics.

How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies.

PubMed

Gagat, Przemysław; Bodył, Andrzej; Mackiewicz, Paweł

2013-07-11

It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host proteins, which already were targeted through the ES, later were adapted to reach the plastid via the vesicular trafficking. They represent a derived class of higher plant plastid-targeted proteins with an unusual evolutionary history.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions.

PubMed

Du, Jinping; Rehm, Bernd H A

2017-11-02

Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction. By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b). Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Inside Job: Methods for Delivering Proteins to the Interior of Mammalian Cells.

PubMed

Bruce, Virginia J; McNaughton, Brian R

2017-08-17

Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research. Copyright © 2017. Published by Elsevier Ltd.

Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

PubMed

Ahn, Yeong Hee; Shin, Park Min; Kim, Yong-Sam; Oh, Na Ree; Ji, Eun Sun; Kim, Kwang Hoe; Lee, Yeon Jung; Kim, Sung Ho; Yoo, Jong Shin

2013-11-07

A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins.

Selective Targeting of Proteins within Secretory Pathway for Endoplasmic Reticulum-associated Degradation

PubMed Central

Vecchi, Lara; Petris, Gianluca; Bestagno, Marco; Burrone, Oscar R.

2012-01-01

The endoplasmic reticulum-associated degradation (ERAD) is a cellular quality control mechanism to dispose of misfolded proteins of the secretory pathway via proteasomal degradation. SEL1L is an ER-resident protein that participates in identification of misfolded molecules as ERAD substrates, therefore inducing their ER-to-cytosol retrotranslocation and degradation. We have developed a novel class of fusion proteins, termed degradins, composed of a fragment of SEL1L fused to a target-specific binding moiety located on the luminal side of the ER. The target-binding moiety can be a ligand of the target or derived from specific mAbs. Here, we describe the ability of degradins with two different recognition moieties to promote degradation of a model target. Degradins recognize the target protein within the ER both in secretory and membrane-bound forms, inducing their degradation following retrotranslocation to the cytosol. Thus, degradins represent an effective technique to knock-out proteins within the secretory pathway with high specificity. PMID:22523070

Deubiquitylating enzymes as cancer stem cell therapeutics.

PubMed

Haq, Saba; Suresh, Bharathi; Ramakrishna, Suresh

2018-01-01

The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The Relationship of Novel Plasma Proteins in the Early Neonatal Period With Retinopathy of Prematurity

PubMed Central

Lynch, Anne M.; Wagner, Brandie D.; Mandava, Naresh; Palestine, Alan G.; Mourani, Peter M.; McCourt, Emily A.; Oliver, Scott C. N.; Abman, Steven H.

2016-01-01

Purpose Retinopathy of prematurity (ROP) is a vision-threatening disease associated with abnormal retinal vascular development. Proteins from the insulin-like growth factor pathway are related to ROP. However, there is a paucity of research on the role of other proteins in ROP. The aim of this study was to identify plasma proteins related to clinically significant ROP. Methods We measured 1121 plasma proteins in the early neonatal period in infants at risk for ROP using an aptamer-based proteomic technology. The primary aim of the study was to compare plasma protein concentrations in infants who did (n = 12) and did not (n = 23) subsequently develop clinically significant ROP using logistic regression. As a secondary aim, we examined patterns in the proteins across categories of clinically significant, low-grade, and no ROP groups. Results Lower levels of 16 proteins were associated with an increased risk of clinically significant ROP. In this group, superoxide dismutase (Mn), mitochondrial (MnSOD), and chordin-like protein 1 (CRDL1) were highly ranked. Other proteins in this group included: C-C motif chemokine 14 (HCC-1), prolactin, insulin-like growth factor-binding protein 7 (IGFBP-7), and eotaxin. Higher levels of 12 proteins were associated with a higher risk for ROP. Fibroblast growth factor 19 (FGF-19) was the top-ranked protein target followed by hepatocyte growth factor-like protein (MSP), luteinizing hormone (LH), cystatin M, plasminogen, and proprotein convertase subtilisin/kexin type 9 (PCSK9). We also noted different patterns in the trend of concentrations of proteins across the clinically significant, low-grade, and no ROP groups. Conclusions We discovered plasma proteins with novel associations with clinically significant ROP (MnSOD, CRDL1, PCSK9), proteins with links to established ROP signaling pathways (IGFBP-7), and proteins such as MnSOD that may be a target for future therapeutic interventions. PMID:27679852

Targeting the RAS oncogene

PubMed Central

Takashima, Asami

2013-01-01

Introduction The Ras proteins (K-Ras, N-Ras, H-Ras) are GTPases that function as molecular switches for a variety of critical cellular activities and their function is tightly and temporally regulated in normal cells. Oncogenic mutations in the RAS genes, which create constitutively-active Ras proteins, can result in uncontrolled proliferation or survival in tumor cells. Areas covered The paper discusses three therapeutic approaches targeting the Ras pathway in cancer: 1) Ras itself, 2) Ras downstream pathways, and 3) synthetic lethality. The most adopted approach is targeting Ras downstream signaling, and specifically the PI3K-AKT-mTOR and Raf-MEK pathways, as they are frequently major oncogenic drivers in cancers with high Ras signaling. Although direct targeting of Ras has not been successful clinically, newer approaches being investigated in preclinical studies, such as RNA interference-based and synthetic lethal approaches, promise great potential for clinical application. Expert opinion The challenges of current and emerging therapeutics include the lack of “tumor specificity” and their limitation to those cancers which are “dependent” upon aberrant Ras signaling for survival. While the newer approaches have the potential to overcome these limitations, they also highlight the importance of robust preclinical studies and bidirectional translational research for successful clinical development of Ras-related targeted therapies. PMID:23360111

Mass spectrometric analyses of organophosphate insecticide oxon protein adducts.

PubMed

Thompson, Charles M; Prins, John M; George, Kathleen M

2010-01-01

Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. A number of OP-based insecticides share common structural elements that result in predictable OP-protein adducts. The resultant OP-protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure.

Mass Spectrometric Analyses of Organophosphate Insecticide Oxon Protein Adducts

PubMed Central

Thompson, Charles M.; Prins, John M.; George, Kathleen M.

2010-01-01

Objective Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. Data sources and extraction We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. Data synthesis A number of OP-based insecticides share common structural elements that result in predictable OP–protein adducts. The resultant OP–protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. Conclusions MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure. PMID:20056576

Combination of atomic force microscopy and mass spectrometry for the detection of target protein in the serum samples of children with autism spectrum disorders

NASA Astrophysics Data System (ADS)

Kaysheva, A. L.; Pleshakova, T. O.; Kopylov, A. T.; Shumov, I. D.; Iourov, I. Y.; Vorsanova, S. G.; Yurov, Y. B.; Ziborov, V. S.; Archakov, A. I.; Ivanov, Y. D.

2017-10-01

Possibility of detection of target proteins associated with development of autistic disorders in children with use of combined atomic force microscopy and mass spectrometry (AFM/MS) method is demonstrated. The proposed method is based on the combination of affine enrichment of proteins from biological samples and visualization of these proteins by AFM and MS analysis with quantitative detection of target proteins.

Targeting malaria parasite proteins to the erythrocyte.

PubMed

Templeton, Thomas J; Deitsch, Kirk W

2005-09-01

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Recording information on protein complexes in an information management system

PubMed Central

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

2011-01-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

HIV envelope glycoprotein imaged at high resolution | Center for Cancer Research

Cancer.gov

The outer surface of the human immunodeficiency virus (HIV) is surrounded by an envelope studded with spike-shaped glycoproteins called Env that help the deadly virus identify, bind, and infect cells. When unbound, Env exists in a “closed” conformational state. Upon binding with target cells, such as CD4+ T cells, the protein transitions to an “open” configuration. Given that Env is the only viral protein expressed on HIV’s surface, knowing its detailed structure—especially in the unbound state—may be critical for designing antibodies and vaccines against HIV.

A comparative study of disease genes and drug targets in the human protein interactome

PubMed Central

2015-01-01

Background Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. Results In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. Conclusions The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing. PMID:25861037

A comparative study of disease genes and drug targets in the human protein interactome.

PubMed

Sun, Jingchun; Zhu, Kevin; Zheng, W; Xu, Hua

2015-01-01

Disease genes cause or contribute genetically to the development of the most complex diseases. Drugs are the major approaches to treat the complex disease through interacting with their targets. Thus, drug targets are critical for treatment efficacy. However, the interrelationship between the disease genes and drug targets is not clear. In this study, we comprehensively compared the network properties of disease genes and drug targets for five major disease categories (cancer, cardiovascular disease, immune system disease, metabolic disease, and nervous system disease). We first collected disease genes from genome-wide association studies (GWAS) for five disease categories and collected their corresponding drugs based on drugs' Anatomical Therapeutic Chemical (ATC) classification. Then, we obtained the drug targets for these five different disease categories. We found that, though the intersections between disease genes and drug targets were small, disease genes were significantly enriched in targets compared to their enrichment in human protein-coding genes. We further compared network properties of the proteins encoded by disease genes and drug targets in human protein-protein interaction networks (interactome). The results showed that the drug targets tended to have higher degree, higher betweenness, and lower clustering coefficient in cancer Furthermore, we observed a clear fraction increase of disease proteins or drug targets in the near neighborhood compared with the randomized genes. The study presents the first comprehensive comparison of the disease genes and drug targets in the context of interactome. The results provide some foundational network characteristics for further designing computational strategies to predict novel drug targets and drug repurposing.

"Multiple partial recognitions in dynamic equilibrium" in the binding sites of proteins form the molecular basis of promiscuous recognition of structurally diverse ligands.

PubMed

Kohda, Daisuke

2018-04-01

Promiscuous recognition of ligands by proteins is as important as strict recognition in numerous biological processes. In living cells, many short, linear amino acid motifs function as targeting signals in proteins to specify the final destination of the protein transport. In general, the target signal is defined by a consensus sequence containing wild-characters, and hence represented by diverse amino acid sequences. The classical lock-and-key or induced-fit/conformational selection mechanism may not cover all aspects of the promiscuous recognition. On the basis of our crystallographic and NMR studies on the mitochondrial Tom20 protein-presequence interaction, we proposed a new hypothetical mechanism based on "a rapid equilibrium of multiple states with partial recognitions". This dynamic, multiple recognition mode enables the Tom20 receptor to recognize diverse mitochondrial presequences with nearly equal affinities. The plant Tom20 is evolutionally unrelated to the animal Tom20 in our study, but is a functional homolog of the animal/fungal Tom20. NMR studies by another research group revealed that the presequence binding by the plant Tom20 was not fully explained by simple interaction modes, suggesting the presence of a similar dynamic, multiple recognition mode. Circumstantial evidence also suggested that similar dynamic mechanisms may be applicable to other promiscuous recognitions of signal peptides by the SRP54/Ffh and SecA proteins.

Regulation of Skeletal Muscle Plasticity by Protein Arginine Methyltransferases and Their Potential Roles in Neuromuscular Disorders

PubMed Central

Stouth, Derek W.; vanLieshout, Tiffany L.; Shen, Nicole Y.; Ljubicic, Vladimir

2017-01-01

Protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the methylation of arginine residues on target proteins, thereby mediating a diverse set of intracellular functions that are indispensable for survival. Indeed, full-body knockouts of specific PRMTs are lethal and PRMT dysregulation has been implicated in the most prevalent chronic disorders, such as cancers and cardiovascular disease (CVD). PRMTs are now emerging as important mediators of skeletal muscle phenotype and plasticity. Since their first description in muscle in 2002, a number of studies employing wide varieties of experimental models support the hypothesis that PRMTs regulate multiple aspects of skeletal muscle biology, including development and regeneration, glucose metabolism, as well as oxidative metabolism. Furthermore, investigations in non-muscle cell types strongly suggest that proteins, such as peroxisome proliferator-activated receptor-γ coactivator-1α, E2F transcription factor 1, receptor interacting protein 140, and the tumor suppressor protein p53, are putative downstream targets of PRMTs that regulate muscle phenotype determination and remodeling. Recent studies demonstrating that PRMT function is dysregulated in Duchenne muscular dystrophy (DMD), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS) suggests that altering PRMT expression and/or activity may have therapeutic value for neuromuscular disorders (NMDs). This review summarizes our understanding of PRMT biology in skeletal muscle, and identifies uncharted areas that warrant further investigation in this rapidly expanding field of research. PMID:29163212

CURE Scholar Spotlight - Dr. Brady

Cancer.gov

Donita C. Brady, a Research Associate Senior at the Department of Pharmacology and Cancer Biology at Duke University, is investigating the role that copper plays in cell growth and tumor biology. Inspired by her mentor Christopher Counter, a cancer biologist, and Dennis Thiele, a copper biologist, at Duke University, Brady has a unique interest in the way copper interacts with protein pathways, such as the BRAF (a human gene that directs cell growth)-MEK-ERK pathway, which is a major target for targeted cancer therapies because the BRAF gene is mutated 60% of the time in melanoma.

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

PubMed

Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

Elucidation of the Structure Formation of Polymer-Conjugated Proteins in Solution and Block Copolymer Templates

NASA Astrophysics Data System (ADS)

Ferebee, Rachel L.

The broader technical objective of this work is to contribute to the development of enzyme-functionalized nanoporous membranes that can function as autonomous and target selective dynamic separators. The scientific objective of the research performed within this thesis is to elucidate the parameters that control the mixing of proteins in organic host materials and in block copolymers templates in particular. A "biomimetic" membrane system that uses enzymes to selectively neutralize targets and trigger a change in permeability of nanopores lined with a pH-responsive polymer has been fabricated and characterized. Mechanical and functional stability, as well as scalability, have been demonstrated for this system. Additional research has focused on the role of polymeric ligands on the solubility characteristics of the model protein, Bovine Serum Albumin (BSA). For this purpose BSA was conjugated with poly(ethylene glycol) (PEG) ligands of varied degree of polymerization and grafting density. Combined static and dynamic light scattering was used (in conjunction with MALDI-TOF) to determine the second virial coefficient in PBS solutions. At a given mass fraction PEG or average number of grafts, the solubility of BSA-PEG conjugates is found to increase with the degree of polymerization of conjugated PEG. This result informs the synthesis of protein-conjugate systems that are optimized for the fabrication of block copolymer blend materials with maximum protein loading. Blends of BSA-PEG conjugates and block copolymer (BCP) matrices were fabricated to evaluate the dispersion morphology and solubility limits in a model system. Electron microscopy was used to evaluate the changes in lamellar spacing with increased filling fraction of BSA-PEG conjugates.

Staying Alive: Cancer Cells Expressing Mutant KRas Depend on ERH for Survival | Center for Cancer Research

Cancer.gov

The small G-protein KRas acts like a molecular switch, turning on and off pro-growth signaling pathways within cells when appropriate. In a large number of cancers, KRas is permanently turned on by a variety of mutations and drives the constant growth of these tumor cells. KRas itself has proved to be a poor drug target so researchers in the laboratory of Ji Luo, Ph.D., in

Contributing to Tumor Molecular Characterization Projects with a Global Impact | Office of Cancer Genomics

Cancer.gov

My name is Nicholas Griner and I am the Scientific Program Manager for the Cancer Genome Characterization Initiative (CGCI) in the Office of Cancer Genomics (OCG). Until recently, I spent most of my scientific career working in a cancer research laboratory. In my postdoctoral training, my research focused on identifying novel pathways that contribute to both prostate and breast cancers and studying proteins within these pathways that may be targeted with cancer drugs.