Mining host-pathogen protein interactions to characterize Burkholderia mallei infectivity mechanisms.

PubMed

Memišević, Vesna; Zavaljevski, Nela; Rajagopala, Seesandra V; Kwon, Keehwan; Pieper, Rembert; DeShazer, David; Reifman, Jaques; Wallqvist, Anders

2015-03-01

Burkholderia pathogenicity relies on protein virulence factors to control and promote bacterial internalization, survival, and replication within eukaryotic host cells. We recently used yeast two-hybrid (Y2H) screening to identify a small set of novel Burkholderia proteins that were shown to attenuate disease progression in an aerosol infection animal model using the virulent Burkholderia mallei ATCC 23344 strain. Here, we performed an extended analysis of primarily nine B. mallei virulence factors and their interactions with human proteins to map out how the bacteria can influence and alter host processes and pathways. Specifically, we employed topological analyses to assess the connectivity patterns of targeted host proteins, identify modules of pathogen-interacting host proteins linked to processes promoting infectivity, and evaluate the effect of crosstalk among the identified host protein modules. Overall, our analysis showed that the targeted host proteins generally had a large number of interacting partners and interacted with other host proteins that were also targeted by B. mallei proteins. We also introduced a novel Host-Pathogen Interaction Alignment (HPIA) algorithm and used it to explore similarities between host-pathogen interactions of B. mallei, Yersinia pestis, and Salmonella enterica. We inferred putative roles of B. mallei proteins based on the roles of their aligned Y. pestis and S. enterica partners and showed that up to 73% of the predicted roles matched existing annotations. A key insight into Burkholderia pathogenicity derived from these analyses of Y2H host-pathogen interactions is the identification of eukaryotic-specific targeted cellular mechanisms, including the ubiquitination degradation system and the use of the focal adhesion pathway as a fulcrum for transmitting mechanical forces and regulatory signals. This provides the mechanisms to modulate and adapt the host-cell environment for the successful establishment of host infections and intracellular spread.

Understanding protein-nanoparticle interaction: a new gateway to disease therapeutics.

PubMed

Giri, Karuna; Shameer, Khader; Zimmermann, Michael T; Saha, Sounik; Chakraborty, Prabir K; Sharma, Anirudh; Arvizo, Rochelle R; Madden, Benjamin J; Mccormick, Daniel J; Kocher, Jean-Pierre A; Bhattacharya, Resham; Mukherjee, Priyabrata

2014-06-18

Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses.

Integrating Structure to Protein-Protein Interaction Networks That Drive Metastasis to Brain and Lung in Breast Cancer

PubMed Central

Engin, H. Billur; Guney, Emre; Keskin, Ozlem; Oliva, Baldo; Gursoy, Attila

2013-01-01

Blocking specific protein interactions can lead to human diseases. Accordingly, protein interactions and the structural knowledge on interacting surfaces of proteins (interfaces) have an important role in predicting the genotype-phenotype relationship. We have built the phenotype specific sub-networks of protein-protein interactions (PPIs) involving the relevant genes responsible for lung and brain metastasis from primary tumor in breast cancer. First, we selected the PPIs most relevant to metastasis causing genes (seed genes), by using the “guilt-by-association” principle. Then, we modeled structures of the interactions whose complex forms are not available in Protein Databank (PDB). Finally, we mapped mutations to interface structures (real and modeled), in order to spot the interactions that might be manipulated by these mutations. Functional analyses performed on these sub-networks revealed the potential relationship between immune system-infectious diseases and lung metastasis progression, but this connection was not observed significantly in the brain metastasis. Besides, structural analyses showed that some PPI interfaces in both metastasis sub-networks are originating from microbial proteins, which in turn were mostly related with cell adhesion. Cell adhesion is a key mechanism in metastasis, therefore these PPIs may be involved in similar molecular pathways that are shared by infectious disease and metastasis. Finally, by mapping the mutations and amino acid variations on the interface regions of the proteins in the metastasis sub-networks we found evidence for some mutations to be involved in the mechanisms differentiating the type of the metastasis. PMID:24278371

XLinkDB 2.0: integrated, large-scale structural analysis of protein crosslinking data

PubMed Central

Schweppe, Devin K.; Zheng, Chunxiang; Chavez, Juan D.; Navare, Arti T.; Wu, Xia; Eng, Jimmy K.; Bruce, James E.

2016-01-01

Motivation: Large-scale chemical cross-linking with mass spectrometry (XL-MS) analyses are quickly becoming a powerful means for high-throughput determination of protein structural information and protein–protein interactions. Recent studies have garnered thousands of cross-linked interactions, yet the field lacks an effective tool to compile experimental data or access the network and structural knowledge for these large scale analyses. We present XLinkDB 2.0 which integrates tools for network analysis, Protein Databank queries, modeling of predicted protein structures and modeling of docked protein structures. The novel, integrated approach of XLinkDB 2.0 enables the holistic analysis of XL-MS protein interaction data without limitation to the cross-linker or analytical system used for the analysis. Availability and Implementation: XLinkDB 2.0 can be found here, including documentation and help: http://xlinkdb.gs.washington.edu/. Contact: jimbruce@uw.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153666

Predicting Physical Interactions between Protein Complexes*

PubMed Central

Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

2013-01-01

Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732

Receptor Tyrosine Kinase MET Interactome and Neurodevelopmental Disorder Partners at the Developing Synapse.

PubMed

Xie, Zhihui; Li, Jing; Baker, Jonathan; Eagleson, Kathie L; Coba, Marcelo P; Levitt, Pat

2016-12-15

Atypical synapse development and plasticity are implicated in many neurodevelopmental disorders (NDDs). NDD-associated, high-confidence risk genes have been identified, yet little is known about functional relationships at the level of protein-protein interactions, which are the dominant molecular bases responsible for mediating circuit development. Proteomics in three independent developing neocortical synaptosomal preparations identified putative interacting proteins of the ligand-activated MET receptor tyrosine kinase, an autism risk gene that mediates synapse development. The candidates were translated into interactome networks and analyzed bioinformatically. Additionally, three independent quantitative proximity ligation assays in cultured neurons and four independent immunoprecipitation analyses of synaptosomes validated protein interactions. Approximately 11% (8/72) of MET-interacting proteins, including SHANK3, SYNGAP1, and GRIN2B, are associated with NDDs. Proteins in the MET interactome were translated into a novel MET interactome network based on human protein-protein interaction databases. High-confidence genes from different NDD datasets that encode synaptosomal proteins were analyzed for being enriched in MET interactome proteins. This was found for autism but not schizophrenia, bipolar disorder, major depressive disorder, or attention-deficit/hyperactivity disorder. There is correlated gene expression between MET and its interactive partners in developing human temporal and visual neocortices but not with highly expressed genes that are not in the interactome. Proximity ligation assays and biochemical analyses demonstrate that MET-protein partner interactions are dynamically regulated by receptor activation. The results provide a novel molecular framework for deciphering the functional relations of key regulators of synaptogenesis that contribute to both typical cortical development and to NDDs. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

PubMed Central

Hida, Naoki; Awais, Muhammad; Takeuchi, Masaki; Ueno, Naoto; Tashiro, Mayuri; Takagi, Chiyo; Singh, Tanuja; Hayashi, Makoto; Ohmiya, Yoshihiro; Ozawa, Takeaki

2009-01-01

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. PMID:19536355

Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

DOE PAGES

Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

2006-01-01

The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay.

PubMed

Hudry, Bruno; Viala, Séverine; Graba, Yacine; Merabet, Samir

2011-01-28

Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Interaction between core protein of classical swine fever virus with cellular IQGAP1 proetin appears essential for virulence in swine

USDA-ARS?s Scientific Manuscript database

Here we show that IQGAP1, a cellular protein that plays a pivotal role as a regulator of the cytoskeleton affecting cell adhesion, polarization and migration, interacts with Classical Swine Fever Virus (CSFV) Core protein. Sequence analyses identified a defined set of residues within CSFV Core prote...

Proteomics approach combined with biochemical attributes to elucidate compatible and incompatible plant-virus interactions between Vigna mungo and Mungbean Yellow Mosaic India Virus.

PubMed

Kundu, Subrata; Chakraborty, Dipjyoti; Kundu, Anirban; Pal, Amita

2013-01-01

Vigna mungo, a tropical leguminous plant, highly susceptible to yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus (MYMIV) resulting in high yield penalty. The molecular events occurring during compatible and incompatible interactions between V. mungo and MYMIV pathosystem are yet to be explored. In this study biochemical analyses in conjunction with proteomics of MYMIV-susceptible and -resistant V. mungo genotypes were executed to get an insight in the molecular events during compatible and incompatible plant-virus interactions. Biochemical analysis revealed an increase in phenolics, hydrogen peroxide and carbohydrate contents in both compatible and incompatible interactions; but the magnitudes were higher during incompatible interaction. In the resistant genotype the activities of superoxide dismutase and ascorbate peroxidase increased significantly, while catalase activity decreased. Comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified 109 differentially abundant proteins at 3, 7 and 14 days post MYMIV-inoculation. Proteins of several functional categories were differentially changed in abundance during both compatible and incompatible interactions. Among these, photosynthesis related proteins were mostly affected in the susceptible genotype resulting in reduced photosynthesis rate under MYMIV-stress. Differential intensities of chlorophyll fluorescence and chlorophyll contents are in congruence with proteomics data. It was revealed that Photosystem II electron transports are the primary targets of MYMIV during pathogenesis. Quantitative real time PCR analyses of selected genes corroborates with respective protein abundance during incompatible interaction. The network of various cellular pathways that are involved in inducing defense response contains several conglomerated cores of nodal proteins, of which ascorbate peroxidase, rubisco activase and serine/glycine hydroxymethyl transferase are the three major hubs with high connectivity. These nodal proteins play the crucial role of key regulators in bringing about a coordinated defense response in highly orchestrated manner. Biochemical and proteomic analyses revealed early accumulation of the defense/stress related proteins involved in ROS metabolism during incompatible interaction. The robustness in induction of defense/stress and signal transduction related proteins is the key factor in inducing resistance. The mechanism of MYMIV-resistance in V. mungo involves redirection of carbohydrate flux towards pentose phosphate pathway. Some of these identified, differentially regulated proteins are also conferring abiotic stress responses illustrating harmony amongst different stress responses. To the best of our knowledge, this is the lone study deciphering differential regulations of V. mungo leaf proteome upon MYMIV infection elucidating the mode of resistance response at the biochemical level.

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

PubMed Central

Zhang, Chongxu; Nielsen, Maria E. O.; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J.; Andersen, Jens S.; Yao, Gang

2013-01-01

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1’s defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made. PMID:22836166

Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

PubMed

Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

2012-09-01

Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

SBION: A Program for Analyses of Salt-Bridges from Multiple Structure Files.

PubMed

Gupta, Parth Sarthi Sen; Mondal, Sudipta; Mondal, Buddhadev; Islam, Rifat Nawaz Ul; Banerjee, Shyamashree; Bandyopadhyay, Amal K

2014-01-01

Salt-bridge and network salt-bridge are specific electrostatic interactions that contribute to the overall stability of proteins. In hierarchical protein folding model, these interactions play crucial role in nucleation process. The advent and growth of protein structure database and its availability in public domain made an urgent need for context dependent rapid analysis of salt-bridges. While these analyses on single protein is cumbersome and time-consuming, batch analyses need efficient software for rapid topological scan of a large number of protein for extracting details on (i) fraction of salt-bridge residues (acidic and basic). (ii) Chain specific intra-molecular salt-bridges, (iii) inter-molecular salt-bridges (protein-protein interactions) in all possible binary combinations (iv) network salt-bridges and (v) secondary structure distribution of salt-bridge residues. To the best of our knowledge, such efficient software is not available in public domain. At this juncture, we have developed a program i.e. SBION which can perform all the above mentioned computations for any number of protein with any number of chain at any given distance of ion-pair. It is highly efficient, fast, error-free and user friendly. Finally we would say that our SBION indeed possesses potential for applications in the field of structural and comparative bioinformatics studies. SBION is freely available for non-commercial/academic institutions on formal request to the corresponding author (akbanerjee@biotech.buruniv.ac.in).

Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

PubMed

Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

2017-10-07

The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

MD simulations of papillomavirus DNA-E2 protein complexes hints at a protein structural code for DNA deformation.

PubMed

Falconi, M; Oteri, F; Eliseo, T; Cicero, D O; Desideri, A

2008-08-01

The structural dynamics of the DNA binding domains of the human papillomavirus strain 16 and the bovine papillomavirus strain 1, complexed with their DNA targets, has been investigated by modeling, molecular dynamics simulations, and nuclear magnetic resonance analysis. The simulations underline different dynamical features of the protein scaffolds and a different mechanical interaction of the two proteins with DNA. The two protein structures, although very similar, show differences in the relative mobility of secondary structure elements. Protein structural analyses, principal component analysis, and geometrical and energetic DNA analyses indicate that the two transcription factors utilize a different strategy in DNA recognition and deformation. Results show that the protein indirect DNA readout is not only addressable to the DNA molecule flexibility but it is finely tuned by the mechanical and dynamical properties of the protein scaffold involved in the interaction.

Protein-Protein Interactions in a Crowded Environment: An Analysis via Cross-Docking Simulations and Evolutionary Information

PubMed Central

Lopes, Anne; Sacquin-Mora, Sophie; Dimitrova, Viktoriya; Laine, Elodie; Ponty, Yann; Carbone, Alessandra

2013-01-01

Large-scale analyses of protein-protein interactions based on coarse-grain molecular docking simulations and binding site predictions resulting from evolutionary sequence analysis, are possible and realizable on hundreds of proteins with variate structures and interfaces. We demonstrated this on the 168 proteins of the Mintseris Benchmark 2.0. On the one hand, we evaluated the quality of the interaction signal and the contribution of docking information compared to evolutionary information showing that the combination of the two improves partner identification. On the other hand, since protein interactions usually occur in crowded environments with several competing partners, we realized a thorough analysis of the interactions of proteins with true partners but also with non-partners to evaluate whether proteins in the environment, competing with the true partner, affect its identification. We found three populations of proteins: strongly competing, never competing, and interacting with different levels of strength. Populations and levels of strength are numerically characterized and provide a signature for the behavior of a protein in the crowded environment. We showed that partner identification, to some extent, does not depend on the competing partners present in the environment, that certain biochemical classes of proteins are intrinsically easier to analyze than others, and that small proteins are not more promiscuous than large ones. Our approach brings to light that the knowledge of the binding site can be used to reduce the high computational cost of docking simulations with no consequence in the quality of the results, demonstrating the possibility to apply coarse-grain docking to datasets made of thousands of proteins. Comparison with all available large-scale analyses aimed to partner predictions is realized. We release the complete decoys set issued by coarse-grain docking simulations of both true and false interacting partners, and their evolutionary sequence analysis leading to binding site predictions. Download site: http://www.lgm.upmc.fr/CCDMintseris/ PMID:24339765

Coherent microscopic picture for urea-induced denaturation of proteins.

PubMed

Yang, Zaixing; Xiu, Peng; Shi, Biyun; Hua, Lan; Zhou, Ruhong

2012-08-02

In a previous study, we explored the mechanism of urea-induced denaturation of proteins by performing molecular dynamics (MD) simulations of hen lysozyme in 8 M urea and supported the "direct interaction mechanism" whereby urea denatures protein via dispersion interaction (Hua, L.; Zhou, R. H.; Thirumalai, D.; Berne, B. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16928). Here we perform large scale MD simulations of five representative protein/peptide systems in aqueous urea to investigate if the above mechanism is common to other proteins. In all cases, accumulations of urea around proteins/peptide are observed, suggesting that urea denatures proteins by directly attacking protein backbones and side chains rather than indirectly disrupting water structure as a "water breaker". Consistent with our previous case study of lysozyme, the current energetic analyses with five protein/peptide systems reveal that urea's preferential binding to proteins mainly comes from urea's stronger dispersion interactions with proteins than with bulk solution, whereas the electrostatic (hydrogen-bonded) interactions only play a relatively minor (even negative) role during this denaturation process. Furthermore, the simulations of the peptide system at different urea concentrations (8 and 4.5 M), and with different force fields (CHARMM and OPLSAA) suggest that the above mechanism is robust, independent of the urea concentration and force field used. Last, we emphasize the importance of periodic boundary conditions in pairwise energetic analyses. This article provides a comprehensive study on the physical mechanism of urea-induced protein denaturation and suggests that the "dispersion-interaction-driven" mechanism should be general.

Quantitative Investigation of Protein-Nucleic Acid Interactions by Biosensor Surface Plasmon Resonance.

PubMed

Wang, Shuo; Poon, Gregory M K; Wilson, W David

2015-01-01

Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with low sample requirements and without the need for external probes. A detailed and practical guide for protein-DNA interaction analyses using biosensor-SPR methods is presented. Details of SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, experimental design, quantitative and qualitative data analyses and presentation. A specific example of the interaction of a transcription factor with DNA is provided with results evaluated by both kinetic and steady-state SPR methods.

From protein interaction profile to functional assignment: the human protein Ki-1/57 is associated with pre-mRNA splicing events.

PubMed

Bressan, Gustavo Costa; Kobarg, Jörg

2010-01-01

The mapping of protein-protein interactions of a determined organism is considered fundamental to assign protein function in the post-genomic era. As part of this effort, screenings for pairwise interactions by yeast two-hybrid system have been used popularly to reveal protein interaction networks in different biological systems. Through the identification of protein interaction partners we have successfully obtained interesting functional clues for Ki-1/57, a human protein with no previous functional annotation, in the context of RNA metabolism. We briefly discuss the way we approached protein-protein interaction data to conduct and interpret further molecular biological and cellular studies as well as structural analyses on this protein. Our data suggest that Ki-1/57 belongs to the family of intrinsically unstructured proteins and that the structural flexibility may be crucial for its capacity to interact with many different proteins. A large fraction of these proteins are involved in pre-mRNA splicing control. Finally, Ki-1/57 is localized to several subnuclear domains, all of which have been described to splicing and other RNA processing events.

Characterization of MRP RNA–protein interactions within the perinucleolar compartment

PubMed Central

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-01-01

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA–processing (MRP) RNA, pyrimidine tract–binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA–containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)–PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA–protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC. PMID:21233287

Characterization of MRP RNA-protein interactions within the perinucleolar compartment.

PubMed

Pollock, Callie; Daily, Kelly; Nguyen, Van Trung; Wang, Chen; Lewandowska, Marzena Anna; Bensaude, Olivier; Huang, Sui

2011-03-15

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA-processing (MRP) RNA, pyrimidine tract-binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA-containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)-PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA-protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.

Interaction of Proteins Identified in Human Thyroid Cells

PubMed Central

Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

2013-01-01

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

P-MartCancer-Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets.

PubMed

Webb-Robertson, Bobbie-Jo M; Bramer, Lisa M; Jensen, Jeffrey L; Kobold, Markus A; Stratton, Kelly G; White, Amanda M; Rodland, Karin D

2017-11-01

P-MartCancer is an interactive web-based software environment that enables statistical analyses of peptide or protein data, quantitated from mass spectrometry-based global proteomics experiments, without requiring in-depth knowledge of statistical programming. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification, and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access and the capability to analyze multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium at the peptide, gene, and protein levels. P-MartCancer is deployed as a web service (https://pmart.labworks.org/cptac.html), alternatively available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/). Cancer Res; 77(21); e47-50. ©2017 AACR . ©2017 American Association for Cancer Research.

Integrative analyses of leprosy susceptibility genes indicate a common autoimmune profile.

PubMed

Zhang, Deng-Feng; Wang, Dong; Li, Yu-Ye; Yao, Yong-Gang

2016-04-01

Leprosy is an ancient chronic infection in the skin and peripheral nerves caused by Mycobacterium leprae. The development of leprosy depends on genetic background and the immune status of the host. However, there is no systematic view focusing on the biological pathways, interaction networks and overall expression pattern of leprosy-related immune and genetic factors. To identify the hub genes in the center of leprosy genetic network and to provide an insight into immune and genetic factors contributing to leprosy. We retrieved all reported leprosy-related genes and performed integrative analyses covering gene expression profiling, pathway analysis, protein-protein interaction network, and evolutionary analyses. A list of 123 differentially expressed leprosy related genes, which were enriched in activation and regulation of immune response, was obtained in our analyses. Cross-disorder analysis showed that the list of leprosy susceptibility genes was largely shared by typical autoimmune diseases such as lupus erythematosus and arthritis, suggesting that similar pathways might be affected in leprosy and autoimmune diseases. Protein-protein interaction (PPI) and positive selection analyses revealed a co-evolution network of leprosy risk genes. Our analyses showed that leprosy associated genes constituted a co-evolution network and might undergo positive selection driven by M. leprae. We suggested that leprosy may be a kind of autoimmune disease and the development of leprosy is a matter of defect or over-activation of body immunity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Identification of T1D susceptibility genes within the MHC region by combining protein interaction networks and SNP genotyping data

PubMed Central

Brorsson, C.; Hansen, N. T.; Lage, K.; Bergholdt, R.; Brunak, S.; Pociot, F.

2009-01-01

Aim To develop novel methods for identifying new genes that contribute to the risk of developing type 1 diabetes within the Major Histocompatibility Complex (MHC) region on chromosome 6, independently of the known linkage disequilibrium (LD) between human leucocyte antigen (HLA)-DRB1, -DQA1, -DQB1 genes. Methods We have developed a novel method that combines single nucleotide polymorphism (SNP) genotyping data with protein–protein interaction (ppi) networks to identify disease-associated network modules enriched for proteins encoded from the MHC region. Approximately 2500 SNPs located in the 4 Mb MHC region were analysed in 1000 affected offspring trios generated by the Type 1 Diabetes Genetics Consortium (T1DGC). The most associated SNP in each gene was chosen and genes were mapped to ppi networks for identification of interaction partners. The association testing and resulting interacting protein modules were statistically evaluated using permutation. Results A total of 151 genes could be mapped to nodes within the protein interaction network and their interaction partners were identified. Five protein interaction modules reached statistical significance using this approach. The identified proteins are well known in the pathogenesis of T1D, but the modules also contain additional candidates that have been implicated in β-cell development and diabetic complications. Conclusions The extensive LD within the MHC region makes it important to develop new methods for analysing genotyping data for identification of additional risk genes for T1D. Combining genetic data with knowledge about functional pathways provides new insight into mechanisms underlying T1D. PMID:19143816

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

PubMed Central

2011-01-01

Background Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels. Results Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners. Conclusion Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development. PMID:21276241

RAID: a comprehensive resource for human RNA-associated (RNA–RNA/RNA–protein) interaction

PubMed Central

Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

2014-01-01

Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA–RNA/RNA–protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA–RNA interactions and 1619 RNA–protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA–RNA/RNA–protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA–RNA/RNA–protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

Semantic integration to identify overlapping functional modules in protein interaction networks

PubMed Central

Cho, Young-Rae; Hwang, Woochang; Ramanathan, Murali; Zhang, Aidong

2007-01-01

Background The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms. Results We have developed novel metrics, called semantic similarity and semantic interactivity, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. We presented a flow-based modularization algorithm to efficiently identify overlapping modules in the weighted interaction networks. The experimental results show that the semantic similarity and semantic interactivity of interacting pairs were positively correlated with functional co-occurrence. The effectiveness of the algorithm for identifying modules was evaluated using functional categories from the MIPS database. We demonstrated that our algorithm had higher accuracy compared to other competing approaches. Conclusion The integration of protein interaction networks with GO annotation data and the capability of detecting overlapping modules substantially improve the accuracy of module identification. PMID:17650343

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein-Protein Interaction Network.

PubMed

Alvarez-Ponce, David; Feyertag, Felix; Chakraborty, Sandip

2017-06-01

The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein-protein interaction data set and the human signal transduction network-a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The interaction between NOLC1 and IAV NS1 protein promotes host cell apoptosis and reduces virus replication.

PubMed

Zhu, Chunyu; Zheng, Fangliang; Zhu, Junfeng; Liu, Meichen; Liu, Na; Li, Xue; Zhang, Li; Deng, Zaidong; Zhao, Qi; Liu, Hongsheng

2017-11-07

NS1 of the influenza virus plays an important role in the infection ability of the influenza virus. Our previous research found that NS1 protein interacts with the NOLC1 protein of host cells, however, the function of the interaction is unknown. In the present study, the role of the interaction between the two proteins in infection was further studied. Several analyses, including the use of a pull-down assay, Co-IP, western blot analysis, overexpression, RNAi, flow cytometry, etc., were used to demonstrate that the NS1 protein of H3N2 influenza virus interacts with host protein NOLC1 and reduces the quantity of NOLC1. The interaction also promotes apoptosis in A549 host cells, while the suppression of NOLC1 protein reduces the proliferation of the H3N2 virus. Based on these data, it was concluded that during the process of infection, NS1 protein interacts with NOLC1 protein, reducing the level of NOLC1, and that the interaction between the two proteins promotes apoptosis of host cells, thus reducing the proliferation of the virus. These findings provide new information on the biological function of the interaction between NS1 and NOLC1.

LIL3, a Light-Harvesting Complex Protein, Links Terpenoid and Tetrapyrrole Biosynthesis in Arabidopsis thaliana1[OPEN

PubMed Central

Rothbart, Maxi; Herbst, Josephine; Wittmann, Daniel; Gruhl, Kirsten

2017-01-01

The LIL3 protein of Arabidopsis (Arabidopsis thaliana) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis. PMID:28432258

LIL3, a Light-Harvesting Complex Protein, Links Terpenoid and Tetrapyrrole Biosynthesis in Arabidopsis thaliana.

PubMed

Hey, Daniel; Rothbart, Maxi; Herbst, Josephine; Wang, Peng; Müller, Jakob; Wittmann, Daniel; Gruhl, Kirsten; Grimm, Bernhard

2017-06-01

The LIL3 protein of Arabidopsis ( Arabidopsis thaliana ) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses.

PubMed

Pallás, V; Sánchez-Navarro, J A; Díez, J

1999-01-01

The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

Evolution and function of CAG/polyglutamine repeats in protein–protein interaction networks

PubMed Central

Schaefer, Martin H.; Wanker, Erich E.; Andrade-Navarro, Miguel A.

2012-01-01

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions. PMID:22287626

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.

Combining multiple positive training sets to generate confidence scores for protein-protein interactions.

PubMed

Yu, Jingkai; Finley, Russell L

2009-01-01

High-throughput experimental and computational methods are generating a wealth of protein-protein interaction data for a variety of organisms. However, data produced by current state-of-the-art methods include many false positives, which can hinder the analyses needed to derive biological insights. One way to address this problem is to assign confidence scores that reflect the reliability and biological significance of each interaction. Most previously described scoring methods use a set of likely true positives to train a model to score all interactions in a dataset. A single positive training set, however, may be biased and not representative of true interaction space. We demonstrate a method to score protein interactions by utilizing multiple independent sets of training positives to reduce the potential bias inherent in using a single training set. We used a set of benchmark yeast protein interactions to show that our approach outperforms other scoring methods. Our approach can also score interactions across data types, which makes it more widely applicable than many previously proposed methods. We applied the method to protein interaction data from both Drosophila melanogaster and Homo sapiens. Independent evaluations show that the resulting confidence scores accurately reflect the biological significance of the interactions.

Molecular interaction networks in the analyses of sequence variation and proteomics data.

PubMed

Stelzl, Ulrich

2013-12-01

Protein-protein interaction networks are typically generated in standard cell lines or model organisms as it is prohibitively difficult to record large interaction datasets from specific tissues or disease models at a reasonable pace. Although the interaction data are of high confidence, they thus do not reflect in vivo relationships as such. A wealth of physiologically relevant protein information, obtained under different conditions and from different systems, is available including information on genetic variation, protein levels, and PTMs. However, these data are difficult to assess comprehensively because the relationships between the entities remain elusive from the measurements. Here, we exemplarily highlight recent studies that gained deeper insight from genetic variation, protein, and PTM measurements using interaction information pointing toward the importance and potential of interaction networks for the interpretation of sequencing and proteomics data. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The EICP22 Protein of Equine Herpesvirus 1 Physically Interacts with the Immediate-Early Protein and with Itself To Form Dimers and Higher-Order Complexes

PubMed Central

Derbigny, Wilbert A.; Kim, Seong K.; Caughman, Gretchen B.; O'Callaghan, Dennis J.

2000-01-01

The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP. PMID:10627553

Essential multimeric enzymes in kinetoplastid parasites: A host of potentially druggable protein-protein interactions.

PubMed

Wachsmuth, Leah M; Johnson, Meredith G; Gavenonis, Jason

2017-06-01

Parasitic diseases caused by kinetoplastid parasites of the genera Trypanosoma and Leishmania are an urgent public health crisis in the developing world. These closely related species possess a number of multimeric enzymes in highly conserved pathways involved in vital functions, such as redox homeostasis and nucleotide synthesis. Computational alanine scanning of these protein-protein interfaces has revealed a host of potentially ligandable sites on several established and emerging anti-parasitic drug targets. Analysis of interfaces with multiple clustered hotspots has suggested several potentially inhibitable protein-protein interactions that may have been overlooked by previous large-scale analyses focusing solely on secondary structure. These protein-protein interactions provide a promising lead for the development of new peptide and macrocycle inhibitors of these enzymes.

Analysis of the thermodynamics of binding of an SH3 domain to proline-rich peptides using a chimeric fusion protein.

PubMed

Candel, Adela M; van Nuland, Nico A J; Martin-Sierra, Francisco M; Martinez, Jose C; Conejero-Lara, Francisco

2008-03-14

A complete understanding of the thermodynamic determinants of binding between SH3 domains and proline-rich peptides is crucial to the development of rational strategies for designing ligands for these important domains. Recently we engineered a single-chain chimeric protein by fusing the alpha-spectrin Src homology region 3 (SH3) domain to the decapeptide APSYSPPPPP (p41). This chimera mimics the structural and energetic features of the interaction between SH3 domains and proline-rich peptides. Here we show that analysing the unfolding thermodynamics of single-point mutants of this chimeric fusion protein constitutes a very useful approach to deciphering the thermodynamics of SH3-ligand interactions. To this end, we investigated the contribution of each proline residue of the ligand sequence to the SH3-peptide interaction by producing six single Pro-Ala mutants of the chimeric protein and analysing their unfolding thermodynamics by differential scanning calorimetry (DSC). Structural analyses of the mutant chimeras by circular dichroism, fluorescence and NMR together with NMR-relaxation measurements indicate conformational flexibility at the binding interface, which is strongly affected by the different Pro-Ala mutations. An analysis of the DSC thermograms on the basis of a three-state unfolding model has allowed us to distinguish and separate the thermodynamic magnitudes of the interaction at the binding interface. The model assumes equilibrium between the "unbound" and "bound" states at the SH3-peptide binding interface. The resulting thermodynamic magnitudes classify the different proline residues according to their importance in the interaction as P2 approximately P7 approximately P10>P9 approximately P6>P8, which agrees well with Lim's model for the interaction between SH3 domains and proline-rich peptides. In addition, the thermodynamic signature of the interaction is the same as that usually found for this type of binding, with a strong enthalpy-entropy compensation for all the mutants. This compensation appears to derive from an increase in conformational flexibility concomitant to the weakening of the interactions at the binding interface. We conclude that our approach, based on DSC and site-directed mutagenesis analysis of chimeric fusion proteins, may serve as a suitable tool to analyse the energetics of weak biomolecular interactions such as those involving SH3 domains.

Structural insights of the MLF1/14-3-3 interaction.

PubMed

Molzan, Manuela; Weyand, Michael; Rose, Rolf; Ottmann, Christian

2012-02-01

Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3ε and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3ε/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract •  14-3-3 epsilon and MLF1 bind by x-ray crystallography (View interaction) •  14-3-3 epsilon and MLF1 bind by isothermal titration calorimetry (View Interaction: 1, 2). © 2011 The Authors Journal compilation © 2011 FEBS.

RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

PubMed

Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

2014-07-01

Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. © 2014 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

PubMed Central

Codoñer, Francisco M.; Fares, Mario A.; Elena, Santiago F.

2006-01-01

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions. PMID:16731922

Adaptive covariation between the coat and movement proteins of prunus necrotic ringspot virus.

PubMed

Codoñer, Francisco M; Fares, Mario A; Elena, Santiago F

2006-06-01

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.

Position Matters: Network Centrality Considerably Impacts Rates of Protein Evolution in the Human Protein–Protein Interaction Network

PubMed Central

Feyertag, Felix; Chakraborty, Sandip

2017-01-01

Abstract The proteins of any organism evolve at disparate rates. A long list of factors affecting rates of protein evolution have been identified. However, the relative importance of each factor in determining rates of protein evolution remains unresolved. The prevailing view is that evolutionary rates are dominantly determined by gene expression, and that other factors such as network centrality have only a marginal effect, if any. However, this view is largely based on analyses in yeasts, and accurately measuring the importance of the determinants of rates of protein evolution is complicated by the fact that the different factors are often correlated with each other, and by the relatively poor quality of available functional genomics data sets. Here, we use correlation, partial correlation and principal component regression analyses to measure the contributions of several factors to the variability of the rates of evolution of human proteins. For this purpose, we analyzed the entire human protein–protein interaction data set and the human signal transduction network—a network data set of exceptionally high quality, obtained by manual curation, which is expected to be virtually free from false positives. In contrast with the prevailing view, we observe that network centrality (measured as the number of physical and nonphysical interactions, betweenness, and closeness) has a considerable impact on rates of protein evolution. Surprisingly, the impact of centrality on rates of protein evolution seems to be comparable, or even superior according to some analyses, to that of gene expression. Our observations seem to be independent of potentially confounding factors and from the limitations (biases and errors) of interactomic data sets. PMID:28854629

ISAAC - InterSpecies Analysing Application using Containers.

PubMed

Baier, Herbert; Schultz, Jörg

2014-01-15

Information about genes, transcripts and proteins is spread over a wide variety of databases. Different tools have been developed using these databases to identify biological signals in gene lists from large scale analysis. Mostly, they search for enrichments of specific features. But, these tools do not allow an explorative walk through different views and to change the gene lists according to newly upcoming stories. To fill this niche, we have developed ISAAC, the InterSpecies Analysing Application using Containers. The central idea of this web based tool is to enable the analysis of sets of genes, transcripts and proteins under different biological viewpoints and to interactively modify these sets at any point of the analysis. Detailed history and snapshot information allows tracing each action. Furthermore, one can easily switch back to previous states and perform new analyses. Currently, sets can be viewed in the context of genomes, protein functions, protein interactions, pathways, regulation, diseases and drugs. Additionally, users can switch between species with an automatic, orthology based translation of existing gene sets. As todays research usually is performed in larger teams and consortia, ISAAC provides group based functionalities. Here, sets as well as results of analyses can be exchanged between members of groups. ISAAC fills the gap between primary databases and tools for the analysis of large gene lists. With its highly modular, JavaEE based design, the implementation of new modules is straight forward. Furthermore, ISAAC comes with an extensive web-based administration interface including tools for the integration of third party data. Thus, a local installation is easily feasible. In summary, ISAAC is tailor made for highly explorative interactive analyses of gene, transcript and protein sets in a collaborative environment.

P-MartCancer–Interactive Online Software to Enable Analysis of Shotgun Cancer Proteomic Datasets

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

Webb-Robertson, Bobbie-Jo M.; Bramer, Lisa M.; Jensen, Jeffrey L.

P-MartCancer is a new interactive web-based software environment that enables biomedical and biological scientists to perform in-depth analyses of global proteomics data without requiring direct interaction with the data or with statistical software. P-MartCancer offers a series of statistical modules associated with quality assessment, peptide and protein statistics, protein quantification and exploratory data analyses driven by the user via customized workflows and interactive visualization. Currently, P-MartCancer offers access to multiple cancer proteomic datasets generated through the Clinical Proteomics Tumor Analysis Consortium (CPTAC) at the peptide, gene and protein levels. P-MartCancer is deployed using Azure technologies (http://pmart.labworks.org/cptac.html), the web-service is alternativelymore » available via Docker Hub (https://hub.docker.com/r/pnnl/pmart-web/) and many statistical functions can be utilized directly from an R package available on GitHub (https://github.com/pmartR).« less

An attempt to understand glioma stem cell biology through centrality analysis of a protein interaction network.

PubMed

Mallik, Mrinmay Kumar

2018-02-07

Biological networks can be analyzed using "Centrality Analysis" to identify the more influential nodes and interactions in the network. This study was undertaken to create and visualize a biological network comprising of protein-protein interactions (PPIs) amongst proteins which are preferentially over-expressed in glioma cancer stem cell component (GCSC) of glioblastomas as compared to the glioma non-stem cancer cell (GNSC) component and then to analyze this network through centrality analyses (CA) in order to identify the essential proteins in this network and their interactions. In addition, this study proposes a new centrality analysis method pertaining exclusively to transcription factors (TFs) and interactions amongst them. Moreover the relevant molecular functions, biological processes and biochemical pathways amongst these proteins were sought through enrichment analysis. A protein interaction network was created using a list of proteins which have been shown to be preferentially expressed or over-expressed in GCSCs isolated from glioblastomas as compared to the GNSCs. This list comprising of 38 proteins, created using manual literature mining, was submitted to the Reactome FIViz tool, a web based application integrated into Cytoscape, an open source software platform for visualizing and analyzing molecular interaction networks and biological pathways to produce the network. This network was subjected to centrality analyses utilizing ranked lists of six centrality measures using the FIViz application and (for the first time) a dedicated centrality analysis plug-in ; CytoNCA. The interactions exclusively amongst the transcription factors were nalyzed through a newly proposed centrality analysis method called "Gene Expression Associated Degree Centrality Analysis (GEADCA)". Enrichment analysis was performed using the "network function analysis" tool on Reactome. The CA was able to identify a small set of proteins with consistently high centrality ranks that is indicative of their strong influence in the protein protein interaction network. Similarly the newly proposed GEADCA helped identify the transcription factors with high centrality values indicative of their key roles in transcriptional regulation. The enrichment studies provided a list of molecular functions, biological processes and biochemical pathways associated with the constructed network. The study shows how pathway based databases may be used to create and analyze a relevant protein interaction network in glioma cancer stem cells and identify the essential elements within it to gather insights into the molecular interactions that regulate the properties of glioma stem cells. How these insights may be utilized to help the development of future research towards formulation of new management strategies have been discussed from a theoretical standpoint. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

PubMed Central

Fromont-Racine, Micheline; Mayes, Andrew E.; Brunet-Simon, Adeline; Rain, Jean-Christophe; Colley, Alan; Dix, Ian; Decourty, Laurence; Joly, Nicolas; Ricard, Florence; Beggs, Jean D.

2000-01-01

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale. PMID:10900456

Quantitative analyses of bifunctional molecules.

PubMed

Braun, Patrick D; Wandless, Thomas J

2004-05-11

Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.

R Script Approach to Infer Toxoplasma Infection Mechanisms From Microarrays and Domain-Domain Protein Interactions

PubMed Central

Arenas, Ailan F; Salcedo, Gladys E; Gomez-Marin, Jorge E

2017-01-01

Pathogen-host protein-protein interaction systems examine the interactions between the protein repertoires of 2 distinct organisms. Some of these pathogen proteins interact with the host protein system and may manipulate it for their own advantages. In this work, we designed an R script by concatenating 2 functions called rowDM and rowCVmed to infer pathogen-host interaction using previously reported microarray data, including host gene enrichment analysis and the crossing of interspecific domain-domain interactions. We applied this script to the Toxoplasma-host system to describe pathogen survival mechanisms from human, mouse, and Toxoplasma Gene Expression Omnibus series. Our outcomes exhibited similar results with previously reported microarray analyses, but we found other important proteins that could contribute to toxoplasma pathogenesis. We observed that Toxoplasma ROP38 is the most differentially expressed protein among toxoplasma strains. Enrichment analysis and KEGG mapping indicated that the human retinal genes most affected by Toxoplasma infections are those related to antiapoptotic mechanisms. We suggest that proteins PIK3R1, PRKCA, PRKCG, PRKCB, HRAS, and c-JUN could be the possible substrates for differentially expressed Toxoplasma kinase ROP38. Likewise, we propose that Toxoplasma causes overexpression of apoptotic suppression human genes. PMID:29317802

PLI: a web-based tool for the comparison of protein-ligand interactions observed on PDB structures.

PubMed

Gallina, Anna Maria; Bisignano, Paola; Bergamino, Maurizio; Bordo, Domenico

2013-02-01

A large fraction of the entries contained in the Protein Data Bank describe proteins in complex with low molecular weight molecules such as physiological compounds or synthetic drugs. In many cases, the same molecule is found in distinct protein-ligand complexes. There is an increasing interest in Medicinal Chemistry in comparing protein binding sites to get insight on interactions that modulate the binding specificity, as this structural information can be correlated with other experimental data of biochemical or physiological nature and may help in rational drug design. The web service protein-ligand interaction presented here provides a tool to analyse and compare the binding pockets of homologous proteins in complex with a selected ligand. The information is deduced from protein-ligand complexes present in the Protein Data Bank and stored in the underlying database. Freely accessible at http://bioinformatics.istge.it/pli/.

Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

PubMed

Winzen, S; Schoettler, S; Baier, G; Rosenauer, C; Mailaender, V; Landfester, K; Mohr, K

2015-02-21

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

Network-based study reveals potential infection pathways of hepatitis-C leading to various diseases.

PubMed

Mukhopadhyay, Anirban; Maulik, Ujjwal

2014-01-01

Protein-protein interaction network-based study of viral pathogenesis has been gaining popularity among computational biologists in recent days. In the present study we attempt to investigate the possible pathways of hepatitis-C virus (HCV) infection by integrating the HCV-human interaction network, human protein interactome and human genetic disease association network. We have proposed quasi-biclique and quasi-clique mining algorithms to integrate these three networks to identify infection gateway host proteins and possible pathways of HCV pathogenesis leading to various diseases. Integrated study of three networks, namely HCV-human interaction network, human protein interaction network, and human proteins-disease association network reveals potential pathways of infection by the HCV that lead to various diseases including cancers. The gateway proteins have been found to be biologically coherent and have high degrees in human interactome compared to the other virus-targeted proteins. The analyses done in this study provide possible targets for more effective anti-hepatitis-C therapeutic involvement.

Network-Based Study Reveals Potential Infection Pathways of Hepatitis-C Leading to Various Diseases

PubMed Central

Mukhopadhyay, Anirban; Maulik, Ujjwal

2014-01-01

Protein-protein interaction network-based study of viral pathogenesis has been gaining popularity among computational biologists in recent days. In the present study we attempt to investigate the possible pathways of hepatitis-C virus (HCV) infection by integrating the HCV-human interaction network, human protein interactome and human genetic disease association network. We have proposed quasi-biclique and quasi-clique mining algorithms to integrate these three networks to identify infection gateway host proteins and possible pathways of HCV pathogenesis leading to various diseases. Integrated study of three networks, namely HCV-human interaction network, human protein interaction network, and human proteins-disease association network reveals potential pathways of infection by the HCV that lead to various diseases including cancers. The gateway proteins have been found to be biologically coherent and have high degrees in human interactome compared to the other virus-targeted proteins. The analyses done in this study provide possible targets for more effective anti-hepatitis-C therapeutic involvement. PMID:24743187

Diversity in Genetic In Vivo Methods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

PubMed Central

Stynen, Bram; Tournu, Hélène; Tavernier, Jan

2012-01-01

Summary: The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli, but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays. PMID:22688816

A Laminin G-EGF-Laminin G module in Neurexin IV is essential for the apico-lateral localization of Contactin and organization of septate junctions.

PubMed

Banerjee, Swati; Paik, Raehum; Mino, Rosa E; Blauth, Kevin; Fisher, Elizabeth S; Madden, Victoria J; Fanning, Alan S; Bhat, Manzoor A

2011-01-01

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons.

A Laminin G-EGF-Laminin G Module in Neurexin IV Is Essential for the Apico-Lateral Localization of Contactin and Organization of Septate Junctions

PubMed Central

Banerjee, Swati; Paik, Raehum; Mino, Rosa E.; Blauth, Kevin; Fisher, Elizabeth S.; Madden, Victoria J.; Fanning, Alan S.; Bhat, Manzoor A.

2011-01-01

Septate junctions (SJs) display a unique ultrastructural morphology with ladder-like electron densities that are conserved through evolution. Genetic and molecular analyses have identified a highly conserved core complex of SJ proteins consisting of three cell adhesion molecules Neurexin IV, Contactin, and Neuroglian, which interact with the cytoskeletal FERM domain protein Coracle. How these individual proteins interact to form the septal arrays that create the paracellular barrier is poorly understood. Here, we show that point mutations that map to specific domains of neurexin IV lead to formation of fewer septae and disorganization of SJs. Consistent with these observations, our in vivo domain deletion analyses identified the first Laminin G-EGF-Laminin G module in the extracellular region of Neurexin IV as necessary for the localization of and association with Contactin. Neurexin IV protein that is devoid of its cytoplasmic region is able to create septae, but fails to form a full complement of SJs. These data provide the first in vivo evidence that specific domains in Neurexin IV are required for protein-protein interactions and organization of SJs. Given the molecular conservation of SJ proteins across species, our studies may provide insights into how vertebrate axo-glial SJs are organized in myelinated axons. PMID:22022470

Structure-based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling Through Caveolin-Protein Interactions?

PubMed Central

Collins, Brett M.; Davis, Melissa J.; Hancock, John F.; Parton, Robert G.

2012-01-01

Summary Caveolin proteins drive formation of caveolae, specialized cell-surface microdomains that influence cell signaling. Signaling proteins are proposed to use conserved caveolin-binding motifs (CBMs) to associate with caveolae via the caveolin scaffolding domain (CSD). However, structural and bioinformatic analyses argue against such direct physical interactions: In the majority of signaling proteins, the CBM is buried and inaccessible. Putative CBMs do not form a common structure for caveolin recognition, are not enriched amongst caveolin-binding proteins, and are even more common in yeast, which lack caveolae. We propose that CBM/CSD-dependent interactions are unlikely to mediate caveolar signaling, and the basis for signaling effects should therefore be reassessed. PMID:22814599

Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana.

PubMed

Dortay, Hakan; Mehnert, Nijuscha; Bürkle, Lukas; Schmülling, Thomas; Heyl, Alexander

2006-10-01

The signal of the plant hormone cytokinin is perceived by membrane-located sensor histidine kinases and transduced by other members of the plant two-component system. In Arabidopsis thaliana, 28 two-component system proteins (phosphotransmitters and response regulators) act downstream of three receptors, transmitting the signal from the membrane to the nucleus and modulating the cellular response. Although the principal signaling mechanism has been elucidated, redundancy in the system has made it difficult to understand which of the many components interact to control the downstream biological processes. Here, we present a large-scale interaction study comprising most members of the Arabidopsis cytokinin signaling pathway. Using the yeast two-hybrid system, we detected 42 new interactions, of which more than 90% were confirmed by in vitro coaffinity purification. There are distinct patterns of interaction between protein families, but only a few interactions between proteins of the same family. An interaction map of this signaling pathway shows the Arabidopsis histidine phosphotransfer proteins as hubs, which interact with members from all other protein families, mostly in a redundant fashion. Domain-mapping experiments revealed the interaction domains of the proteins of this pathway. Analyses of Arabidopsis histidine phosphotransfer protein 5 mutant proteins showed that the presence of the canonical phospho-accepting histidine residue is not required for the interactions. Interaction of A-type response regulators with Arabidopsis histidine phosphotransfer proteins but not with B-type response regulators suggests that their known activity in feedback regulation may be realized by interfering at the level of Arabidopsis histidine phosphotransfer protein-mediated signaling. This study contributes to our understanding of the protein interactions of the cytokinin-signaling system and provides a framework for further functional studies in planta.

Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

NASA Astrophysics Data System (ADS)

Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

2011-03-01

Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

Proteomics in the investigation of HIV-1 interactions with host proteins.

PubMed

Li, Ming

2015-02-01

Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the interactions between protein and carbon black.

PubMed

Chen, Tzu-Tao; Chuang, Kai-Jen; Chiang, Ling-Ling; Chen, Chun-Chao; Yeh, Chi-Tai; Wang, Liang-Shun; Gregory, Clive; Jones, Tim; BéruBé, Kelly; Lee, Chun-Nin; Chuang, Hsiao-Chi; Cheng, Tsun-Jen

2014-01-15

A considerable amount of studies have been conducted to investigate the interactions of biological fluids with nanoparticle surfaces, which exhibit a high affinity for proteins and particles. However, the mechanisms underlying these interactions have not been elucidated, particularly as they relate to human health. Using bovine serum albumin (BSA) and mice bronchoalveolar lavage fluid (BALF) as models for protein-particle conjugates, we characterized the physicochemical modifications of carbon blacks (CB) with 23nm or 65nm in diameter after protein treatment. Adsorbed BALF-containing proteins were quantified and identified by pathways, biological analyses and protein classification. Significant modifications of the physicochemistry of CB were induced by the addition of BSA. Enzyme modulators and hydrolase predominately interacted with CB, with protein-to-CB interactions that were associated with the coagulation pathways. Additionally, our results revealed that an acute-phase response could be activated by these proteins. With regard to human health, the present study revealed that the CB can react with proteins (∼55kDa and 70kDa) after inhalation and may modify the functional structures of lung proteins, leading to the activation of acute-inflammatory responses in the lungs. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiplex single-molecule interaction profiling of DNA barcoded proteins

PubMed Central

Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

2014-01-01

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

PubMed Central

Goodin, Michael M.; Austin, Jennifer; Tobias, Renée; Fujita, Miki; Morales, Christina; Jackson, Andrew O.

2001-01-01

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization. PMID:11533202

A rice kinase-protein interaction map.

PubMed

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses

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

Camuzeaux, Barbara; Spriet, Corentin; Heliot, Laurent

2005-07-15

Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg-Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixedmore » and living cells. FRET between YFP-Erg and CFP-Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP-Erg and CFP-Jun. This is stressed by loss of FRET with an YFP-Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction.« less

APID interactomes: providing proteome-based interactomes with controlled quality for multiple species and derived networks

PubMed Central

Alonso-López, Diego; Gutiérrez, Miguel A.; Lopes, Katia P.; Prieto, Carlos; Santamaría, Rodrigo; De Las Rivas, Javier

2016-01-01

APID (Agile Protein Interactomes DataServer) is an interactive web server that provides unified generation and delivery of protein interactomes mapped to their respective proteomes. This resource is a new, fully redesigned server that includes a comprehensive collection of protein interactomes for more than 400 organisms (25 of which include more than 500 interactions) produced by the integration of only experimentally validated protein–protein physical interactions. For each protein–protein interaction (PPI) the server includes currently reported information about its experimental validation to allow selection and filtering at different quality levels. As a whole, it provides easy access to the interactomes from specific species and includes a global uniform compendium of 90,379 distinct proteins and 678,441 singular interactions. APID integrates and unifies PPIs from major primary databases of molecular interactions, from other specific repositories and also from experimentally resolved 3D structures of protein complexes where more than two proteins were identified. For this purpose, a collection of 8,388 structures were analyzed to identify specific PPIs. APID also includes a new graph tool (based on Cytoscape.js) for visualization and interactive analyses of PPI networks. The server does not require registration and it is freely available for use at http://apid.dep.usal.es. PMID:27131791

Nitrogenase of Klebsiella pneumoniae. Interaction of the component proteins studied by ultracentrifugation

PubMed Central

Eady, Robert R.

1973-01-01

Sedimentation-velocity analyses of mixtures of the component proteins of nitrogenase of Klebsiella pneumoniae at a 1:1 molar ratio, showed a single peak of sedimentation coefficient (12.4S) considerably greater than that obtained for the larger (Fe+Mo-containing) protein centrifuged alone (10.4S). When the ratio exceeded 1:1 (the smaller Fe-containing protein in excess) an additional peak corresponding in sedimentation coefficient (about 4.5S) to free Fe-containing protein appeared. When proteins, which had been inactivated by exposure to air were used, no interaction occurred. Na2S2O4 at 2mm both reversed and prevented interaction between the two proteins; sedimentation coefficients corresponded to those of the proteins when centrifuged alone. These results demonstrate the formation of a complex between the nitrogenase proteins, and, together with data of activity titration curves, are consistent with the formulation of the nitrogenase complex of K. pneumoniae as (Fe-containing protein)–(Fe+Mo-containing protein). ImagesFig. 1. PMID:4589392

A nano-bio interfacial protein corona on silica nanoparticle.

PubMed

Zhang, Hongyan; Peng, Jiaxi; Li, Xin; Liu, Shengju; Hu, Zhengyan; Xu, Guiju; Wu, Ren'an

2018-07-01

Nano-bio interaction takes the crucial role in bio-application of nanoparticles. The systematic mapping of interfacial proteins remains the big challenge as low level of proteins within interface regions and lack of appropriate technology. Here, a facile proteomic strategy was developed to characterize the interfacial protein corona (noted as IPC) that has strong interactions with silica nanoparticle, via the combination of the vigorous elution with high concentration sodium dodecyl sulfate (SDS) and the pre-isolation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The trace level IPCs for silica nanoparticle were thus qualitatively and quantitatively identified. Bioinformatics analyses revealed the intrinsic compositions, relevance and potential regularity addressing the strong interactions between IPC and nanoparticle. This strategy in determining IPCs is opening an avenue to give a deep insight to understand the interaction between proteins and not only nanoparticles but also other bulk materials. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of proteins interacting with lactate dehydrogenase in claw muscle of the porcelain crab Petrolisthes cinctipes

PubMed Central

Cayenne, Andrea P.; Gabert, Beverly; Stillman, Jonathon H.

2011-01-01

Biochemical adaptation of enzymes involves conservation of activity, stability and affinity across a wide range of intracellular and environmental conditions. Enzyme adaptation by alteration of primary structure is well known, but the roles of protein-protein interactions in enzyme adaptation are less well understood. Interspecific differences in thermal stability of lactate dehydrogenase (LDH) in porcelain crabs (genus Petrolisthes) are related to intrinsic differences among LDH molecules and by interactions with other stabilizing proteins. Here, we identified proteins that interact with LDH in porcelain crab claw muscle tissue using co-immunoprecipitation, and showed LDH exists in high molecular weight complexes using size exclusion chromatography and Western blot analyses. Co-immunoprecipitated proteins were separated using 2D SDS PAGE and analyzed by LC/ESI using peptide MS/MS. Peptide MS/MS ions were compared to an EST database for Petrolisthes cinctipes to identify proteins. Identified proteins included cytoskeletal elements, glycolytic enzymes, a phosphagen kinase, and the respiratory protein hemocyanin. Our results support the hypothesis that LDH interacts with glycolytic enzymes in a metabolon structured by cytoskeletal elements that may also include the enzyme for transfer of the adenylate charge in glycolytically produced ATP. Those interactions may play specific roles in biochemical adaptation of glycolytic enzymes. PMID:21968246

Proteomic characterization of larval and adult developmental stages in Echinococcus granulosus reveals novel insight into host-parasite interactions.

PubMed

Cui, Shu-Jian; Xu, Lei-Lei; Zhang, Ting; Xu, Ming; Yao, Jun; Fang, Cai-Yun; Feng, Zheng; Yang, Peng-Yuan; Hu, Wei; Liu, Feng

2013-06-12

Cystic hydatid disease is an important zoonosis caused by Echinococcus granulosus infection. The expression profiles of its parasitic life stages and host-Echinococcus interactions remain to be elucidated. Here, we identified 157 adult and 1588 protoscolex proteins (1610 in all), including 1290 novel identifications. Paramyosins and an antigen B (AgB) were the dominant adult proteins. Dog proteins (30) identified in adults indicated diminished local inflammation caused by adult infection. The protoscolex expresses proteins that have been reported to be antigens in other parasites, such as 6-phosphofructokinase and calcineurin B. Pathway analyses suggested that E. granulosus uses both aerobic and anaerobic carbohydrate metabolisms to generate ATP. E. granulosus expresses proteins involved in synthesis and metabolism of lipids or steroids. At least 339 of 390 sheep proteins identified in protoscolex were novel identifications not seen in previous analyses. IgGs and lambda light chains were the most abundant antibody species. Sheep proteins were enriched for detoxification pathways, implying that host detoxification effects play a central role during host-parasite interactions. Our study provides valuable data on E. granulosus expression characteristics, allowing novel insights into the molecular mechanisms involved in host-parasite interactions. In this study, the Echinococcus granulosus adult worm proteome was analyzed for the first time. The protein identification of E. granulosus protoscoleces was extended dramatically. We also identified the most abundant host proteins co-purified with Echinococcus. The results provide useful information pertaining to the molecular mechanisms behind host-Echinococcus interaction and Echinococcus biology. This data also increases the potential for identifying vaccine candidates and new therapeutic targets, and may aid in the development of protein probes for selective and sensitive diagnosis of echinococcosis infection. In addition, the data collected here represents a valuable proteomic resource for subsequent genome annotation. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of novel interactors and potential phosphorylation substrates of GsSnRK1 from wild soybean (Glycine soja).

PubMed

Song, Yu; Zhang, Hang; You, Hongguang; Liu, Yuanming; Chen, Chao; Feng, Xu; Yu, Xingyu; Wu, Shengyang; Wang, Libo; Zhong, Shihua; Li, Qiang; Zhu, Yanming; Ding, Xiaodong

2018-04-17

The plant sucrose nonfermenting kinase 1 (SnRK1) kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth, and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library-scale screens by the means of yeast two-hybrid to identify its interacting proteins. The putative interactions were verified by yeast retransformation and β-galactosidase assays, and the selected interactions were further confirmed in planta by bimolecular fluorescence complementation and biochemical Co-IP assays. Protein phosphorylation analyses were carried out by phos-tag assay and anti-phospho-(Ser/Thr) substrate antibodies. Finally, we obtained 24 GsSnRK1 interactors and several putative substrates that can be categorized into SnRK1 regulatory β subunit, protein modification, biotic and abiotic stress-related, hormone perception and signalling, gene expression regulation, water and nitrogen transport, metabolism, and unknown proteins. Intriguingly, we first discovered that GsSnRK1 interacted with and phosphorylated the components of soybean nodulation and symbiotic nitrogen fixation. The interactions and potential functions of GsSnRK1 and its associated proteins were extensively discussed and analysed. This work provides plausible clues to elucidate the novel functions of SnRK1 in response to variable environmental, metabolic, and physiological requirements. © 2018 John Wiley & Sons Ltd.

Electrostatic interactions as governing the fouling in protein microfiltration

NASA Astrophysics Data System (ADS)

Ouammou, M.; Tijani, N.; Calvo, J. I.; Palacio, L.; Prádanos, P.; Hernández, A.

2005-03-01

The influence of pH and electrostatic interactions on the fouling mechanism during protein dead-end microfiltration (MF) has been investigated for two charged membranes. Polyethersulfone acidic membranes (ICE-450), being negatively charged, and basic ones (SB-6407), these positively charged, both from Pall Co., have been used in the investigations. BSA and Lysozyme solutions at different pH values (3.0, 5.0, 7.0, 8.5 and 10.0) were microfiltered through the membranes at a constant applied transmembrane pressure. Results have been analysed in terms of usual blocking filtration laws and a substantial change in the fouling behaviour has been observed when solution pH and/or membrane charge as the pressure was changed, this change being clearly related with the specific membrane-protein and protein-protein interactions.

Integration of Structural Dynamics and Molecular Evolution via Protein Interaction Networks: A New Era in Genomic Medicine

PubMed Central

Kumar, Avishek; Butler, Brandon M.; Kumar, Sudhir; Ozkan, S. Banu

2016-01-01

Summary Sequencing technologies are revealing many new non-synonymous single nucleotide variants (nsSNVs) in each personal exome. To assess their functional impacts, comparative genomics is frequently employed to predict if they are benign or not. However, evolutionary analysis alone is insufficient, because it misdiagnoses many disease-associated nsSNVs, such as those at positions involved in protein interfaces, and because evolutionary predictions do not provide mechanistic insights into functional change or loss. Structural analyses can aid in overcoming both of these problems by incorporating conformational dynamics and allostery in nSNV diagnosis. Finally, protein-protein interaction networks using systems-level methodologies shed light onto disease etiology and pathogenesis. Bridging these network approaches with structurally resolved protein interactions and dynamics will advance genomic medicine. PMID:26684487

Combining NMR and Molecular Dynamics Studies for Insights into the Allostery of Small GTPase–Protein Interactions

PubMed Central

Zhang, Liqun; Bouguet-Bonnet, Sabine; Buck, Matthias

2014-01-01

Combinations of experimentally derived data from nuclear magnetic resonance spectroscopy and analyses of molecular dynamics trajectories increasingly allow us to obtain a detailed description of the molecular mechanisms by which proteins function in signal transduction. This chapter provides an introduction into these two methodologies, illustrated by example of a small GTPase–effector interaction. It is increasingly becoming clear that new insights are provided by the combination of experimental and computational methods. Understanding the structural and protein dynamical contributions to allostery will be useful for the engineering of new binding interfaces and protein functions, as well as for the design/in silico screening of chemical agents that can manipulate the function of small GTPase–protein interactions in diseases such as cancer. PMID:22052494

Differentially co-expressed interacting protein pairs discriminate samples under distinct stages of HIV type 1 infection.

PubMed

Yoon, Dukyong; Kim, Hyosil; Suh-Kim, Haeyoung; Park, Rae Woong; Lee, KiYoung

2011-01-01

Microarray analyses based on differentially expressed genes (DEGs) have been widely used to distinguish samples across different cellular conditions. However, studies based on DEGs have not been able to clearly determine significant differences between samples of pathophysiologically similar HIV-1 stages, e.g., between acute and chronic progressive (or AIDS) or between uninfected and clinically latent stages. We here suggest a novel approach to allow such discrimination based on stage-specific genetic features of HIV-1 infection. Our approach is based on co-expression changes of genes known to interact. The method can identify a genetic signature for a single sample as contrasted with existing protein-protein-based analyses with correlational designs. Our approach distinguishes each sample using differentially co-expressed interacting protein pairs (DEPs) based on co-expression scores of individual interacting pairs within a sample. The co-expression score has positive value if two genes in a sample are simultaneously up-regulated or down-regulated. And the score has higher absolute value if expression-changing ratios are similar between the two genes. We compared characteristics of DEPs with that of DEGs by evaluating their usefulness in separation of HIV-1 stage. And we identified DEP-based network-modules and their gene-ontology enrichment to find out the HIV-1 stage-specific gene signature. Based on the DEP approach, we observed clear separation among samples from distinct HIV-1 stages using clustering and principal component analyses. Moreover, the discrimination power of DEPs on the samples (70-100% accuracy) was much higher than that of DEGs (35-45%) using several well-known classifiers. DEP-based network analysis also revealed the HIV-1 stage-specific network modules; the main biological processes were related to "translation," "RNA splicing," "mRNA, RNA, and nucleic acid transport," and "DNA metabolism." Through the HIV-1 stage-related modules, changing stage-specific patterns of protein interactions could be observed. DEP-based method discriminated the HIV-1 infection stages clearly, and revealed a HIV-1 stage-specific gene signature. The proposed DEP-based method might complement existing DEG-based approaches in various microarray expression analyses.

PPI-IRO: a two-stage method for protein-protein interaction extraction based on interaction relation ontology.

PubMed

Li, Chuan-Xi; Chen, Peng; Wang, Ru-Jing; Wang, Xiu-Jie; Su, Ya-Ru; Li, Jinyan

2014-01-01

Mining Protein-Protein Interactions (PPIs) from the fast-growing biomedical literature resources has been proven as an effective approach for the identification of biological regulatory networks. This paper presents a novel method based on the idea of Interaction Relation Ontology (IRO), which specifies and organises words of various proteins interaction relationships. Our method is a two-stage PPI extraction method. At first, IRO is applied in a binary classifier to determine whether sentences contain a relation or not. Then, IRO is taken to guide PPI extraction by building sentence dependency parse tree. Comprehensive and quantitative evaluations and detailed analyses are used to demonstrate the significant performance of IRO on relation sentences classification and PPI extraction. Our PPI extraction method yielded a recall of around 80% and 90% and an F1 of around 54% and 66% on corpora of AIMed and BioInfer, respectively, which are superior to most existing extraction methods.

Proteomic Analyses of NF1-Interacting Proteins in Keratinocytes

DTIC Science & Technology

2015-04-01

and knockout mice further confirmed the interactions suggested by the proteomic analyses. In relation to the development of psoriasis -like symptoms...in the NF1 null epidermis, we analyzed NF1 expression in a mouse model of psoriasis (imiquimod-induced psoriasis -like skin inflammation) and...knockout of epidermal NF1 to elucidate the molecular underpinnings of psoriasis . 15. SUBJECT TERMS neurofibromin-1 (NF1), psoriasis , inflammation

Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed.

PubMed

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

2016-03-04

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

JET2 Viewer: a database of predicted multiple, possibly overlapping, protein-protein interaction sites for PDB structures.

PubMed

Ripoche, Hugues; Laine, Elodie; Ceres, Nicoletta; Carbone, Alessandra

2017-01-04

The database JET2 Viewer, openly accessible at http://www.jet2viewer.upmc.fr/, reports putative protein binding sites for all three-dimensional (3D) structures available in the Protein Data Bank (PDB). This knowledge base was generated by applying the computational method JET 2 at large-scale on more than 20 000 chains. JET 2 strategy yields very precise predictions of interacting surfaces and unravels their evolutionary process and complexity. JET2 Viewer provides an online intelligent display, including interactive 3D visualization of the binding sites mapped onto PDB structures and suitable files recording JET 2 analyses. Predictions were evaluated on more than 15 000 experimentally characterized protein interfaces. This is, to our knowledge, the largest evaluation of a protein binding site prediction method. The overall performance of JET 2 on all interfaces are: Sen = 52.52, PPV = 51.24, Spe = 80.05, Acc = 75.89. The data can be used to foster new strategies for protein-protein interactions modulation and interaction surface redesign. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deciphering RNA-Recognition Patterns of Intrinsically Disordered Proteins.

PubMed

Srivastava, Ambuj; Ahmad, Shandar; Gromiha, M Michael

2018-05-29

Intrinsically disordered regions (IDRs) and protein (IDPs) are highly flexible owing to their lack of well-defined structures. A subset of such proteins interacts with various substrates; including RNA; frequently adopting regular structures in the final complex. In this work; we have analysed a dataset of protein⁻RNA complexes undergoing disorder-to-order transition (DOT) upon binding. We found that DOT regions are generally small in size (less than 3 residues) for RNA binding proteins. Like structured proteins; positively charged residues are found to interact with RNA molecules; indicating the dominance of electrostatic and cation-π interactions. However, a comparison of binding frequency shows that interface hydrophobic and aromatic residues have more interactions in only DOT regions than in a protein. Further; DOT regions have significantly higher exposure to water than their structured counterparts. Interactions of DOT regions with RNA increase the sheet formation with minor changes in helix forming residues. We have computed the interaction energy for amino acids⁻nucleotide pairs; which showed the preference of His⁻G; Asn⁻U and Ser⁻U at for the interface of DOT regions. This study provides insights to understand protein⁻RNA interactions and the results could also be used for developing a tool for identifying DOT regions in RNA binding proteins.

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

PubMed

Wallqvist, Anders; Wang, Hao; Zavaljevski, Nela; Memišević, Vesna; Kwon, Keehwan; Pieper, Rembert; Rajagopala, Seesandra V; Reifman, Jaques

2017-01-01

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs-the largest collection of unbiased Coxiella host-pathogen interactions to date-represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.

Multi-level machine learning prediction of protein-protein interactions in Saccharomyces cerevisiae.

PubMed

Zubek, Julian; Tatjewski, Marcin; Boniecki, Adam; Mnich, Maciej; Basu, Subhadip; Plewczynski, Dariusz

2015-01-01

Accurate identification of protein-protein interactions (PPI) is the key step in understanding proteins' biological functions, which are typically context-dependent. Many existing PPI predictors rely on aggregated features from protein sequences, however only a few methods exploit local information about specific residue contacts. In this work we present a two-stage machine learning approach for prediction of protein-protein interactions. We start with the carefully filtered data on protein complexes available for Saccharomyces cerevisiae in the Protein Data Bank (PDB) database. First, we build linear descriptions of interacting and non-interacting sequence segment pairs based on their inter-residue distances. Secondly, we train machine learning classifiers to predict binary segment interactions for any two short sequence fragments. The final prediction of the protein-protein interaction is done using the 2D matrix representation of all-against-all possible interacting sequence segments of both analysed proteins. The level-I predictor achieves 0.88 AUC for micro-scale, i.e., residue-level prediction. The level-II predictor improves the results further by a more complex learning paradigm. We perform 30-fold macro-scale, i.e., protein-level cross-validation experiment. The level-II predictor using PSIPRED-predicted secondary structure reaches 0.70 precision, 0.68 recall, and 0.70 AUC, whereas other popular methods provide results below 0.6 threshold (recall, precision, AUC). Our results demonstrate that multi-scale sequence features aggregation procedure is able to improve the machine learning results by more than 10% as compared to other sequence representations. Prepared datasets and source code for our experimental pipeline are freely available for download from: http://zubekj.github.io/mlppi/ (open source Python implementation, OS independent).

Systematic identification of phosphorylation-mediated protein interaction switches

PubMed Central

Wichmann, Oliver; Utz, Mathias; Andre, Timon; Minguez, Pablo; Parca, Luca; Roth, Frederick P.; Gavin, Anne-Claude; Bork, Peer; Russell, Robert B.

2017-01-01

Proteomics techniques can identify thousands of phosphorylation sites in a single experiment, the majority of which are new and lack precise information about function or molecular mechanism. Here we present a fast method to predict potential phosphorylation switches by mapping phosphorylation sites to protein-protein interactions of known structure and analysing the properties of the protein interface. We predict 1024 sites that could potentially enable or disable particular interactions. We tested a selection of these switches and showed that phosphomimetic mutations indeed affect interactions. We estimate that there are likely thousands of phosphorylation mediated switches yet to be uncovered, even among existing phosphorylation datasets. The results suggest that phosphorylation sites on globular, as distinct from disordered, parts of the proteome frequently function as switches, which might be one of the ancient roles for kinase phosphorylation. PMID:28346509

Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

PubMed

Ramya, L; Ramakrishnan, Vigneshwar

2016-07-01

Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel protein-protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani.

PubMed

Mishra, Arjun K; Agnihotri, Pragati; Srivastava, Vijay Kumar; Pratap, J Venkatesh

2015-01-09

Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethionine (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein-protein interaction between SpdSyn and AdoMetDc. The protein-protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes. Copyright © 2014 Elsevier Inc. All rights reserved.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

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

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

Protein-protein interaction networks: unraveling the wiring of molecular machines within the cell.

PubMed

De Las Rivas, Javier; Fontanillo, Celia

2012-11-01

Mapping and understanding of the protein interaction networks with their key modules and hubs can provide deeper insights into the molecular machinery underlying complex phenotypes. In this article, we present the basic characteristics and definitions of protein networks, starting with a distinction of the different types of associations between proteins. We focus the review on protein-protein interactions (PPIs), a subset of associations defined as physical contacts between proteins that occur by selective molecular docking in a particular biological context. We present such definition as opposed to other types of protein associations derived from regulatory, genetic, structural or functional relations. To determine PPIs, a variety of binary and co-complex methods exist; however, not all the technologies provide the same information and data quality. A way of increasing confidence in a given protein interaction is to integrate orthogonal experimental evidences. The use of several complementary methods testing each single interaction assesses the accuracy of PPI data and tries to minimize the occurrence of false interactions. Following this approach there have been important efforts to unify primary databases of experimentally proven PPIs into integrated databases. These meta-databases provide a measure of the confidence of interactions based on the number of experimental proofs that report them. As a conclusion, we can state that integrated information allows the building of more reliable interaction networks. Identification of communities, cliques, modules and hubs by analysing the topological parameters and graph properties of the protein networks allows the discovery of central/critical nodes, which are candidates to regulate cellular flux and dynamics.

Cytoscape: a software environment for integrated models of biomolecular interaction networks.

PubMed

Shannon, Paul; Markiel, Andrew; Ozier, Owen; Baliga, Nitin S; Wang, Jonathan T; Ramage, Daniel; Amin, Nada; Schwikowski, Benno; Ideker, Trey

2003-11-01

Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

Developing advanced X-ray scattering methods combined with crystallography and computation.

PubMed

Perry, J Jefferson P; Tainer, John A

2013-03-01

The extensive use of small angle X-ray scattering (SAXS) over the last few years is rapidly providing new insights into protein interactions, complex formation and conformational states in solution. This SAXS methodology allows for detailed biophysical quantification of samples of interest. Initial analyses provide a judgment of sample quality, revealing the potential presence of aggregation, the overall extent of folding or disorder, the radius of gyration, maximum particle dimensions and oligomerization state. Structural characterizations include ab initio approaches from SAXS data alone, and when combined with previously determined crystal/NMR, atomistic modeling can further enhance structural solutions and assess validity. This combination can provide definitions of architectures, spatial organizations of protein domains within a complex, including those not determined by crystallography or NMR, as well as defining key conformational states of a protein interaction. SAXS is not generally constrained by macromolecule size, and the rapid collection of data in a 96-well plate format provides methods to screen sample conditions. This includes screening for co-factors, substrates, differing protein or nucleotide partners or small molecule inhibitors, to more fully characterize the variations within assembly states and key conformational changes. Such analyses may be useful for screening constructs and conditions to determine those most likely to promote crystal growth of a complex under study. Moreover, these high throughput structural determinations can be leveraged to define how polymorphisms affect assembly formations and activities. This is in addition to potentially providing architectural characterizations of complexes and interactions for systems biology-based research, and distinctions in assemblies and interactions in comparative genomics. Thus, SAXS combined with crystallography/NMR and computation provides a unique set of tools that should be considered as being part of one's repertoire of biophysical analyses, when conducting characterizations of protein and other macromolecular interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

Engineering Aromatic-Aromatic Interactions To Nucleate Folding in Intrinsically Disordered Regions of Proteins.

PubMed

Balakrishnan, Swati; Sarma, Siddhartha P

2017-08-22

Aromatic interactions are an important force in protein folding as they combine the stability of a hydrophobic interaction with the selectivity of a hydrogen bond. Much of our understanding of aromatic interactions comes from "bioinformatics" based analyses of protein structures and from the contribution of these interactions to stabilizing secondary structure motifs in model peptides. In this study, the structural consequences of aromatic interactions on protein folding have been explored in engineered mutants of the molten globule protein apo-cytochrome b 5 . Structural changes from disorder to order due to aromatic interactions in two variants of the protein, viz., WF-cytb5 and FF-cytb5, result in significant long-range secondary and tertiary structure. The results show that 54 and 52% of the residues in WF-cytb5 and FF-cytb5, respectively, occupy ordered regions versus 26% in apo-cytochrome b 5 . The interactions between the aromatic groups are offset-stacked and edge-to-face for the Trp-Phe and Phe-Phe mutants, respectively. Urea denaturation studies indicate that both mutants have a C m higher than that of apo-cytochrome b 5 and are more stable to chaotropic agents than apo-cytochrome b 5 . The introduction of these aromatic residues also results in "trimer" interactions with existing aromatic groups, reaffirming the selectivity of the aromatic interactions. These studies provide insights into the aromatic interactions that drive disorder-to-order transitions in intrinsically disordered regions of proteins and will aid in de novo protein design beyond small peptide scaffolds.

A structural insight into the inhibitory mechanism of an orally active PI3K/mTOR dual inhibitor, PKI-179 using computational approaches.

PubMed

Mohd Rehan

2015-11-01

The PI3K/AKT/mTOR signaling pathway has been identified as an important target for cancer therapy. Attempts are increasingly made to design the inhibitors against the key proteins of this pathway for anti-cancer therapy. The PI3K/mTOR dual inhibitors have proved more effective than the inhibitors against only single protein targets. Recently discovered PKI-179, an orally effective compound, is one such dual inhibitor targeting both PI3K and mTOR. This anti-cancer compound is efficacious both in vitro and in vivo. However, the binding mechanisms and the molecular interactions of PKI-179 with PI3K and mTOR are not yet available. The current study investigated the exact binding mode and the molecular interactions of PKI-179 with PI3Kγ and mTOR using molecular docking and (un)binding simulation analyses. The study identified PKI-179 interacting residues of both the proteins and their importance in binding was ranked by the loss in accessible surface area, number of molecular interactions of the residue, and consistent appearance of the residue in (un)binding simulation analysis. The key residues involved in binding of PKI-179 were Ala-805 in PI3Kγ and Ile-2163 in mTOR as they have lost maximum accessible surface area due to binding. In addition, the residues which played a role in binding of the drug but were away from the catalytic site were also identified using (un)binding simulation analyses. Finally, comparison of the interacting residues in the respective catalytic sites was done for the difference in the binding of the drug to the two proteins. Thus, the pairs of the residues falling at the similar location with respect to the docked drug were identified. The striking similarity in the interacting residues of the catalytic site explains the concomitant inhibition of both proteins by a number of inhibitors. In conclusion, the docking and (un)binding simulation analyses of dual inhibitor PKI-179 with PI3K and mTOR will provide a suitable multi-target model for studying drug-protein interactions and thus help in designing the novel drugs with higher potency. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

PubMed

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Bacteriophage Protein–Protein Interactions

PubMed Central

Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian

2012-01-01

Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage–host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

High-throughput analysis of peptide binding modules

PubMed Central

Liu, Bernard A.; Engelmann, Brett; Nash, Piers D.

2014-01-01

Modular protein interaction domains that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some protein interaction domains such as SH2, 14-3-3, Chromo and Bromo domains serve to recognize post-translational modification of amino acids (such as phosphorylation, acetylation, methylation etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PDZ domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High throughput analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls of high-throughput analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of protein interaction domains and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions. PMID:22610655

Oligomerisation status and evolutionary conservation of interfaces of protein structural domain superfamilies.

PubMed

Sukhwal, Anshul; Sowdhamini, Ramanathan

2013-07-01

Protein-protein interactions are important in carrying out many biological processes and functions. These interactions may be either permanent or of temporary nature. Several studies have employed tools like solvent accessibility and graph theory to identify these interactions, but still more studies need to be performed to quantify and validate them. Although we now have many databases available with predicted and experimental results on protein-protein interactions, we still do not have many databases which focus on providing structural details of the interacting complexes, their oligomerisation state and homologues. In this work, protein-protein interactions have been thoroughly investigated within the structural regime and quantified for their strength using calculated pseudoenergies. The PPCheck server, an in-house webserver, has been used for calculating the pseudoenergies like van der Waals, hydrogen bonds and electrostatic energy based on distances between atoms of amino acids from two interacting proteins. PPCheck can be visited at . Based on statistical data, as obtained by studying established protein-protein interacting complexes from earlier studies, we came to a conclusion that an average protein-protein interface consisted of about 51 to 150 amino acid residues and the generalized energy per residue ranged from -2 kJ mol(-1) to -6 kJ mol(-1). We found that some of the proteins have an exceptionally higher number of amino acids at the interface and it was purely because of their elaborate interface or extended topology i.e. some of their secondary structure regions or loops were either inter-mixing or running parallel to one another or they were taking part in domain swapping. Residue networks were prepared for all the amino acids of the interacting proteins involved in different types of interactions (like van der Waals, hydrogen-bonding, electrostatic or intramolecular interactions) and were analysed between the query domain-interacting partner pair and its remote homologue-interacting partner pair. We found that, in exceptional cases, homologous proteins belonging to the same superfamily, but with remote sequence similarity, can share similar interfaces.

Dissortativity and duplications in oral cancer

NASA Astrophysics Data System (ADS)

Shinde, Pramod; Yadav, Alok; Rai, Aparna; Jalan, Sarika

2015-08-01

More than 300 000 new cases worldwide are being diagnosed with oral cancer annually. Complexity of oral cancer renders designing drug targets very difficult. We analyse protein-protein interaction network for the normal and oral cancer tissue and detect crucial changes in the structural properties of the networks in terms of the interactions of the hub proteins and the degree-degree correlations. Further analysis of the spectra of both the networks, while exhibiting universal statistical behaviour, manifest distinction in terms of the zero degeneracy, providing insight to the complexity of the underlying system.

Integration of structural dynamics and molecular evolution via protein interaction networks: a new era in genomic medicine.

PubMed

Kumar, Avishek; Butler, Brandon M; Kumar, Sudhir; Ozkan, S Banu

2015-12-01

Sequencing technologies are revealing many new non-synonymous single nucleotide variants (nsSNVs) in each personal exome. To assess their functional impacts, comparative genomics is frequently employed to predict if they are benign or not. However, evolutionary analysis alone is insufficient, because it misdiagnoses many disease-associated nsSNVs, such as those at positions involved in protein interfaces, and because evolutionary predictions do not provide mechanistic insights into functional change or loss. Structural analyses can aid in overcoming both of these problems by incorporating conformational dynamics and allostery in nSNV diagnosis. Finally, protein-protein interaction networks using systems-level methodologies shed light onto disease etiology and pathogenesis. Bridging these network approaches with structurally resolved protein interactions and dynamics will advance genomic medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Abnormally high expression of POLD1, MCM2, and PLK4 promotes relapse of acute lymphoblastic leukemia.

PubMed

Li, Sheng; Wang, Chengzhong; Wang, Weikai; Liu, Weidong; Zhang, Guiqin

2018-05-01

This study aimed to explore the underlying mechanism of relapsed acute lymphoblastic leukemia (ALL).Datasets of GSE28460 and GSE18497 were downloaded from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) between diagnostic and relapsed ALL samples were identified using Limma package in R, and a Venn diagram was drawn. Next, functional enrichment analyses of co-regulated DEGs were performed. Based on the String database, protein-protein interaction network and module analyses were also conducted. Moreover, transcription factors and miRNAs targeting co-regulated DEGs were predicted using the WebGestalt online tool.A total of 71 co-regulated DEGs were identified, including 56 co-upregulated genes and 15 co-downregulated genes. Functional enrichment analyses showed that upregulated DEGs were significantly enriched in the cell cycle, and DNA replication, and repair related pathways. POLD1, MCM2, and PLK4 were hub proteins in both protein-protein interaction network and module, and might be potential targets of E2F. Additionally, POLD1 and MCM2 were found to be regulated by miR-520H via E2F1.High expression of POLD1, MCM2, and PLK4 might play positive roles in the recurrence of ALL, and could serve as potential therapeutic targets for the treatment of relapsed ALL.

The effect of high pressure on the functional properties of pork myofibrillar proteins.

PubMed

Grossi, Alberto; Olsen, Karsten; Bolumar, Tomas; Rinnan, Åsmund; Øgendal, Lars H; Orlien, Vibeke

2016-04-01

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments. Copyright © 2015 Elsevier Ltd. All rights reserved.

A fluorescent bimolecular complementation screen reveals MAF1, RNF7 and SETD3 as PCNA-associated proteins in human cells

PubMed Central

Cooper, Simon E; Hodimont, Elsie; Green, Catherine M

2015-01-01

The proliferating cell nuclear antigen (PCNA) is a conserved component of DNA replication factories, and interactions with PCNA mediate the recruitment of many essential DNA replication enzymes to these sites of DNA synthesis. A complete description of the structure and composition of these factories remains elusive, and a better knowledge of them will improve our understanding of how the maintenance of genome and epigenetic stability is achieved. To fully characterize the set of proteins that interact with PCNA we developed a bimolecular fluorescence complementation (BiFC) screen for PCNA-interactors in human cells. This 2-hybrid type screen for interactors from a human cDNA library is rapid and efficient. The fluorescent read-out for protein interaction enables facile selection of interacting clones, and we combined this with next generation sequencing to identify the cDNAs encoding the interacting proteins. This method was able to reproducibly identify previously characterized PCNA-interactors but importantly also identified RNF7, Maf1 and SetD3 as PCNA-interacting proteins. We validated these interactions by co-immunoprecipitation from human cell extracts and by interaction analyses using recombinant proteins. These results show that the BiFC screen is a valuable method for the identification of protein-protein interactions in living mammalian cells. This approach has potentially wide application as it is high throughput and readily automated. We suggest that, given this interaction with PCNA, Maf1, RNF7, and SetD3 are potentially involved in DNA replication, DNA repair, or associated processes. PMID:26030842

Application of the fragment molecular orbital method analysis to fragment-based drug discovery of BET (bromodomain and extra-terminal proteins) inhibitors.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Ueda, Kazuyoshi

2017-06-01

The molecular interactions of inhibitors of bromodomains (BRDs) were investigated. BRDs are protein interaction modules that recognizing ε-N-acetyl-lysine (εAc-Lys) motifs found in histone tails and are promising protein-protein interaction (PPI) targets. First, we analyzed a peptide ligand containing εAc-Lys to evaluate native PPIs. We then analyzed tetrahydroquinazoline-6-yl-benzensulfonamide derivatives found by fragment-based drug design (FBDD) and examined their interactions with the protein compared with the peptide ligand in terms of the inter-fragment interaction energy. In addition, we analyzed benzodiazepine derivatives that are high-affinity ligands for BRDs and examined differences in the CH/π interactions of the amino acid residues. We further surveyed changes in the charges of the amino acid residues among individual ligands, performed pair interaction energy decomposition analysis and estimated the water profile within the ligand binding site. Thus, useful insights for drug design were provided. Through these analyses and considerations, we show that the FMO method is a useful drug design tool to evaluate the process of FBDD and to explore PPI inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

DTIC Science & Technology

the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

PubMed Central

Kuo, Lili; Hurst-Hess, Kelley R.; Koetzner, Cheri A.

2016-01-01

ABSTRACT The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins. PMID:26889024

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus

PubMed Central

Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations. PMID:26180540

Evolutionary Influenced Interaction Pattern as Indicator for the Investigation of Natural Variants Causing Nephrogenic Diabetes Insipidus.

PubMed

Grunert, Steffen; Labudde, Dirk

2015-01-01

The importance of short membrane sequence motifs has been shown in many works and emphasizes the related sequence motif analysis. Together with specific transmembrane helix-helix interactions, the analysis of interacting sequence parts is helpful for understanding the process during membrane protein folding and in retaining the three-dimensional fold. Here we present a simple high-throughput analysis method for deriving mutational information of interacting sequence parts. Applied on aquaporin water channel proteins, our approach supports the analysis of mutational variants within different interacting subsequences and finally the investigation of natural variants which cause diseases like, for example, nephrogenic diabetes insipidus. In this work we demonstrate a simple method for massive membrane protein data analysis. As shown, the presented in silico analyses provide information about interacting sequence parts which are constrained by protein evolution. We present a simple graphical visualization medium for the representation of evolutionary influenced interaction pattern pairs (EIPPs) adapted to mutagen investigations of aquaporin-2, a protein whose mutants are involved in the rare endocrine disorder known as nephrogenic diabetes insipidus, and membrane proteins in general. Furthermore, we present a new method to derive new evolutionary variations within EIPPs which can be used for further mutagen laboratory investigations.

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes.

PubMed

Sonntag, Yonathan; Musgaard, Maria; Olesen, Claus; Schiøtt, Birgit; Møller, Jesper Vuust; Nissen, Poul; Thøgersen, Lea

2011-01-01

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

Models of S/π interactions in protein structures: Comparison of the H2S–benzene complex with PDB data

PubMed Central

Ringer, Ashley L.; Senenko, Anastasia; Sherrill, C. David

2007-01-01

S/π interactions are prevalent in biochemistry and play an important role in protein folding and stabilization. Geometries of cysteine/aromatic interactions found in crystal structures from the Brookhaven Protein Data Bank (PDB) are analyzed and compared with the equilibrium configurations predicted by high-level quantum mechanical results for the H2S–benzene complex. A correlation is observed between the energetically favorable configurations on the quantum mechanical potential energy surface of the H2S–benzene model and the cysteine/aromatic configurations most frequently found in crystal structures of the PDB. In contrast to some previous PDB analyses, configurations with the sulfur over the aromatic ring are found to be the most important. Our results suggest that accurate quantum computations on models of noncovalent interactions may be helpful in understanding the structures of proteins and other complex systems. PMID:17766371

The determinants of bond angle variability in protein/peptide backbones: A comprehensive statistical/quantum mechanics analysis.

PubMed

Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

2015-11-01

The elucidation of the mutual influence between peptide bond geometry and local conformation has important implications for protein structure refinement, validation, and prediction. To gain insights into the structural determinants and the energetic contributions associated with protein/peptide backbone plasticity, we here report an extensive analysis of the variability of the peptide bond angles by combining statistical analyses of protein structures and quantum mechanics calculations on small model peptide systems. Our analyses demonstrate that all the backbone bond angles strongly depend on the peptide conformation and unveil the existence of regular trends as function of ψ and/or φ. The excellent agreement of the quantum mechanics calculations with the statistical surveys of protein structures validates the computational scheme here employed and demonstrates that the valence geometry of protein/peptide backbone is primarily dictated by local interactions. Notably, for the first time we show that the position of the H(α) hydrogen atom, which is an important parameter in NMR structural studies, is also dependent on the local conformation. Most of the trends observed may be satisfactorily explained by invoking steric repulsive interactions; in some specific cases the valence bond variability is also influenced by hydrogen-bond like interactions. Moreover, we can provide a reliable estimate of the energies involved in the interplay between geometry and conformations. © 2015 Wiley Periodicals, Inc.

On the Importance of Polar Interactions for Complexes Containing Intrinsically Disordered Proteins

PubMed Central

Wong, Eric T. C.; Na, Dokyun; Gsponer, Jörg

2013-01-01

There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions. PMID:23990768

Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

PubMed Central

Kozakova, Lucie; Liao, Chunyan; Guerineau, Marc; Colnaghi, Rita; Vidot, Susanne; Marek, Jaromir; Bathula, Sreenivas R.; Lehmann, Alan R.; Palecek, Jan

2011-01-01

Background The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6–8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. Methodology/Principal Findings Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins. Conclusions/Significance We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins. PMID:21364888

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses.

PubMed

Rohman, Muhammad S; Tadokoro, Takashi; Angkawidjaja, Clement; Abe, Yumi; Matsumura, Hiroyoshi; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2009-01-01

The Arg97 --> Gly and Asp136 --> His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 degrees C, respectively, in T(m), and 3.5 and 6.1 kJ x mol(-1), respectively, in DeltaG(H2O). These mutations also stabilized the So-RNase HI derivative (4x-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6x-RNase HI) was more stable than the wild-type protein by 28.8 degrees C in T(m) and 27.0 kJ x mol(-1) in DeltaG(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6x-RNase HI protein was determined at 2.5 A resolution. The main chain fold and interactions of the side-chains of the 6x-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6x-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.

Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

PubMed

Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

2013-12-13

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system.

PubMed

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. This database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

Amyloid precursor protein interaction network in human testis: sentinel proteins for male reproduction.

PubMed

Silva, Joana Vieira; Yoon, Sooyeon; Domingues, Sara; Guimarães, Sofia; Goltsev, Alexander V; da Cruz E Silva, Edgar Figueiredo; Mendes, José Fernando F; da Cruz E Silva, Odete Abreu Beirão; Fardilha, Margarida

2015-01-16

Amyloid precursor protein (APP) is widely recognized for playing a central role in Alzheimer's disease pathogenesis. Although APP is expressed in several tissues outside the human central nervous system, the functions of APP and its family members in other tissues are still poorly understood. APP is involved in several biological functions which might be potentially important for male fertility, such as cell adhesion, cell motility, signaling, and apoptosis. Furthermore, APP superfamily members are known to be associated with fertility. Knowledge on the protein networks of APP in human testis and spermatozoa will shed light on the function of APP in the male reproductive system. We performed a Yeast Two-Hybrid screen and a database search to study the interaction network of APP in human testis and sperm. To gain insights into the role of APP superfamily members in fertility, the study was extended to APP-like protein 2 (APLP2). We analyzed several topological properties of the APP interaction network and the biological and physiological properties of the proteins in the APP interaction network were also specified by gene ontologyand pathways analyses. We classified significant features related to the human male reproduction for the APP interacting proteins and identified modules of proteins with similar functional roles which may show cooperative behavior for male fertility. The present work provides the first report on the APP interactome in human testis. Our approach allowed the identification of novel interactions and recognition of key APP interacting proteins for male reproduction, particularly in sperm-oocyte interaction.

Calponin-Like Chd64 Is Partly Disordered

PubMed Central

Jakób, Michał; Szpotkowski, Kamil; Wojtas, Magdalena; Rymarczyk, Grzegorz; Ożyhar, Andrzej

2014-01-01

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function. PMID:24805353

Enhancing the prediction of protein pairings between interacting families using orthology information

PubMed Central

Izarzugaza, Jose MG; Juan, David; Pons, Carles; Pazos, Florencio; Valencia, Alfonso

2008-01-01

Background It has repeatedly been shown that interacting protein families tend to have similar phylogenetic trees. These similarities can be used to predicting the mapping between two families of interacting proteins (i.e. which proteins from one family interact with which members of the other). The correct mapping will be that which maximizes the similarity between the trees. The two families may eventually comprise orthologs and paralogs, if members of the two families are present in more than one organism. This fact can be exploited to restrict the possible mappings, simply by impeding links between proteins of different organisms. We present here an algorithm to predict the mapping between families of interacting proteins which is able to incorporate information regarding orthologues, or any other assignment of proteins to "classes" that may restrict possible mappings. Results For the first time in methods for predicting mappings, we have tested this new approach on a large number of interacting protein domains in order to statistically assess its performance. The method accurately predicts around 80% in the most favourable cases. We also analysed in detail the results of the method for a well defined case of interacting families, the sensor and kinase components of the Ntr-type two-component system, for which up to 98% of the pairings predicted by the method were correct. Conclusion Based on the well established relationship between tree similarity and interactions we developed a method for predicting the mapping between two interacting families using genomic information alone. The program is available through a web interface. PMID:18215279

The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions.

PubMed

Won, Jung Hee; Park, Jung Sik; Ju, Hyun Hee; Kim, Soyeon; Suh-Kim, Haeyoung; Ghil, Sung Ho

2008-05-01

Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gbetagamma dimer; downstream effectors for its alpha subunit (Goalpha) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goalpha-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goalpha. Interactions between PLZF and Goalpha were confirmed using in vitro and in vivo affinity binding assays. Activated Goalpha interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/ialpha-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goalpha modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goalpha and its coupled receptor.

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

PubMed Central

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

2013-01-01

Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

NASA Astrophysics Data System (ADS)

Gnedenko, O. V.; Yablokov, E. O.; Usanov, S. A.; Mukha, D. V.; Sergeev, G. V.; Bulko, T. V.; Kuzikov, A. V.; Moskaleva, N. E.; Shumyantseva, V. V.; Ivanov, A. S.; Archakov, A. I.

2014-02-01

The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

Sunitinib: from charge-density studies to interaction with proteins.

PubMed

Malińska, Maura; Jarzembska, Katarzyna N; Goral, Anna M; Kutner, Andrzej; Woźniak, Krzysztof; Dominiak, Paulina M

2014-05-01

Protein kinases are targets for the treatment of a number of diseases. Sunitinib malate is a type I inhibitor of tyrosine kinases and was approved as a drug in 2006. This contribution constitutes the first comprehensive analysis of the crystal structures of sunitinib malate and of complexes of sunitinib with a series of protein kinases. The high-resolution single-crystal X-ray measurement and aspherical atom databank approach served as a basis for reconstruction of the charge-density distribution of sunitinib and its protein complexes. Hirshfeld surface and topological analyses revealed a similar interaction pattern in the sunitinib malate crystal structure to that in the protein binding pockets. Sunitinib forms nine preserved bond paths corresponding to hydrogen bonds and also to the C-H···O and C-H···π contacts common to the VEGRF2, CDK2, G2, KIT and IT kinases. In general, sunitinib interacts with the studied proteins with a similar electrostatic interaction energy and can adjust its conformation to fit the binding pocket in such a way as to enhance the electrostatic interactions, e.g. hydrogen bonds in ligand-kinase complexes. Such behaviour may be responsible for the broad spectrum of action of sunitinib as a kinase inhibitor.

Proteomic Identification of the Galectin-1-Involved Molecular Pathways in Urinary Bladder Urothelial Carcinoma.

PubMed

Li, Chien-Feng; Shen, Kun-Hung; Chien, Lan-Hsiang; Huang, Cheng-Hao; Wu, Ting-Feng; He, Hong-Lin

2018-04-19

Among various heterogeneous types of bladder tumors, urothelial carcinoma is the most prevalent lesion. Some of the urinary bladder urothelial carcinomas (UBUCs) develop local recurrence and may cause distal invasion. Galectin-1 de-regulation significantly affects cell transformation, cell proliferation, angiogenesis, and cell invasiveness. In continuation of our previous investigation on the role of galectin-1 in UBUC tumorigenesis, in this study, proteomics strategies were implemented in order to find more galectin-1-associated signaling pathways. The results of this study showed that galectin-1 knockdown could induce 15 down-regulated proteins and two up-regulated proteins in T24 cells. These de-regulated proteins might participate in lipid/amino acid/energy metabolism, cytoskeleton, cell proliferation, cell-cell interaction, cell apoptosis, metastasis, and protein degradation. The aforementioned dys-regulated proteins were confirmed by western immunoblotting. Proteomics results were further translated to prognostic markers by analyses of biopsy samples. Results of cohort studies demonstrated that over-expressions of glutamine synthetase, alcohol dehydrogenase (NADP⁺), fatty acid binding protein 4, and toll interacting protein in clinical specimens were all significantly associated with galectin-1 up-regulation. Univariate analyses showed that de-regulations of glutamine synthetase and fatty acid binding protein 4 in clinical samples were respectively linked to disease-specific survival and metastasis-free survival.

Analysis of Protein–Protein Interactions in MCF-7 and MDA-MB-231 Cell Lines Using Phthalic Acid Chemical Probes

PubMed Central

Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

2014-01-01

Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein–protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

SpidermiR: An R/Bioconductor Package for Integrative Analysis with miRNA Data.

PubMed

Cava, Claudia; Colaprico, Antonio; Bertoli, Gloria; Graudenzi, Alex; Silva, Tiago C; Olsen, Catharina; Noushmehr, Houtan; Bontempi, Gianluca; Mauri, Giancarlo; Castiglioni, Isabella

2017-01-27

Gene Regulatory Networks (GRNs) control many biological systems, but how such network coordination is shaped is still unknown. GRNs can be subdivided into basic connections that describe how the network members interact e.g., co-expression, physical interaction, co-localization, genetic influence, pathways, and shared protein domains. The important regulatory mechanisms of these networks involve miRNAs. We developed an R/Bioconductor package, namely SpidermiR, which offers an easy access to both GRNs and miRNAs to the end user, and integrates this information with differentially expressed genes obtained from The Cancer Genome Atlas. Specifically, SpidermiR allows the users to: (i) query and download GRNs and miRNAs from validated and predicted repositories; (ii) integrate miRNAs with GRNs in order to obtain miRNA-gene-gene and miRNA-protein-protein interactions, and to analyze miRNA GRNs in order to identify miRNA-gene communities; and (iii) graphically visualize the results of the analyses. These analyses can be performed through a single interface and without the need for any downloads. The full data sets are then rapidly integrated and processed locally.

Glycan array data management at Consortium for Functional Glycomics.

PubMed

Venkataraman, Maha; Sasisekharan, Ram; Raman, Rahul

2015-01-01

Glycomics or the study of structure-function relationships of complex glycans has reshaped post-genomics biology. Glycans mediate fundamental biological functions via their specific interactions with a variety of proteins. Recognizing the importance of glycomics, large-scale research initiatives such as the Consortium for Functional Glycomics (CFG) were established to address these challenges. Over the past decade, the Consortium for Functional Glycomics (CFG) has generated novel reagents and technologies for glycomics analyses, which in turn have led to generation of diverse datasets. These datasets have contributed to understanding glycan diversity and structure-function relationships at molecular (glycan-protein interactions), cellular (gene expression and glycan analysis), and whole organism (mouse phenotyping) levels. Among these analyses and datasets, screening of glycan-protein interactions on glycan array platforms has gained much prominence and has contributed to cross-disciplinary realization of the importance of glycomics in areas such as immunology, infectious diseases, cancer biomarkers, etc. This manuscript outlines methodologies for capturing data from glycan array experiments and online tools to access and visualize glycan array data implemented at the CFG.

Characterization of binding preference of polyhydroxyalkanoate biosynthesis-related multifunctional protein PhaM from Ralstonia eutropha.

PubMed

Ushimaru, Kazunori; Tsuge, Takeharu

2016-05-01

The binding preference of a polyhydroxyalkanoate (PHA) biosynthesis-related multifunctional protein from Ralstonia eutropha (PhaMRe) was characterized. In vitro activity assay showed that PHA synthase from R. eutropha (PhaCRe) was activated by the presence of PhaMRe but PHA synthase from Aeromonas caviae (PhaCAc) was not. Additionally, in vitro assays of protein-protein interactions demonstrated that PhaMRe interacted with PhaCRe directly, but did not interact with PhaCAc. These results suggest that the protein-protein interaction is important for the activation of PhaC by PhaMRe. Further analyses indicated that PhaMRe has little or no direct interaction with the PHA polymer chain. Subsequently, PHA biosynthesis genes (phaA Re, phaB Re, and phaC Re/phaC Ac) and the phaM Re gene were introduced into recombinant Escherichia coli and cultivated for PHA accumulation. Contrary to our expectations, the expression of PhaMRe decreased PHA accumulation and changed the morphology of PHA granules to be microscopically obscure shape in PhaCRe-expressing E. coli. No change in the amount of P(3HB) or the morphology of granules by PhaMRe expression was observed in PhaCAc-expressing E. coli. These observations suggest that PhaMRe affects cellular physiology through the PhaM-PhaC interaction.

The CCDC55 couples cannabinoid receptor CNR1 to a putative DISC1 schizophrenia pathway.

PubMed

Xie, J; Gizatullin, R; Vukojevic, V; Leopardi, R

2015-12-03

Our previous study suggested that the coiled coil domain-containing 55 gene (CCDC55), also named as NSRP1 (nuclear speckle splicing regulatory protein 1 (NSRP1)), was encompassed in a haplotype block spanning over the serotonin transporter (5-HTT) gene in patients with schizophrenia (SCZ). However, the neurobiological function of CCDC55 gene remains unknown. This study aims to uncover the potential role of CCDC55 in SCZ-associated molecular pathways. Using molecular cloning, sequencing and immune blotting to identify basic properties, yeast two-hybrid screening and glutathione S-transferase (GST) pull-down assay to test protein-protein interaction, and confocal laser scanning microscopy (CSLM) to show intracellular interaction of proteins. (i) CCDC55 is expressed as a nuclear protein in human neuronal cells; (ii) Protein-protein interaction analyses showed CCDC55 physically interacted with Ran binding protein 9 (RanBP9) and disrupted in schizophrenia 1 (DISC1); (iii) CCDC55 and RanBP9 co-localized in the nucleus of human neuronal cells; (iv) CCDC55 also interacted with the cannabinoid receptor 1 (CNR1), and with the brain cannabinoid receptor-interacting protein 1a (CNRIP1a); (v) CNR1 activation in differentiated human neuronal cells resulted in an altered RanBP9 localization. CCDC55 may be involved in a functional bridging between the CNR1 activation and the DISC1/RanBP9-associated pathways. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Ebola virus VP24 interacts with NP to facilitate nucleocapsid assembly and genome packaging.

PubMed

Banadyga, Logan; Hoenen, Thomas; Ambroggio, Xavier; Dunham, Eric; Groseth, Allison; Ebihara, Hideki

2017-08-09

Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.

Interactions of different carrageenan isoforms and flour components in breadmaking.

PubMed

León, A E; Ribotta, P D; Ausar, S F; Fernández, C; Lanada, C A; Beltramo, D M

2000-07-01

The aim of this study was to compare the effects of carrageenans with different sulfate contents on bread volume and dough rheological properties. Results showed that only lambda carrageenan, the most sulfated isoform, produced a significant increase in bread volume. In contrast, the different carrageenans induced a negative effect on the cookie factor. Alveographic and farinographic analyses indicated that dough rheological properties were differentially modified depending on whether lambda carrageenan was added to flour and then hydrated or vice versa. Analysis of the interaction between lambda carrageenan and flour components by infrared spectroscopy and SDS-PAGE indicated that a pool of low molecular weight hydrophobic gluten proteins interact with carrageenan. This interaction drastically changes their physicochemical properties since carrageenan-gluten protein complexes show a hydrophilic behavior. In addition, the results indicate that carrageenan sulfate groups and probably the amino groups of glutamines present in the primary structure of gluten proteins are involved in the interaction.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Architecture of the human interactome defines protein communities and disease networks

PubMed Central

Huttlin, Edward L.; Bruckner, Raphael J.; Paulo, Joao A.; Cannon, Joe R.; Ting, Lily; Baltier, Kurt; Colby, Greg; Gebreab, Fana; Gygi, Melanie P.; Parzen, Hannah; Szpyt, John; Tam, Stanley; Zarraga, Gabriela; Pontano-Vaites, Laura; Swarup, Sharan; White, Anne E.; Schweppe, Devin K.; Rad, Ramin; Erickson, Brian K.; Obar, Robert A.; Guruharsha, K.G.; Li, Kejie; Artavanis-Tsakonas, Spyros; Gygi, Steven P.; Harper, J. Wade

2017-01-01

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidation of how genome variation contributes to disease1–3. Here, we present BioPlex 2.0 (Biophysical Interactions of ORFEOME-derived complexes), which employs robust affinity purification-mass spectrometry (AP-MS) methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein coding genes from the human genome, and constitutes the largest such network to date. With >56,000 candidate interactions, BioPlex 2.0 contains >29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering (MCL)5 of interacting proteins identified more than 1300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization. PMID:28514442

Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

PubMed Central

Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

2016-01-01

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200.

PubMed

Morselli, Eugenia; Shen, Shensi; Ruckenstuhl, Christoph; Bauer, Maria Anna; Mariño, Guillermo; Galluzzi, Lorenzo; Criollo, Alfredo; Michaud, Mickael; Maiuri, Maria Chiara; Chano, Tokuhiro; Madeo, Frank; Kroemer, Guido

2011-08-15

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53 (K382R) failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

Environmental Light and Its Relationship with Electromagnetic Resonances of Biomolecular Interactions, as Predicted by the Resonant Recognition Model.

PubMed

Cosic, Irena; Cosic, Drasko; Lazar, Katarina

2016-06-29

The meaning and influence of light to biomolecular interactions, and consequently to health, has been analyzed using the Resonant Recognition Model (RRM). The RRM proposes that biological processes/interactions are based on electromagnetic resonances between interacting biomolecules at specific electromagnetic frequencies within the infra-red, visible and ultra-violet frequency ranges, where each interaction can be identified by the certain frequency critical for resonant activation of specific biological activities of proteins and DNA. We found that: (1) the various biological interactions could be grouped according to their resonant frequency into super families of these functions, enabling simpler analyses of these interactions and consequently analyses of influence of electromagnetic frequencies to health; (2) the RRM spectrum of all analyzed biological functions/interactions is the same as the spectrum of the sun light on the Earth, which is in accordance with fact that life is sustained by the sun light; (3) the water is transparent to RRM frequencies, enabling proteins and DNA to interact without loss of energy; (4) the spectrum of some artificial sources of light, as opposed to the sun light, do not cover the whole RRM spectrum, causing concerns for disturbance to some biological functions and consequently we speculate that it can influence health.

Environmental Light and Its Relationship with Electromagnetic Resonances of Biomolecular Interactions, as Predicted by the Resonant Recognition Model

PubMed Central

Cosic, Irena; Cosic, Drasko; Lazar, Katarina

2016-01-01

The meaning and influence of light to biomolecular interactions, and consequently to health, has been analyzed using the Resonant Recognition Model (RRM). The RRM proposes that biological processes/interactions are based on electromagnetic resonances between interacting biomolecules at specific electromagnetic frequencies within the infra-red, visible and ultra-violet frequency ranges, where each interaction can be identified by the certain frequency critical for resonant activation of specific biological activities of proteins and DNA. We found that: (1) the various biological interactions could be grouped according to their resonant frequency into super families of these functions, enabling simpler analyses of these interactions and consequently analyses of influence of electromagnetic frequencies to health; (2) the RRM spectrum of all analyzed biological functions/interactions is the same as the spectrum of the sun light on the Earth, which is in accordance with fact that life is sustained by the sun light; (3) the water is transparent to RRM frequencies, enabling proteins and DNA to interact without loss of energy; (4) the spectrum of some artificial sources of light, as opposed to the sun light, do not cover the whole RRM spectrum, causing concerns for disturbance to some biological functions and consequently we speculate that it can influence health. PMID:27367714

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

PARPs database: A LIMS systems for protein-protein interaction data mining or laboratory information management system

PubMed Central

Droit, Arnaud; Hunter, Joanna M; Rouleau, Michèle; Ethier, Chantal; Picard-Cloutier, Aude; Bourgais, David; Poirier, Guy G

2007-01-01

Background In the "post-genome" era, mass spectrometry (MS) has become an important method for the analysis of proteins and the rapid advancement of this technique, in combination with other proteomics methods, results in an increasing amount of proteome data. This data must be archived and analysed using specialized bioinformatics tools. Description We herein describe "PARPs database," a data analysis and management pipeline for liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics. PARPs database is a web-based tool whose features include experiment annotation, protein database searching, protein sequence management, as well as data-mining of the peptides and proteins identified. Conclusion Using this pipeline, we have successfully identified several interactions of biological significance between PARP-1 and other proteins, namely RFC-1, 2, 3, 4 and 5. PMID:18093328

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection*

PubMed Central

Gold, Matthew G.; Fowler, Douglas M.; Means, Christopher K.; Pawson, Catherine T.; Stephany, Jason J.; Langeberg, Lorene K.; Fields, Stanley; Scott, John D.

2013-01-01

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces. PMID:23625929

Heating and reduction affect the reaction with tannins of wine protein fractions differing in hydrophobicity.

PubMed

Marangon, Matteo; Vincenzi, Simone; Lucchetta, Marco; Curioni, Andrea

2010-02-15

During the storage, bottled white wines can manifest haziness due to the insolubilisation of the grape proteins that may 'survive' in the fermentation process. Although the exact mechanism of this occurrence is not fully understood, proteins and tannins are considered two of the key factors involved in wine hazing, since their aggregation leads to the formation of insoluble particles. To better understand this complex interaction, proteins and tannins from the same unfined Pinot grigio wine were separated. Wine proteins were then fractionated by hydrophobic interaction chromatography (HIC). A significant correlation between hydrophobicity of the wine protein fractions and the haze formed after reacting with wine tannins was found, with the most reactive fractions revealing (by SDS-PAGE and RP-HPLC analyses) the predominant presence of thaumatin-like proteins. Moreover, the effects of both protein heating and disulfide bonds reduction (with dithiotreithol) on haze formation in the presence of tannins were assessed. These treatments generally resulted in an improved reactivity with tannins, and this phenomenon was related to both the surface hydrophobicity and composition of the protein fractions. Therefore, haze formation in wines seems to be related to hydrophobic interactions occurring among proteins and tannins. These interactions should occur on hydrophobic tannin-binding sites, whose exposition on the proteins can depend on both protein heating and reduction. Copyright 2009 Elsevier B.V. All rights reserved.

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

PubMed

O'Neill, Sharon; Mathis, Magalie; Kovačič, Lidija; Zhang, Suisheng; Reinhardt, Jürgen; Scholz, Dimitri; Schopfer, Ulrich; Bouhelal, Rochdi; Knaus, Ulla G

2018-06-08

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22 phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22 phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22 phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Analyses of pea necrotic yellow dwarf virus-encoded proteins.

PubMed

Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

2017-06-01

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

Truncation of the C-terminal region of Toscana Virus NSs protein is critical for interferon-β antagonism and protein stability.

PubMed

Gori Savellini, Gianni; Gandolfo, Claudia; Cusi, Maria Grazia

2015-12-01

Toscana Virus (TOSV) is a Phlebovirus responsible for central nervous system (CNS) injury in humans. The TOSV non-structural protein (NSs), which interacting with RIG-I leads to its degradation, was analysed in the C terminus fragment in order to identify its functional domains. To this aim, two C-terminal truncated NSs proteins, Δ1C-NSs (aa 1-284) and Δ2C-NSs (aa 1-287) were tested. Only Δ1C-NSs did not present any inhibitory effect on RIG-I and it showed a greater stability than the whole NSs protein. Moreover, the deletion of the TLQ aa sequence interposed between the two ΔC constructs caused a greater accumulation of the protein with a weak inhibitory effect on RIG-I, indicating some involvement of these amino acids in the NSs activity. Nevertheless, all the truncated proteins were still able to interact with RIG-I, suggesting that the domains responsible for RIG-I signaling and RIG-I interaction are mapped on different regions of the protein. Copyright © 2015 Elsevier Inc. All rights reserved.

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation

PubMed Central

Upadhyay, Srijana; Xu, Xinping

2016-01-01

ABSTRACT Melanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism. PMID:27879337

Identification of lactoferricin B intracellular targets using an Escherichia coli proteome chip.

PubMed

Tu, Yu-Hsuan; Ho, Yu-Hsuan; Chuang, Ying-Chih; Chen, Po-Chung; Chen, Chien-Sheng

2011-01-01

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach.

Identification of Lactoferricin B Intracellular Targets Using an Escherichia coli Proteome Chip

PubMed Central

Chen, Po-Chung; Chen, Chien-Sheng

2011-01-01

Lactoferricin B (LfcinB) is a well-known antimicrobial peptide. Several studies have indicated that it can inhibit bacteria by affecting intracellular activities, but the intracellular targets of this antimicrobial peptide have not been identified. Therefore, we used E. coli proteome chips to identify the intracellular target proteins of LfcinB in a high-throughput manner. We probed LfcinB with E. coli proteome chips and further conducted normalization and Gene Ontology (GO) analyses. The results of the GO analyses showed that the identified proteins were associated with metabolic processes. Moreover, we validated the interactions between LfcinB and chip assay-identified proteins with fluorescence polarization (FP) assays. Sixteen proteins were identified, and an E. coli interaction database (EcID) analysis revealed that the majority of the proteins that interact with these 16 proteins affected the tricarboxylic acid (TCA) cycle. Knockout assays were conducted to further validate the FP assay results. These results showed that phosphoenolpyruvate carboxylase was a target of LfcinB, indicating that one of its mechanisms of action may be associated with pyruvate metabolism. Thus, we used pyruvate assays to conduct an in vivo validation of the relationship between LfcinB and pyruvate level in E. coli. These results showed that E. coli exposed to LfcinB had abnormal pyruvate amounts, indicating that LfcinB caused an accumulation of pyruvate. In conclusion, this study successfully revealed the intracellular targets of LfcinB using an E. coli proteome chip approach. PMID:22164243

A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein

PubMed Central

Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

2012-01-01

Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering. PMID:22792305

Investigating the effect of key mutations on the conformational dynamics of toll-like receptor dimers through molecular dynamics simulations and protein structure networks.

PubMed

Mahita, Jarjapu; Sowdhamini, Ramanathan

2018-04-01

The Toll-like receptors (TLRs) are critical components of the innate immune system due to their ability to detect conserved pathogen-associated molecular patterns, present in bacteria, viruses, and other microorganisms. Ligand detection by TLRs leads to a signaling cascade, mediated by interactions among TIR domains present in the receptors, the bridging adaptors and sorting adaptors. The BB loop is a highly conserved region present in the TIR domain and is crucial for mediating interactions among TIR domain-containing proteins. Mutations in the BB loop of the Toll-like receptors, such as the A795P mutation in TLR3 and the P712H mutation (Lps d mutation) in TLR4, have been reported to disrupt or alter downstream signaling. While the phenotypic effect of these mutations is known, the underlying effect of these mutations on the structure, dynamics and interactions with other TIR domain-containing proteins is not well understood. Here, we have attempted to investigate the effect of the BB loop mutations on the dimer form of TLRs, using TLR2 and TLR3 as case studies. Our results based on molecular dynamics simulations, protein-protein interaction analyses and protein structure network analyses highlight significant differences between the dimer interfaces of the wild-type and mutant forms and provide a logical reasoning for the effect of these mutations on adaptor binding to TLRs. Furthermore, it also leads us to propose a hypothesis for the differential requirement of signaling and bridging adaptors by TLRs. This could aid in further understanding of the mechanisms governing such signaling pathways. © 2018 Wiley Periodicals, Inc.

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

PDBsum new things.

PubMed

Laskowski, Roman A

2009-01-01

PDBsum (http://www.ebi.ac.uk/pdbsum) provides summary information about each experimentally determined structural model in the Protein Data Bank (PDB). Here we describe some of its most recent features, including figures from the structure's key reference, citation data, Pfam domain diagrams, topology diagrams and protein-protein interactions. Furthermore, it now accepts users' own PDB format files and generates a private set of analyses for each uploaded structure.

ESBRI: a web server for evaluating salt bridges in proteins.

PubMed

Costantini, Susan; Colonna, Giovanni; Facchiano, Angelo M

2008-01-01

Salt bridges can play important roles in protein structure and function and have stabilizing and destabilizing effects in protein folding. ESBRI is a software available as web tool which analyses the salt bridges in a protein structure, starting from the atomic coordinates. In the case of protein complexes, the salt bridges between protein chains can be evaluated, as well as those among specific charged amino acids and the different protein subunits, in order to obtain useful information regard the protein-protein interaction. The service is available at the URL: http://bioinformatica.isa.cnr.it/ESBRI/

Structural Basis for the Interaction of the Golgi-Associated Retrograde Protein (GARP) Complex with the t-SNARE Syntaxin 6

PubMed Central

Abascal-Palacios, Guillermo; Schindler, Christina; Rojas, Adriana L; Bonifacino, Juan S.; Hierro, Aitor

2016-01-01

Summary The Golgi-Associated Retrograde Protein (GARP) is a tethering complex involved in the fusion of endosome-derived transport vesicles to the trans-Golgi network through interaction with components of the Syntaxin 6/Syntaxin 16/Vti1a/VAMP4 SNARE complex. The mechanisms by which GARP and other tethering factors engage the SNARE fusion machinery are poorly understood. Herein we report the structural basis for the interaction of the human Ang2 subunit of GARP with Syntaxin 6 and the closely related Syntaxin 10. The crystal structure of Syntaxin 6 Habc domain in complex with a peptide from the N terminus of Ang2 shows a novel binding mode in which a di-tyrosine motif of Ang2 interacts with a highly conserved groove in Syntaxin 6. Structure-based mutational analyses validate the crystal structure and support the phylogenetic conservation of this interaction. The same binding determinants are found in other tethering proteins and syntaxins, suggesting a general interaction mechanism. PMID:23932592

Supramolecular Structures with Blood Plasma Proteins, Sugars and Nanosilica

NASA Astrophysics Data System (ADS)

Turov, V. V.; Gun'ko, V. M.; Galagan, N. P.; Rugal, A. A.; Barvinchenko, V. M.; Gorbyk, P. P.

Supramolecular structures with blood plasma proteins (albumin, immunoglobulin and fibrinogen (HPF)), protein/water/silica and protein/water/ silica/sugar (glucose, fructose and saccharose) were studied by NMR, adsorption, IR and UV spectroscopy methods. Hydration parameters, amounts of weakly and strongly bound waters and interfacial energy (γ S) were determined over a wide range of component concentrations. The γ S(C protein,C silica) graphs were used to estimate the energy of protein-protein, protein-surface and particle-particle interactions. It was shown that interfacial energy of self-association (γ as) of protein molecules depends on a type of proteins. A large fraction of water bound to proteins can be displaced by sugars, and the effect of disaccharide (saccharose) was greater than that of monosugars. Changes in the structural parameters of cavities in HPF molecules and complexes with HPF/silica nanoparticles filled by bound water were analysed using NMR-cryoporometry showing that interaction of proteins with silica leads to a significant decrease in the amounts of water bound to both protein and silica surfaces. Bionanocomposites with BSA/nanosilica/sugar can be used to influence states of living cells and tissues after cryopreservation or other treatments. It was shown that interaction of proteins with silica leads to strong decrease in the volume of all types of internal cavities filled by water.

Mapping the miRNA interactome by crosslinking ligation and sequencing of hybrids (CLASH)

PubMed Central

Helwak, Aleksandra; Tollervey, David

2014-01-01

RNA-RNA interactions play critical roles in many cellular processes but studying them is difficult and laborious. Here, we describe an experimental procedure, termed crosslinking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein is UV crosslinked in vivo to stabilise RNA interactions and purified under denaturing conditions. RNAs associated with the bait protein are partially truncated, and the ends of RNA-duplexes are ligated together. Following linker addition, cDNA library preparation and high-throughput sequencing, the ligated duplexes give rise to chimeric cDNAs, which unambiguously identify RNA-RNA interaction sites independent of bioinformatic predictions. This protocol is optimized for studying miRNA targets bound by Argonaute proteins, but should be easily adapted for other RNA-binding proteins and classes of RNA. The protocol requires around 5 days to complete, excluding the time required for high-throughput sequencing and bioinformatic analyses. PMID:24577361

The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

PubMed Central

García-López, M. Carmen; Pelechano, Vicent; Mirón-García, M. Carmen; Garrido-Godino, Ana I.; García, Alicia; Calvo, Olga; Werner, Michel; Pérez-Ortín, José E.; Navarro, Francisco

2011-01-01

RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. PMID:21954159

Hox Proteins Display a Common and Ancestral Ability to Diversify Their Interaction Mode with the PBC Class Cofactors

PubMed Central

Hudry, Bruno; Remacle, Sophie; Delfini, Marie-Claire; Rezsohazy, René; Graba, Yacine; Merabet, Samir

2012-01-01

Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes. PMID:22745600

Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

PubMed Central

Pandit, Shatakshi; Zhou, Yu; Shiue, Lily; Coutinho-Mansfield, Gabriela; Li, Hairi; Qiu, Jinsong; Huang, Jie; Yeo, Gene W.; Ares, Manuel; Fu, Xiang-Dong

2013-01-01

Summary SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins SRSF1 (SF2/ASF) and SRSF2 (SC35) using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes. PMID:23562324

CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

PubMed

Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

2017-09-01

Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

Hydrophobic environment is a key factor for the stability of thermophilic proteins.

PubMed

Gromiha, M Michael; Pathak, Manish C; Saraboji, Kadhirvel; Ortlund, Eric A; Gaucher, Eric A

2013-04-01

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins. Copyright © 2013 Wiley Periodicals, Inc.

Protein discovery: combined transcriptomic and proteomic analyses of venom from the endoparasitoid Cotesia chilonis (Hymenoptera: Brachonidae)

USDA-ARS?s Scientific Manuscript database

Background: Many species of endoparasitoid wasps provide biological control services in agroecosystems. Although there is a great deal of information on the ecology and physiology of host/parasitoid interactions, relatively little is known on the protein composition of venom and how specific venom p...

Analysis of functional redundancies within the Arabidopsis TCP transcription factor family.

PubMed

Danisman, Selahattin; van Dijk, Aalt D J; Bimbo, Andrea; van der Wal, Froukje; Hennig, Lars; de Folter, Stefan; Angenent, Gerco C; Immink, Richard G H

2013-12-01

Analyses of the functions of TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 (TCP) transcription factors have been hampered by functional redundancy between its individual members. In general, putative functionally redundant genes are predicted based on sequence similarity and confirmed by genetic analysis. In the TCP family, however, identification is impeded by relatively low overall sequence similarity. In a search for functionally redundant TCP pairs that control Arabidopsis leaf development, this work performed an integrative bioinformatics analysis, combining protein sequence similarities, gene expression data, and results of pair-wise protein-protein interaction studies for the 24 members of the Arabidopsis TCP transcription factor family. For this, the work completed any lacking gene expression and protein-protein interaction data experimentally and then performed a comprehensive prediction of potential functional redundant TCP pairs. Subsequently, redundant functions could be confirmed for selected predicted TCP pairs by genetic and molecular analyses. It is demonstrated that the previously uncharacterized class I TCP19 gene plays a role in the control of leaf senescence in a redundant fashion with TCP20. Altogether, this work shows the power of combining classical genetic and molecular approaches with bioinformatics predictions to unravel functional redundancies in the TCP transcription factor family.

A theoretical study on predicted protein targets of apple polyphenols and possible mechanisms of chemoprevention in colorectal cancer

PubMed Central

Scafuri, Bernardina; Marabotti, Anna; Carbone, Virginia; Minasi, Paola; Dotolo, Serena; Facchiano, Angelo

2016-01-01

We investigated the potential role of apple phenolic compounds in human pathologies by integrating chemical characterization of phenolic compounds in three apple varieties, computational approaches to identify potential protein targets of the compounds, bioinformatics analyses on data from public archive of gene expression data, and functional analyses to hypothesize the effects of the selected compounds in molecular pathways. Starting by the analytic characterization of phenolic compounds in three apple varieties, i.e. Annurca, Red Delicious, and Golden Delicious, we used computational approaches to verify by reverse docking the potential protein targets of the identified compounds. Direct docking validation of the potential protein-ligand interactions has generated a short list of human proteins potentially bound by the apple phenolic compounds. By considering the known chemo-preventive role of apple antioxidants’ extracts against some human pathologies, we performed a functional analysis by comparison with experimental gene expression data and interaction networks, obtained from public repositories. The results suggest the hypothesis that chemo-preventive effects of apple extracts in human pathologies, in particular for colorectal cancer, may be the interference with the activity of nucleotide metabolism and methylation enzymes, similarly to some classes of anticancer drugs. PMID:27587238

Functional and Genomic Features of Human Genes Mutated in Neuropsychiatric Disorders.

PubMed

Forero, Diego A; Prada, Carlos F; Perry, George

2016-01-01

In recent years, a large number of studies around the world have led to the identification of causal genes for hereditary types of common and rare neurological and psychiatric disorders. To explore the functional and genomic features of known human genes mutated in neuropsychiatric disorders. A systematic search was used to develop a comprehensive catalog of genes mutated in neuropsychiatric disorders (NPD). Functional enrichment and protein-protein interaction analyses were carried out. A false discovery rate approach was used for correction for multiple testing. We found several functional categories that are enriched among NPD genes, such as gene ontologies, protein domains, tissue expression, signaling pathways and regulation by brain-expressed miRNAs and transcription factors. Sixty six of those NPD genes are known to be druggable. Several topographic parameters of protein-protein interaction networks and the degree of conservation between orthologous genes were identified as significant among NPD genes. These results represent one of the first analyses of enrichment of functional categories of genes known to harbor mutations for NPD. These findings could be useful for a future creation of computational tools for prioritization of novel candidate genes for NPD.

Functional and Genomic Features of Human Genes Mutated in Neuropsychiatric Disorders

PubMed Central

Forero, Diego A.; Prada, Carlos F.; Perry, George

2016-01-01

Background: In recent years, a large number of studies around the world have led to the identification of causal genes for hereditary types of common and rare neurological and psychiatric disorders. Objective: To explore the functional and genomic features of known human genes mutated in neuropsychiatric disorders. Methods: A systematic search was used to develop a comprehensive catalog of genes mutated in neuropsychiatric disorders (NPD). Functional enrichment and protein-protein interaction analyses were carried out. A false discovery rate approach was used for correction for multiple testing. Results: We found several functional categories that are enriched among NPD genes, such as gene ontologies, protein domains, tissue expression, signaling pathways and regulation by brain-expressed miRNAs and transcription factors. Sixty six of those NPD genes are known to be druggable. Several topographic parameters of protein-protein interaction networks and the degree of conservation between orthologous genes were identified as significant among NPD genes. Conclusion: These results represent one of the first analyses of enrichment of functional categories of genes known to harbor mutations for NPD. These findings could be useful for a future creation of computational tools for prioritization of novel candidate genes for NPD. PMID:27990183

Calcium/calmodulin-dependent serine protein kinase (CASK), a protein implicated in mental retardation and autism-spectrum disorders, interacts with T-Brain-1 (TBR1) to control extinction of associative memory in male mice.

PubMed

Huang, Tzyy-Nan; Hsueh, Yi-Ping

2017-01-01

Human genetic studies have indicated that mutations in calcium/calmodulin-dependent serine protein kinase ( CASK ) result in X-linked mental retardation and autism-spectrum disorders. We aimed to establish a mouse model to study how Cask regulates mental ability. Because Cask encodes a multidomain scaffold protein, a possible strategy to dissect how CASK regulates mental ability and cognition is to disrupt specific protein-protein interactions of CASK in vivo and then investigate the impact of individual specific protein interactions. Previous in vitro analyses indicated that a rat CASK T724A mutation reduces the interaction between CASK and T-brain-1 (TBR1) in transfected COS cells. Because TBR1 is critical for glutamate receptor, ionotropic, N -methyl-D-aspartate receptor subunit 2B ( Grin2b ) expression and is a causative gene for autism and intellectual disability, we then generated CASK T740A (corresponding to rat CASK T724A) mutant mice using a gene-targeting approach. Immunoblotting, coimmunoprecipitation, histological methods and behavioural assays (including home cage, open field, auditory and contextual fear conditioning and conditioned taste aversion) were applied to investigate expression of CASK and its related proteins, the protein-protein interactions of CASK, and anatomic and behavioural features of CASK T740A mice. The CASK T740A mutation attenuated the interaction between CASK and TBR1 in the brain. However, CASK T740A mice were generally healthy, without obvious defects in brain morphology. The most dramatic defect among the mutant mice was in extinction of associative memory, though acquisition was normal. The functions of other CASK protein interactions cannot be addressed using CASK T740A mice. Disruption of the CASK and TBR1 interaction impairs extinction, suggesting the involvement of CASK in cognitive flexibility.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

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

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

DOE PAGES

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

Buried chloride stereochemistry in the Protein Data Bank

PubMed Central

2014-01-01

Background Despite the chloride anion is involved in fundamental biological processes, its interactions with proteins are little known. In particular, we lack a systematic survey of its coordination spheres. Results The analysis of a non-redundant set (pairwise sequence identity?

Buried chloride stereochemistry in the Protein Data Bank.

PubMed

Carugo, Oliviero

2014-09-23

Despite the chloride anion is involved in fundamental biological processes, its interactions with proteins are little known. In particular, we lack a systematic survey of its coordination spheres. The analysis of a non-redundant set (pairwise sequence identity < 30%) of 1739 high resolution (<2 Å) crystal structures that contain at least one chloride anion shows that the first coordination spheres of the chlorides are essentially constituted by hydrogen bond donors. Amongst the side-chains positively charged, arginine interacts with chlorides much more frequently than lysine. Although the most common coordination number is 4, the coordination stereochemistry is closer to the expected geometry when the coordination number is 5, suggesting that this is the coordination number towards which the chlorides tend when they interact with proteins. The results of these analyses are useful in interpreting, describing, and validating new protein crystal structures that contain chloride anions.

Structural Bioinformatics of the Interactome

PubMed Central

Petrey, Donald; Honig, Barry

2014-01-01

The last decade has seen a dramatic expansion in the number and range of techniques available to obtain genome-wide information, and to analyze this information so as to infer both the function of individual molecules and how they interact to modulate the behavior of biological systems. Here we review these techniques, focusing on the construction of physical protein-protein interaction networks, and highlighting approaches that incorporate protein structure which is becoming an increasingly important component of systems-level computational techniques. We also discuss how network analyses are being applied to enhance the basic understanding of biological systems and their disregulation, and how they are being applied in drug development. PMID:24895853

The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity

PubMed Central

Stein, Matthias; Pilli, Manohar; Bernauer, Sabine; Habermann, Bianca H.; Zerial, Marino; Wade, Rebecca C.

2012-01-01

Background Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood. Methodology/Principal Findings Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics. Conclusions/Significance We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity. PMID:22523562

The RING 2.0 web server for high quality residue interaction networks.

PubMed

Piovesan, Damiano; Minervini, Giovanni; Tosatto, Silvio C E

2016-07-08

Residue interaction networks (RINs) are an alternative way of representing protein structures where nodes are residues and arcs physico-chemical interactions. RINs have been extensively and successfully used for analysing mutation effects, protein folding, domain-domain communication and catalytic activity. Here we present RING 2.0, a new version of the RING software for the identification of covalent and non-covalent bonds in protein structures, including π-π stacking and π-cation interactions. RING 2.0 is extremely fast and generates both intra and inter-chain interactions including solvent and ligand atoms. The generated networks are very accurate and reliable thanks to a complex empirical re-parameterization of distance thresholds performed on the entire Protein Data Bank. By default, RING output is generated with optimal parameters but the web server provides an exhaustive interface to customize the calculation. The network can be visualized directly in the browser or in Cytoscape. Alternatively, the RING-Viz script for Pymol allows visualizing the interactions at atomic level in the structure. The web server and RING-Viz, together with an extensive help and tutorial, are available from URL: http://protein.bio.unipd.it/ring. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Systematic analyses of the ultraviolet radiation resistance-associated gene product (UVRAG) protein interactome by tandem affinity purification.

PubMed

Son, Ji-Hye; Hwang, Eurim C; Kim, Joungmok

2016-03-01

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions.

Comparative analysis of protein-protein interactions in the defense response of rice and wheat.

PubMed

Cantu, Dario; Yang, Baoju; Ruan, Randy; Li, Kun; Menzo, Virginia; Fu, Daolin; Chern, Mawsheng; Ronald, Pamela C; Dubcovsky, Jorge

2013-03-12

Despite the importance of wheat as a major staple crop and the negative impact of diseases on its production worldwide, the genetic mechanisms and gene interactions involved in the resistance response in wheat are still poorly understood. The complete sequence of the rice genome has provided an extremely useful parallel road map for genetic and genomics studies in wheat. The recent construction of a defense response interactome in rice has the potential to further enhance the translation of advances in rice to wheat and other grasses. The objective of this study was to determine the degree of conservation in the protein-protein interactions in the rice and wheat defense response interactomes. As entry points we selected proteins that serve as key regulators of the rice defense response: the RAR1/SGT1/HSP90 protein complex, NPR1, XA21, and XB12 (XA21 interacting protein 12). Using available wheat sequence databases and phylogenetic analyses we identified and cloned the wheat orthologs of these four rice proteins, including recently duplicated paralogs, and their known direct interactors and tested 86 binary protein interactions using yeast-two-hybrid (Y2H) assays. All interactions between wheat proteins were further tested using in planta bimolecular fluorescence complementation (BiFC). Eighty three percent of the known rice interactions were confirmed when wheat proteins were tested with rice interactors and 76% were confirmed using wheat protein pairs. All interactions in the RAR1/SGT1/ HSP90, NPR1 and XB12 nodes were confirmed for the identified orthologous wheat proteins, whereas only forty four percent of the interactions were confirmed in the interactome node centered on XA21. We hypothesize that this reduction may be associated with a different sub-functionalization history of the multiple duplications that occurred in this gene family after the divergence of the wheat and rice lineages. The observed high conservation of interactions between proteins that serve as key regulators of the rice defense response suggests that the existing rice interactome can be used to predict interactions in wheat. Such predictions are less reliable for nodes that have undergone a different history of duplications and sub-functionalization in the two lineages.

Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO3)2 stress.

PubMed

Du, Jing; Guo, Shirong; Sun, Jin; Shu, Sheng

2018-05-01

The mechanism of exogenous Spd-induced Ca(NO 3 ) 2 stress tolerance in cucumber was studied by proteomics and physiological analyses. Protein-protein interaction network revealed 13 key proteins involved in Spd-induced Ca(NO 3 ) 2 stress resistance. Ca(NO 3 ) 2 stress is one of the major reasons for secondary salinization that limits cucumber plant development in greenhouse. The conferred protective role of exogenous Spd on cucumber in response to Ca(NO 3 ) 2 stress cues involves changes at the cellular and physiological levels. To investigate the molecular foundation of exogenous Spd in Ca(NO 3 ) 2 stress tolerance, a proteomic approach was performed in our work. After a 9 days period of Ca(NO 3 ) 2 stress and/or exogenous Spd, 71 differential protein spots were confidently identified. The resulting proteins were enriched in seven different categories of biological processes, including protein metabolism, carbohydrate and energy metabolism, ROS homeostasis and stress defense, cell wall related, transcription, others and unknown. Protein metabolism (31.2%), carbohydrate and energy metabolism (15.6%), ROS homeostasis and stress defense (32.5%) were the three largest functional categories in cucumber root and most of them were significantly increased by exogenous Spd. The Spd-responsive protein interaction network revealed 13 key proteins, whose accumulation changes could be critical for Spd-induced resistance; all 13 proteins were upregulated by Spd at transcriptional and protein levels in response to Ca(NO 3 ) 2 stress. Furthermore, accumulation of antioxidant enzymes, non-enzymatic antioxidant and polyamines, along with reduction of H 2 O 2 and MDA, were detected after exogenous Spd application during Ca(NO 3 ) 2 stress. The results of these proteomic and physiological analyses in cucumber root may facilitate a better understanding of the underlying mechanism of Ca(NO 3 ) 2 stress tolerance mediated by exogenous Spd.

Translating Current Bioanalytical Techniques for Studying Corona Activity.

PubMed

Wang, Chunming; Wang, Zhenzhen; Dong, Lei

2018-07-01

The recent discovery of the biological corona is revolutionising our understanding of the in vivo behaviour of nanomaterials. Accurate analysis of corona bioactivity is essential for predicting the fate of nanomaterials and thereby improving nanomedicine design. Nevertheless, current biotechniques for protein analysis are not readily adaptable for analysing corona proteins, given that their conformation, activity, and interaction may largely differ from those of the native proteins. Here, we introduce and propose tailor-made modifications to five types of mainstream bioanalytical methodologies. We specifically illustrate how these modifications can translate existing techniques for protein analysis into competent tools for dissecting the composition, bioactivity, and interaction (with both nanomaterials and the tissue) of corona formed on specific nanomaterial surfaces. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

PubMed

Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

2017-10-01

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Direct interaction of the Usher syndrome 1G protein SANS and myomegalin in the retina.

PubMed

Overlack, Nora; Kilic, Dilek; Bauss, Katharina; Märker, Tina; Kremer, Hannie; van Wijk, Erwin; Wolfrum, Uwe

2011-10-01

The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically heterogeneous with at least 11 chromosomal loci assigned to 3 clinical types, USH1-3. We have previously demonstrated that all USH1 and 2 proteins in the eye and the inner ear are organized into protein networks by scaffold proteins. This has contributed essentially to our current understanding of the function of USH proteins and explains why defects in proteins of different families cause very similar phenotypes. We have previously shown that the USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) contributes to the periciliary protein network in retinal photoreceptor cells. This study aimed to further elucidate the role of SANS by identifying novel interaction partners. In yeast two-hybrid screens of retinal cDNA libraries we identified 30 novel putative interacting proteins binding to the central domain of SANS (CENT). We confirmed the direct binding of the phosphodiesterase 4D interacting protein (PDE4DIP), a Golgi associated protein synonymously named myomegalin, to the CENT domain of SANS by independent assays. Correlative immunohistochemical and electron microscopic analyses showed a co-localization of SANS and myomegalin in mammalian photoreceptor cells in close association with microtubules. Based on the present results we propose a role of the SANS-myomegalin complex in microtubule-dependent inner segment cargo transport towards the ciliary base of photoreceptor cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of reference genome selection on the performance of computational methods for genome-wide protein-protein interaction prediction.

PubMed

Muley, Vijaykumar Yogesh; Ranjan, Akash

2012-01-01

Recent progress in computational methods for predicting physical and functional protein-protein interactions has provided new insights into the complexity of biological processes. Most of these methods assume that functionally interacting proteins are likely to have a shared evolutionary history. This history can be traced out for the protein pairs of a query genome by correlating different evolutionary aspects of their homologs in multiple genomes known as the reference genomes. These methods include phylogenetic profiling, gene neighborhood and co-occurrence of the orthologous protein coding genes in the same cluster or operon. These are collectively known as genomic context methods. On the other hand a method called mirrortree is based on the similarity of phylogenetic trees between two interacting proteins. Comprehensive performance analyses of these methods have been frequently reported in literature. However, very few studies provide insight into the effect of reference genome selection on detection of meaningful protein interactions. We analyzed the performance of four methods and their variants to understand the effect of reference genome selection on prediction efficacy. We used six sets of reference genomes, sampled in accordance with phylogenetic diversity and relationship between organisms from 565 bacteria. We used Escherichia coli as a model organism and the gold standard datasets of interacting proteins reported in DIP, EcoCyc and KEGG databases to compare the performance of the prediction methods. Higher performance for predicting protein-protein interactions was achievable even with 100-150 bacterial genomes out of 565 genomes. Inclusion of archaeal genomes in the reference genome set improves performance. We find that in order to obtain a good performance, it is better to sample few genomes of related genera of prokaryotes from the large number of available genomes. Moreover, such a sampling allows for selecting 50-100 genomes for comparable accuracy of predictions when computational resources are limited.

The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

PubMed Central

Galetto, Luciana; Bosco, Domenico; Balestrini, Raffaella; Genre, Andrea; Fletcher, Jacqueline; Marzachì, Cristina

2011-01-01

Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity. PMID:21799902

Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues

DOE PAGES

Nikolaienko, Roman M.; Hammel, Michal; Dubreuil, Véronique; ...

2016-08-18

Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3-6 (CNTN3-6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptormore » cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling.« less

Structural basis for spectrin recognition by ankyrin.

PubMed

Ipsaro, Jonathan J; Mondragón, Alfonso

2010-05-20

Maintenance of membrane integrity and organization in the metazoan cell is accomplished through intracellular tethering of membrane proteins to an extensive, flexible protein network. Spectrin, the principal component of this network, is anchored to membrane proteins through the adaptor protein ankyrin. To elucidate the atomic basis for this interaction, we determined a crystal structure of human betaI-spectrin repeats 13 to 15 in complex with the ZU5-ANK domain of human ankyrin R. The structure reveals the role of repeats 14 to 15 in binding, the electrostatic and hydrophobic contributions along the interface, and the necessity for a particular orientation of the spectrin repeats. Using structural and biochemical data as a guide, we characterized the individual proteins and their interactions by binding and thermal stability analyses. In addition to validating the structural model, these data provide insight into the nature of some mutations associated with cell morphology defects, including those found in human diseases such as hereditary spherocytosis and elliptocytosis. Finally, analysis of the ZU5 domain suggests it is a versatile protein-protein interaction module with distinct interaction surfaces. The structure represents not only the first of a spectrin fragment in complex with its binding partner, but also that of an intermolecular complex involving a ZU5 domain.

Low-temperature protein dynamics: a simulation analysis of interprotein vibrations and the boson peak at 150 k.

PubMed

Kurkal-Siebert, Vandana; Smith, Jeremy C

2006-02-22

An understanding of low-frequency, collective protein dynamics at low temperatures can furnish valuable information on functional protein energy landscapes, on the origins of the protein glass transition and on protein-protein interactions. Here, molecular dynamics (MD) simulations and normal-mode analyses are performed on various models of crystalline myoglobin in order to characterize intra- and interprotein vibrations at 150 K. Principal component analysis of the MD trajectories indicates that the Boson peak, a broad peak in the dynamic structure factor centered at about approximately 2-2.5 meV, originates from approximately 10(2) collective, harmonic vibrations. An accurate description of the environment is found to be essential in reproducing the experimental Boson peak form and position. At lower energies other strong peaks are found in the calculated dynamic structure factor. Characterization of these peaks shows that they arise from harmonic vibrations of proteins relative to each other. These vibrations are likely to furnish valuable information on the physical nature of protein-protein interactions.

BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis.

PubMed

Peng, Yuancheng; Chen, Liangliang; Li, Shengjun; Zhang, Yueying; Xu, Ran; Liu, Zupei; Liu, Wuxia; Kong, Jingjing; Huang, Xiahe; Wang, Yingchun; Cheng, Beijiu; Zheng, Leiying; Li, Yunhai

2018-04-18

Sugars function as signal molecules to regulate growth, development, and gene expression in plants, yeasts, and animals. A coordination of sugar availability with phytohormone signals is crucial for plant growth and development. The molecular link between sugar availability and hormone-dependent plant growth are largely unknown. Here we report that BRI1 and BAK1 are involved in sugar-responsive growth and development. Glucose influences the physical interactions and phosphorylations of BRI1 and BAK1 in a concentration-dependent manner. BRI1 and BAK1 physically interact with G proteins that are essential for mediating sugar signaling. Biochemical data show that BRI1 can phosphorylate G protein β subunit and γ subunits, and BAK1 can phosphorylate G protein γ subunits. Genetic analyses suggest that BRI1 and BAK1 function in a common pathway with G-protein subunits to regulate sugar responses. Thus, our findings reveal an important genetic and molecular mechanism by which BR receptors associate with G proteins to regulate sugar-responsive growth and development.

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

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

PubMed

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

2017-08-01

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

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.

Live-Cell Imaging of Filoviruses.

PubMed

Schudt, Gordian; Dolnik, Olga; Becker, Stephan

2017-01-01

Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.

Optimization of non-denaturing protein extraction conditions for plant PPR proteins.

PubMed

Andrés-Colás, Nuria; Van Der Straeten, Dominique

2017-01-01

Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non-denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pI, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pI/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.

An improved size exclusion-HPLC method for molecular size distribution analysis of immunoglobulin G using sodium perchlorate in the eluent.

PubMed

Wang, Hsiaoling; Levi, Mark S; Del Grosso, Alfred V; McCormick, William M; Bhattacharyya, Lokesh

2017-05-10

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular size distribution (MSD) analyses of various therapeutic proteins. We report development and validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a TSKgel SuperSW3000 column and eluting it with 0.4M NaClO 4 , a chaotropic salt, in 40mM phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run time (12min), better peak resolution, improved peak shape and more stable base-line compared to HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A comparison of MSD analysis results between our method and the EP method shows interactions between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the stationary phase, when the latter method is used. Thus, the EP method shows lower percent of aggregates and tetramer than are actually present in the products. In view of the fact that aggregates have been attributed to playing a critical role in adverse reactions due to IgG products, our observation raises a major concern regarding the actual aggregate content in these products since the EP method is widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the interactions between the proteins and stationary phase as well as the adsorption of proteins onto the column. Our results also show that NaClO 4 in the eluent is more effective in overcoming the protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO 4 is shown not to affect the molecular size and relative distribution of different molecular forms of IgG. The method validated as per ICH Q2(R1) guideline using IgG products, shows good specificity, accuracy, precision and a linear concentration dependence of peak areas for different molecular forms. In summary, our method gives more reliable results than the SE-HPLC methods for MSD analyses of IgG reported in the literature, including the EP, particularly for aggregates and tetramer. The results are interpreted in terms of ionic (polar) and hydrophobic interactions between the stationary phase and the IgG protein. Published by Elsevier B.V.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

PubMed

Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

2015-12-28

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

PubMed Central

Mir, Riyaz A.; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A.; Ammons, Shalis A.; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B.; Qiu, Fang; Band, Hamid

2015-01-01

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. PMID:26711270

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

Competition-cooperation relationship networks characterize the competition and cooperation between proteins

PubMed Central

Li, Hong; Zhou, Yuan; Zhang, Ziding

2015-01-01

By analyzing protein-protein interaction (PPI) networks, one can find that a protein may have multiple binding partners. However, it is difficult to determine whether the interactions with these partners occur simultaneously from binary PPIs alone. Here, we construct the yeast and human competition-cooperation relationship networks (CCRNs) based on protein structural interactomes to clearly exhibit the relationship (competition or cooperation) between two partners of the same protein. If two partners compete for the same interaction interface, they would be connected by a competitive edge; otherwise, they would be connected by a cooperative edge. The properties of three kinds of hubs (i.e., competitive, modest, and cooperative hubs) are analyzed in the CCRNs. Our results show that competitive hubs have higher clustering coefficients and form clusters in the human CCRN, but these tendencies are not observed in the yeast CCRN. We find that the human-specific proteins contribute significantly to these differences. Subsequently, we conduct a series of computational experiments to investigate the regulatory mechanisms that avoid competition between proteins. Our comprehensive analyses reveal that for most yeast and human protein competitors, transcriptional regulation plays an important role. Moreover, the human-specific proteins have a particular preference for other regulatory mechanisms, such as alternative splicing. PMID:26108281

Protein Discovery: Combined Transcriptomic and Proteomic Analyses of Venom from the Endoparasitoid Cotesia chilonis (Hymenoptera: Braconidae)

PubMed Central

Teng, Zi-Wen; Xiong, Shi-Jiao; Xu, Gang; Gan, Shi-Yu; Chen, Xuan; Stanley, David; Yan, Zhi-Chao; Ye, Gong-Yin; Fang, Qi

2017-01-01

Many species of endoparasitoid wasps provide biological control services in agroecosystems. Although there is a great deal of information on the ecology and physiology of host/parasitoid interactions, relatively little is known about the protein composition of venom and how specific venom proteins influence physiological systems within host insects. This is a crucial gap in our knowledge because venom proteins act in modulating host physiology in ways that favor parasitoid development. Here, we identified 37 possible venom proteins from the polydnavirus-carrying endoparasitoid Cotesia chilonis by combining transcriptomic and proteomic analyses. The most abundant proteins were hydrolases, such as proteases, peptidases, esterases, glycosyl hydrolase, and endonucleases. Some components are classical parasitoid venom proteins with known functions, including extracellular superoxide dismutase 3, serine protease inhibitor and calreticulin. The venom contains novel proteins, not recorded from any other parasitoid species, including tolloid-like proteins, chitooligosaccharidolytic β-N-acetylglucosaminidase, FK506-binding protein 14, corticotropin-releasing factor-binding protein and vascular endothelial growth factor receptor 2. These new data generate hypotheses and provide a platform for functional analysis of venom components. PMID:28417942

PSSMSearch: a server for modeling, visualization, proteome-wide discovery and annotation of protein motif specificity determinants.

PubMed

Krystkowiak, Izabella; Manguy, Jean; Davey, Norman E

2018-06-05

There is a pressing need for in silico tools that can aid in the identification of the complete repertoire of protein binding (SLiMs, MoRFs, miniMotifs) and modification (moiety attachment/removal, isomerization, cleavage) motifs. We have created PSSMSearch, an interactive web-based tool for rapid statistical modeling, visualization, discovery and annotation of protein motif specificity determinants to discover novel motifs in a proteome-wide manner. PSSMSearch analyses proteomes for regions with significant similarity to a motif specificity determinant model built from a set of aligned motif-containing peptides. Multiple scoring methods are available to build a position-specific scoring matrix (PSSM) describing the motif specificity determinant model. This model can then be modified by a user to add prior knowledge of specificity determinants through an interactive PSSM heatmap. PSSMSearch includes a statistical framework to calculate the significance of specificity determinant model matches against a proteome of interest. PSSMSearch also includes the SLiMSearch framework's annotation, motif functional analysis and filtering tools to highlight relevant discriminatory information. Additional tools to annotate statistically significant shared keywords and GO terms, or experimental evidence of interaction with a motif-recognizing protein have been added. Finally, PSSM-based conservation metrics have been created for taxonomic range analyses. The PSSMSearch web server is available at http://slim.ucd.ie/pssmsearch/.

Sliding of proteins non-specifically bound to DNA: Brownian dynamics studies with coarse-grained protein and DNA models.

PubMed

Ando, Tadashi; Skolnick, Jeffrey

2014-12-01

DNA binding proteins efficiently search for their cognitive sites on long genomic DNA by combining 3D diffusion and 1D diffusion (sliding) along the DNA. Recent experimental results and theoretical analyses revealed that the proteins show a rotation-coupled sliding along DNA helical pitch. Here, we performed Brownian dynamics simulations using newly developed coarse-grained protein and DNA models for evaluating how hydrodynamic interactions between the protein and DNA molecules, binding affinity of the protein to DNA, and DNA fluctuations affect the one dimensional diffusion of the protein on the DNA. Our results indicate that intermolecular hydrodynamic interactions reduce 1D diffusivity by 30%. On the other hand, structural fluctuations of DNA give rise to steric collisions between the CG-proteins and DNA, resulting in faster 1D sliding of the protein. Proteins with low binding affinities consistent with experimental estimates of non-specific DNA binding show hopping along the CG-DNA. This hopping significantly increases sliding speed. These simulation studies provide additional insights into the mechanism of how DNA binding proteins find their target sites on the genome.

In Silico Analyses of Substrate Interactions with Human Serum Paraoxonase 1

DTIC Science & Technology

2008-01-01

substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the...mod- eling; docking; molecular dynamics simulations ; binding free energy decomposition. 486 PROTEINS Published 2008 WILEY-LISS, INC. yThis article is a...apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The

Salts employed in hydrophobic interaction chromatography can change protein structure - insights from protein-ligand interaction thermodynamics, circular dichroism spectroscopy and small angle X-ray scattering.

PubMed

Komaromy, Andras Z; Kulsing, Chadin; Boysen, Reinhard I; Hearn, Milton T W

2015-03-01

Key requirements of protein purification by hydrophobic interaction chromatography (HIC) are preservation of the tertiary/quaternary structure, maintenance of biological function, and separation of the correctly folded protein from its unfolded forms or aggregates. This study examines the relationship between the HIC retention behavior of hen egg white lysozyme (HEWL) in high concentrations of several kosmotropic salts and its conformation, assessed by circular dichroism (CD) spectroscopy. Further, the physicochemical properties of HEWL in the presence of high concentrations of ammonium sulfate, sodium chloride and magnesium chloride were investigated by small angle X-ray scattering (SAXS) at different temperatures. Radii of gyration were extrapolated from Guinier approximations and the indirect transform program GNOM with protein-protein interaction and contrast variation taken into account. A bead model simulation provided information on protein structural changes using ab initio reconstruction with GASBOR. These results correlated to the secondary structure content obtained from CD spectroscopy of HEWL. These changes in SAXS and CD data were consistent with heat capacity ΔCp -values obtained from van't Hoff plot analyses of the retention data. Collectively, these insights enable informed decisions to be made on the choice of chromatographic conditions, leading to improved separation selectivity and opportunities for innovative column-assisted protein refolding methods. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modelling protein functional domains in signal transduction using Maude

NASA Technical Reports Server (NTRS)

Sriram, M. G.

2003-01-01

Modelling of protein-protein interactions in signal transduction is receiving increased attention in computational biology. This paper describes recent research in the application of Maude, a symbolic language founded on rewriting logic, to the modelling of functional domains within signalling proteins. Protein functional domains (PFDs) are a critical focus of modern signal transduction research. In general, Maude models can simulate biological signalling networks and produce specific testable hypotheses at various levels of abstraction. Developing symbolic models of signalling proteins containing functional domains is important because of the potential to generate analyses of complex signalling networks based on structure-function relationships.

Effects of multiple enzyme-substrate interactions in basic units of cellular signal processing

NASA Astrophysics Data System (ADS)

Seaton, D. D.; Krishnan, J.

2012-08-01

Covalent modification cycles are a ubiquitous feature of cellular signalling networks. In these systems, the interaction of an active enzyme with the unmodified form of its substrate is essential for signalling to occur. However, this interaction is not necessarily the only enzyme-substrate interaction possible. In this paper, we analyse the behaviour of a basic model of signalling in which additional, non-essential enzyme-substrate interactions are possible. These interactions include those between the inactive form of an enzyme and its substrate, and between the active form of an enzyme and its product. We find that these additional interactions can result in increased sensitivity and biphasic responses, respectively. The dynamics of the responses are also significantly altered by the presence of additional interactions. Finally, we evaluate the consequences of these interactions in two variations of our basic model, involving double modification of substrate and scaffold-mediated signalling, respectively. We conclude that the molecular details of protein-protein interactions are important in determining the signalling properties of enzymatic signalling pathways.

Gene expression patterns combined with network analysis identify hub genes associated with bladder cancer.

PubMed

Bi, Dongbin; Ning, Hao; Liu, Shuai; Que, Xinxiang; Ding, Kejia

2015-06-01

To explore molecular mechanisms of bladder cancer (BC), network strategy was used to find biomarkers for early detection and diagnosis. The differentially expressed genes (DEGs) between bladder carcinoma patients and normal subjects were screened using empirical Bayes method of the linear models for microarray data package. Co-expression networks were constructed by differentially co-expressed genes and links. Regulatory impact factors (RIF) metric was used to identify critical transcription factors (TFs). The protein-protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and clusters were obtained through molecular complex detection (MCODE) algorithm. Centralities analyses for complex networks were performed based on degree, stress and betweenness. Enrichment analyses were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Co-expression networks and TFs (based on expression data of global DEGs and DEGs in different stages and grades) were identified. Hub genes of complex networks, such as UBE2C, ACTA2, FABP4, CKS2, FN1 and TOP2A, were also obtained according to analysis of degree. In gene enrichment analyses of global DEGs, cell adhesion, proteinaceous extracellular matrix and extracellular matrix structural constituent were top three GO terms. ECM-receptor interaction, focal adhesion, and cell cycle were significant pathways. Our results provide some potential underlying biomarkers of BC. However, further validation is required and deep studies are needed to elucidate the pathogenesis of BC. Copyright © 2015 Elsevier Ltd. All rights reserved.

DIBS: a repository of disordered binding sites mediating interactions with ordered proteins.

PubMed

Schad, Eva; Fichó, Erzsébet; Pancsa, Rita; Simon, István; Dosztányi, Zsuzsanna; Mészáros, Bálint

2018-02-01

Intrinsically Disordered Proteins (IDPs) mediate crucial protein-protein interactions, most notably in signaling and regulation. As their importance is increasingly recognized, the detailed analyses of specific IDP interactions opened up new opportunities for therapeutic targeting. Yet, large scale information about IDP-mediated interactions in structural and functional details are lacking, hindering the understanding of the mechanisms underlying this distinct binding mode. Here, we present DIBS, the first comprehensive, curated collection of complexes between IDPs and ordered proteins. DIBS not only describes by far the highest number of cases, it also provides the dissociation constants of their interactions, as well as the description of potential post-translational modifications modulating the binding strength and linear motifs involved in the binding. Together with the wide range of structural and functional annotations, DIBS will provide the cornerstone for structural and functional studies of IDP complexes. DIBS is freely accessible at http://dibs.enzim.ttk.mta.hu/. The DIBS application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. dosztanyi@caesar.elte.hu or bmeszaros@caesar.elte.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

d-Omix: a mixer of generic protein domain analysis tools.

PubMed

Wichadakul, Duangdao; Numnark, Somrak; Ingsriswang, Supawadee

2009-07-01

Domain combination provides important clues to the roles of protein domains in protein function, interaction and evolution. We have developed a web server d-Omix (a Mixer of Protein Domain Analysis Tools) aiming as a unified platform to analyze, compare and visualize protein data sets in various aspects of protein domain combinations. With InterProScan files for protein sets of interest provided by users, the server incorporates four services for domain analyses. First, it constructs protein phylogenetic tree based on a distance matrix calculated from protein domain architectures (DAs), allowing the comparison with a sequence-based tree. Second, it calculates and visualizes the versatility, abundance and co-presence of protein domains via a domain graph. Third, it compares the similarity of proteins based on DA alignment. Fourth, it builds a putative protein network derived from domain-domain interactions from DOMINE. Users may select a variety of input data files and flexibly choose domain search tools (e.g. hmmpfam, superfamily) for a specific analysis. Results from the d-Omix could be interactively explored and exported into various formats such as SVG, JPG, BMP and CSV. Users with only protein sequences could prepare an InterProScan file using a service provided by the server as well. The d-Omix web server is freely available at http://www.biotec.or.th/isl/Domix.

Biokinetics of zinc oxide nanoparticles: toxicokinetics, biological fates, and protein interaction

PubMed Central

Choi, Soo-Jin; Choy, Jin-Ho

2014-01-01

Biokinetic studies of zinc oxide (ZnO) nanoparticles involve systematic and quantitative analyses of absorption, distribution, metabolism, and excretion in plasma and tissues of whole animals after exposure. A full understanding of the biokinetics provides basic information about nanoparticle entry into systemic circulation, target organs of accumulation and toxicity, and elimination time, which is important for predicting the long-term toxic potential of nanoparticles. Biokinetic behaviors can be dependent on physicochemical properties, dissolution property in biological fluids, and nanoparticle–protein interaction. Moreover, the determination of biological fates of ZnO nanoparticles in the systemic circulation and tissues is critical in interpreting biokinetic behaviors and predicting toxicity potential as well as mechanism. This review focuses on physicochemical factors affecting the biokinetics of ZnO nanoparticles, in concert with understanding bioavailable fates and their interaction with proteins. PMID:25565844

Interactions between soy protein from water-soluble soy extract and polysaccharides in solutions with polydextrose.

PubMed

Spada, Jordana C; Marczak, Ligia D F; Tessaro, Isabel C; Cardozo, Nilo S M

2015-12-10

This study focuses on the investigation of the interactions between polysaccharides (carrageenan and carboxymethylcellulose--CMC) and soy proteins from the water-soluble soy extract. The influence of pH (2-7) and protein-polysaccharide ratio (5:1-40:1) on the interaction between these polyelectrolytes was investigated in aqueous solutions with 10% of polydextrose and without polydextrose. The studied systems were analyzed in terms of pH-solubility profile of protein, ζ-potential, methylene blue-polysaccharide interactions, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and confocal laser scanning microscopy. Although the mixtures of soy extract with both carrageenan and CMC showed dependency on the pH and protein-polysaccharide ratio, they did not present the same behavior. Both polysaccharides modified the pH-solubility profile of the soy protein, shifting the pH range in which the coacervate is formed to a lower pH region with the decrease of the soy extract-polysaccharide ratio. The samples also presented detectable differences regarding to ζ-potential, DSC, FTIR and microscopy analyses. The complex formation was also detected even in a pH range where both biopolymers were net-negatively charged. The changes promoted by the presence of polydextrose were mainly detected by blue-polysaccharide interactions measures and confocal microscopy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Deriving Heterospecific Self-Assembling Protein-Protein Interactions Using a Computational Interactome Screen.

PubMed

Crooks, Richard O; Baxter, Daniel; Panek, Anna S; Lubben, Anneke T; Mason, Jody M

2016-01-29

Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein-protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel

PubMed Central

Iscla, Irene; Wray, Robin; Blount, Paul

2011-01-01

The bacterial mechanosensitive channel MscL is the best-studied mechanosensor, thus serving as a paradigm of how a protein senses and responds to mechanical force. Models for the transition of Escherichia coli MscL from closed to open states propose a tilting of the transmembrane domains in the plane of the membrane, suggesting dynamic protein-lipid interactions. Here, we used a rapid in vivo assay to assess the function of channels that were post-translationally modified at several different sites in a region just distal to the cytoplasmic end of the second transmembrane helix. We utilized multiple probes with various affinities for the membrane environment. The in vivo functional data, combined with site-directed mutagenesis, single-channel analyses, and tryptophan fluorescence measurements, confirmed that lipid interactions within this region are critical for MscL gating. The data suggest a model in which this region acts as an anchor for the transmembrane domain tilting during gating. Furthermore, the conservation of analogous motifs among many other channels suggests a conserved protein-lipid dynamic mechanism.—Iscla, I., Wray, R., Blount, P. An in vivo screen reveals protein-lipid interactions crucial for gating a mechanosensitive channel. PMID:21068398

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

Characterization of protein-protein interaction domains within the baculovirus Autographa californica multiple nucleopolyhedrovirus late expression factor LEF-3.

PubMed

Downie, Kelsey; Adetola, Gbolagade; Carstens, Eric B

2013-11-01

Autographa californica nucleopolyhedrovirus late expression factor 3 (LEF-3) is required for late viral gene expression probably through its numerous functions related to DNA replication, including nuclear localization of the virus helicase P143 and binding to ssDNA. LEF-3 appears to interact with itself as a homo-oligomer, although the details of this oligomeric structure are not yet known. To examine LEF-3-LEF-3 interactions, a bimolecular fluorescent protein complementation assay was used. Pairs of recombinant plasmids expressing full-length LEF-3 fused to one of two complementary fragments (V1 or V2) of a variant of yellow fluorescent protein named 'Venus' were constructed. Plasmids expressing fusions with complementary fragments of Venus were co-transfected into Sf21 cells and analysed by fluorescence microscopy. Co-transfected plasmids expressing full-length V1-LEF-3 and V2-LEF-3 showed positive fluorescence, confirming the formation of homo-oligomers. A series of truncated V1/V2-LEF-3 fusions was constructed and used to investigate interactions with one another as well as with full-length LEF-3.

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

2012-01-01

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

Bioinformatics and variability in drug response: a protein structural perspective

PubMed Central

Lahti, Jennifer L.; Tang, Grace W.; Capriotti, Emidio; Liu, Tianyun; Altman, Russ B.

2012-01-01

Marketed drugs frequently perform worse in clinical practice than in the clinical trials on which their approval is based. Many therapeutic compounds are ineffective for a large subpopulation of patients to whom they are prescribed; worse, a significant fraction of patients experience adverse effects more severe than anticipated. The unacceptable risk–benefit profile for many drugs mandates a paradigm shift towards personalized medicine. However, prior to adoption of patient-specific approaches, it is useful to understand the molecular details underlying variable drug response among diverse patient populations. Over the past decade, progress in structural genomics led to an explosion of available three-dimensional structures of drug target proteins while efforts in pharmacogenetics offered insights into polymorphisms correlated with differential therapeutic outcomes. Together these advances provide the opportunity to examine how altered protein structures arising from genetic differences affect protein–drug interactions and, ultimately, drug response. In this review, we first summarize structural characteristics of protein targets and common mechanisms of drug interactions. Next, we describe the impact of coding mutations on protein structures and drug response. Finally, we highlight tools for analysing protein structures and protein–drug interactions and discuss their application for understanding altered drug responses associated with protein structural variants. PMID:22552919

Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L.).

PubMed

Sun, Tao; Wang, Yan; Wang, Meng; Li, Tingting; Zhou, Yi; Wang, Xiatian; Wei, Shuya; He, Guangyuan; Yang, Guangxiao

2015-11-04

Calcineurin B-like (CBL) proteins belong to a unique group of calcium sensors in plant that decode the Ca(2+) signature by interacting with CBL-interacting protein kinases (CIPKs). Although CBL-CIPK complexes have been shown to play important roles in the responses to various stresses in plants, little is known about their functions in wheat. A total of seven TaCBL and 20 TaCIPK genes were amplified from bread wheat, Triticum aestivum cv. Chinese Spring. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and in silico expression analyses showed that TaCBL and TaCIPK genes were expressed at different levels in different tissues, or maintained at nearly constant expression levels during the whole life cycle of the wheat plant. Some TaCBL and TaCIPK genes showed up- or down-regulated expressions during seed germination. Preferential interactions between TaCBLs and TaCIPKs were observed in yeast two-hybrid and bimolecular fluorescence complementation experiments. Analyses of a deletion series of TaCIPK proteins with amino acid variations at the C-terminus provided new insights into the specificity of the interactions between TaCIPKs and TaCBLs, and indicated that the TaCBL-TaCIPK signaling pathway is very complex in wheat because of its hexaploid genome. The expressions of many TaCBLs and TaCIPKs were responsive to abiotic stresses (salt, cold, and simulated drought) and abscisic acid treatment. Transgenic Arabidopsis plants overexpressing TaCIPK24 exhibited improved salt tolerance through increased Na(+) efflux and an enhanced reactive oxygen species scavenging capacity. These results contribute to our understanding of the functions of CBL-CIPK complexes and provide the basis for selecting appropriate genes for in-depth functional studies of CBL-CIPK in wheat.

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

PubMed

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of URI nuclear interaction with RPB5 and components of the R2TP/prefoldin-like complex.

PubMed

Mita, Paolo; Savas, Jeffrey N; Ha, Susan; Djouder, Nabil; Yates, John R; Logan, Susan K

2013-01-01

Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function.

Analysis of URI Nuclear Interaction with RPB5 and Components of the R2TP/Prefoldin-Like Complex

PubMed Central

Mita, Paolo; Savas, Jeffrey N.; Ha, Susan; Djouder, Nabil; Yates, John R.; Logan, Susan K.

2013-01-01

Unconventional prefoldin RPB5 Interactor (URI) was identified as a transcriptional repressor that binds RNA polymerase II (pol II) through interaction with the RPB5/POLR2E subunit. Despite the fact that many other proteins involved in transcription regulation have been shown to interact with URI, its nuclear function still remains elusive. Previous mass spectrometry analyses reported that URI is part of a novel protein complex called R2TP/prefoldin-like complex responsible for the cytoplasmic assembly of RNA polymerase II. We performed a mass spectrometry (MS)-based proteomic analysis to identify nuclear proteins interacting with URI in prostate cells. We identified all the components of the R2TP/prefoldin-like complex as nuclear URI interactors and we showed that URI binds and regulates RPB5 protein stability and transcription. Moreover, we validated the interaction of URI to the P53 and DNA damage-Regulated Gene 1 (PDRG1) and show that PDRG1 protein is also stabilized by URI binding. We present data demonstrating that URI nuclear/cytoplasmic shuttling is affected by compounds that stall pol II on the DNA (α-amanitin and actinomycin-D) and by leptomycin B, an inhibitor of the CRM1 exportin that mediates the nuclear export of pol II subunits. These data suggest that URI, and probably the entire R2TP/prefoldin-like complex is exported from the nucleus through CRM1. Finally we identified putative URI sites of phosphorylation and acetylation and confirmed URI sites of post-transcriptional modification identified in previous large-scale analyses the importance of which is largely unknown. However URI post-transcriptional modification was shown to be essential for URI function and therefore characterization of novel sites of URI modification will be important to the understanding of URI function. PMID:23667685

Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

PubMed

Kim, Mirim; Kim, Min-Jung; Pandey, Shashank; Kim, Jungmook

2016-11-01

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions.

PubMed

Beld, Joris; Blatti, Jillian L; Behnke, Craig; Mendez, Michael; Burkart, Michael D

2014-08-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes.

Evolution of acyl-ACP-thioesterases and β-ketoacyl-ACP-synthases revealed by protein-protein interactions

PubMed Central

Beld, Joris; Blatti, Jillian L.; Behnke, Craig; Mendez, Michael; Burkart, Michael D.

2014-01-01

The fatty acid synthase (FAS) is a conserved primary metabolic enzyme complex capable of tolerating cross-species engineering of domains for the development of modified and overproduced fatty acids. In eukaryotes, acyl-acyl carrier protein thioesterases (TEs) off-load mature cargo from the acyl carrier protein (ACP), and plants have developed TEs for short/medium-chain fatty acids. We showed that engineering plant TEs into the green microalga Chlamydomonas reinhardtii does not result in the predicted shift in fatty acid profile. Since fatty acid biosynthesis relies on substrate recognition and protein-protein interactions between the ACP and its partner enzymes, we hypothesized that plant TEs and algal ACP do not functionally interact. Phylogenetic analysis revealed major evolutionary differences between FAS enzymes, including TEs and ketoacyl synthases (KSs), in which the former is present only in some species, whereas the latter is present in all, and has a common ancestor. In line with these results, TEs appeared to be selective towards their ACP partners whereas KSs showed promiscuous behavior across bacterial, plant and algal species. Based on phylogenetic analyses, in silico docking, in vitro mechanistic crosslinking and in vivo algal engineering, we propose that phylogeny can predict effective interactions between ACPs and partner enzymes. PMID:25110394

Mass Spectrometry Based Proteomic Analysis of Salivary Glands of Urban Malaria Vector Anopheles stephensi

PubMed Central

Vijay, Sonam

2014-01-01

Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes. PMID:25126571

Mass spectrometry based proteomic analysis of salivary glands of urban malaria vector Anopheles stephensi.

PubMed

Vijay, Sonam; Rawat, Manmeet; Sharma, Arun

2014-01-01

Salivary gland proteins of Anopheles mosquitoes offer attractive targets to understand interactions with sporozoites, blood feeding behavior, homeostasis, and immunological evaluation of malaria vectors and parasite interactions. To date limited studies have been carried out to elucidate salivary proteins of An. stephensi salivary glands. The aim of the present study was to provide detailed analytical attributives of functional salivary gland proteins of urban malaria vector An. stephensi. A proteomic approach combining one-dimensional electrophoresis (1DE), ion trap liquid chromatography mass spectrometry (LC/MS/MS), and computational bioinformatic analysis was adopted to provide the first direct insight into identification and functional characterization of known salivary proteins and novel salivary proteins of An. stephensi. Computational studies by online servers, namely, MASCOT and OMSSA algorithms, identified a total of 36 known salivary proteins and 123 novel proteins analysed by LC/MS/MS. This first report describes a baseline proteomic catalogue of 159 salivary proteins belonging to various categories of signal transduction, regulation of blood coagulation cascade, and various immune and energy pathways of An. stephensi sialotranscriptome by mass spectrometry. Our results may serve as basis to provide a putative functional role of proteins in concept of blood feeding, biting behavior, and other aspects of vector-parasite host interactions for parasite development in anopheline mosquitoes.

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed Central

2014-01-01

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis. PMID:25011628

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed

Giulietti, Matteo; Vivenzio, Viviana; Piva, Francesco; Principato, Giovanni; Bellantuono, Cesario; Nardi, Bernardo

2014-07-10

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis.

Proteomic and Genomic Analyses of the Rvb1 and Rvb2 Interaction Network upon Deletion of R2TP Complex Components*

PubMed Central

Lakshminarasimhan, Mahadevan; Boanca, Gina; Banks, Charles A. S.; Hattem, Gaye L.; Gabriel, Ana E.; Groppe, Brad D.; Smoyer, Christine; Malanowski, Kate E.; Peak, Allison; Florens, Laurence; Washburn, Michael P.

2016-01-01

The highly conserved yeast R2TP complex, consisting of Rvb1, Rvb2, Pih1, and Tah1, participates in diverse cellular processes ranging from assembly of protein complexes to apoptosis. Rvb1 and Rvb2 are closely related proteins belonging to the AAA+ superfamily and are essential for cell survival. Although Rvbs have been shown to be associated with various protein complexes including the Ino80 and Swr1chromatin remodeling complexes, we performed a systematic quantitative proteomic analysis of their associated proteins and identified two additional complexes that associate with Rvb1 and Rvb2: the chaperonin-containing T-complex and the 19S regulatory particle of the proteasome complex. We also analyzed Rvb1 and Rvb2 purified from yeast strains devoid of PIH1 and TAH1. These analyses revealed that both Rvb1 and Rvb2 still associated with Hsp90 and were highly enriched with RNA polymerase II complex components. Our analyses also revealed that both Rvb1 and Rvb2 were recruited to the Ino80 and Swr1 chromatin remodeling complexes even in the absence of Pih1 and Tah1 proteins. Using further biochemical analysis, we showed that Rvb1 and Rvb2 directly interacted with Hsp90 as well as with the RNA polymerase II complex. RNA-Seq analysis of the deletion strains compared with the wild-type strains revealed an up-regulation of ribosome biogenesis and ribonucleoprotein complex biogenesis genes, down-regulation of response to abiotic stimulus genes, and down-regulation of response to temperature stimulus genes. A Gene Ontology analysis of the 80 proteins whose protein associations were altered in the PIH1 or TAH1 deletion strains found ribonucleoprotein complex proteins to be the most enriched category. This suggests an important function of the R2TP complex in ribonucleoprotein complex biogenesis at both the proteomic and genomic levels. Finally, these results demonstrate that deletion network analyses can provide novel insights into cellular systems. PMID:26831523

Lessons on RNA Silencing Mechanisms in Plants from Eukaryotic Argonaute Structures[W

PubMed Central

Poulsen, Christian; Vaucheret, Hervé; Brodersen, Peter

2013-01-01

RNA silencing refers to a collection of gene regulatory mechanisms that use small RNAs for sequence specific repression. These mechanisms rely on ARGONAUTE (AGO) proteins that directly bind small RNAs and thereby constitute the central component of the RNA-induced silencing complex (RISC). AGO protein function has been probed extensively by mutational analyses, particularly in plants where large allelic series of several AGO proteins have been isolated. Structures of entire human and yeast AGO proteins have only very recently been obtained, and they allow more precise analyses of functional consequences of mutations obtained by forward genetics. To a large extent, these analyses support current models of regions of particular functional importance of AGO proteins. Interestingly, they also identify previously unrecognized parts of AGO proteins with profound structural and functional importance and provide the first hints at structural elements that have important functions specific to individual AGO family members. A particularly important outcome of the analysis concerns the evidence for existence of Gly-Trp (GW) repeat interactors of AGO proteins acting in the plant microRNA pathway. The parallel analysis of AGO structures and plant AGO mutations also suggests that such interactions with GW proteins may be a determinant of whether an endonucleolytically competent RISC is formed. PMID:23303917

Lessons on RNA silencing mechanisms in plants from eukaryotic argonaute structures.

PubMed

Poulsen, Christian; Vaucheret, Hervé; Brodersen, Peter

2013-01-01

RNA silencing refers to a collection of gene regulatory mechanisms that use small RNAs for sequence specific repression. These mechanisms rely on ARGONAUTE (AGO) proteins that directly bind small RNAs and thereby constitute the central component of the RNA-induced silencing complex (RISC). AGO protein function has been probed extensively by mutational analyses, particularly in plants where large allelic series of several AGO proteins have been isolated. Structures of entire human and yeast AGO proteins have only very recently been obtained, and they allow more precise analyses of functional consequences of mutations obtained by forward genetics. To a large extent, these analyses support current models of regions of particular functional importance of AGO proteins. Interestingly, they also identify previously unrecognized parts of AGO proteins with profound structural and functional importance and provide the first hints at structural elements that have important functions specific to individual AGO family members. A particularly important outcome of the analysis concerns the evidence for existence of Gly-Trp (GW) repeat interactors of AGO proteins acting in the plant microRNA pathway. The parallel analysis of AGO structures and plant AGO mutations also suggests that such interactions with GW proteins may be a determinant of whether an endonucleolytically competent RISC is formed.

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

PubMed Central

Akay, Alper; Craig, Ashley; Lehrbach, Nicolas; Larance, Mark; Pourkarimi, Ehsan; Wright, Jane E.; Lamond, Angus; Miska, Eric; Gartner, Anton

2013-01-01

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways. PMID:24258276

MPact: the MIPS protein interaction resource on yeast.

PubMed

Güldener, Ulrich; Münsterkötter, Martin; Oesterheld, Matthias; Pagel, Philipp; Ruepp, Andreas; Mewes, Hans-Werner; Stümpflen, Volker

2006-01-01

In recent years, the Munich Information Center for Protein Sequences (MIPS) yeast protein-protein interaction (PPI) dataset has been used in numerous analyses of protein networks and has been called a gold standard because of its quality and comprehensiveness [H. Yu, N. M. Luscombe, H. X. Lu, X. Zhu, Y. Xia, J. D. Han, N. Bertin, S. Chung, M. Vidal and M. Gerstein (2004) Genome Res., 14, 1107-1118]. MPact and the yeast protein localization catalog provide information related to the proximity of proteins in yeast. Beside the integration of high-throughput data, information about experimental evidence for PPIs in the literature was compiled by experts adding up to 4300 distinct PPIs connecting 1500 proteins in yeast. As the interaction data is a complementary part of CYGD, interactive mapping of data on other integrated data types such as the functional classification catalog [A. Ruepp, A. Zollner, D. Maier, K. Albermann, J. Hani, M. Mokrejs, I. Tetko, U. Güldener, G. Mannhaupt, M. Münsterkötter and H. W. Mewes (2004) Nucleic Acids Res., 32, 5539-5545] is possible. A survey of signaling proteins and comparison with pathway data from KEGG demonstrates that based on these manually annotated data only an extensive overview of the complexity of this functional network can be obtained in yeast. The implementation of a web-based PPI-analysis tool allows analysis and visualization of protein interaction networks and facilitates integration of our curated data with high-throughput datasets. The complete dataset as well as user-defined sub-networks can be retrieved easily in the standardized PSI-MI format. The resource can be accessed through http://mips.gsf.de/genre/proj/mpact.

Applying graph theory to protein structures: an atlas of coiled coils.

PubMed

Heal, Jack W; Bartlett, Gail J; Wood, Christopher W; Thomson, Andrew R; Woolfson, Derek N

2018-05-02

To understand protein structure, folding and function fully and to design proteins de novo reliably, we must learn from natural protein structures that have been characterised experimentally. The number of protein structures available is large and growing exponentially, which makes this task challenging. Indeed, computational resources are becoming increasingly important for classifying and analysing this resource. Here, we use tools from graph theory to define an atlas classification scheme for automatically categorising certain protein substructures. Focusing on the α-helical coiled coils, which are ubiquitous protein-structure and protein-protein interaction motifs, we present a suite of computational resources designed for analysing these assemblies. iSOCKET enables interactive analysis of side-chain packing within proteins to identify coiled coils automatically and with considerable user control. Applying a graph theory-based atlas classification scheme to structures identified by iSOCKET gives the Atlas of Coiled Coils, a fully automated, updated overview of extant coiled coils. The utility of this approach is illustrated with the first formal classification of an emerging subclass of coiled coils called α-helical barrels. Furthermore, in the Atlas, the known coiled-coil universe is presented alongside a partial enumeration of the 'dark matter' of coiled-coil structures; i.e., those coiled-coil architectures that are theoretically possible but have not been observed to date, and thus present defined targets for protein design. iSOCKET is available as part of the open-source GitHub repository associated with this work (https://github.com/woolfson-group/isocket). This repository also contains all the data generated when classifying the protein graphs. The Atlas of Coiled Coils is available at: http://coiledcoils.chm.bris.ac.uk/atlas/app.

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

PubMed

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Electrostatic Similarities between Protein and Small Molecule Ligands Facilitate the Design of Protein-Protein Interaction Inhibitors

PubMed Central

Zhang, Kam Y. J.

2013-01-01

One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs). We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research) SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs. PMID:24130741

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

Cross dimerization of amyloid-β and αsynuclein proteins in aqueous environment: a molecular dynamics simulations study.

PubMed

Jose, Jaya C; Chatterjee, Prathit; Sengupta, Neelanjana

2014-01-01

Self-assembly of the intrinsically unstructured proteins, amyloid beta (Aβ) and alpha synclein (αSyn), are associated with Alzheimer's Disease, and Parkinson's and Lewy Body Diseases, respectively. Importantly, pathological overlaps between these neurodegenerative diseases, and the possibilities of interactions between Aβ and αSyn in biological milieu emerge from several recent clinical reports and in vitro studies. Nevertheless, there are very few molecular level studies that have probed the nature of spontaneous interactions between these two sequentially dissimilar proteins and key characteristics of the resulting cross complexes. In this study, we have used atomistic molecular dynamics simulations to probe the possibility of cross dimerization between αSyn1-95 and Aβ1-42, and thereby gain insights into their plausible early assembly pathways in aqueous environment. Our analyses indicate a strong probability of association between the two sequences, with inter-protein attractive electrostatic interactions playing dominant roles. Principal component analysis revealed significant heterogeneity in the strength and nature of the associations in the key interaction modes. In most, the interactions of repeating Lys residues, mainly in the imperfect repeats 'KTKEGV' present in αSyn1-95 were found to be essential for cross interactions and formation of inter-protein salt bridges. Additionally, a hydrophobicity driven interaction mode devoid of salt bridges, where the non-amyloid component (NAC) region of αSyn1-95 came in contact with the hydrophobic core of Aβ1-42 was observed. The existence of such hetero complexes, and therefore hetero assembly pathways may lead to polymorphic aggregates with variations in pathological attributes. Our results provide a perspective on development of therapeutic strategies for preventing pathogenic interactions between these proteins.

Prediction of Ras-effector interactions using position energy matrices.

PubMed

Kiel, Christina; Serrano, Luis

2007-09-01

One of the more challenging problems in biology is to determine the cellular protein interaction network. Progress has been made to predict protein-protein interactions based on structural information, assuming that structural similar proteins interact in a similar way. In a previous publication, we have determined a genome-wide Ras-effector interaction network based on homology models, with a high accuracy of predicting binding and non-binding domains. However, for a prediction on a genome-wide scale, homology modelling is a time-consuming process. Therefore, we here successfully developed a faster method using position energy matrices, where based on different Ras-effector X-ray template structures, all amino acids in the effector binding domain are sequentially mutated to all other amino acid residues and the effect on binding energy is calculated. Those pre-calculated matrices can then be used to score for binding any Ras or effector sequences. Based on position energy matrices, the sequences of putative Ras-binding domains can be scanned quickly to calculate an energy sum value. By calibrating energy sum values using quantitative experimental binding data, thresholds can be defined and thus non-binding domains can be excluded quickly. Sequences which have energy sum values above this threshold are considered to be potential binding domains, and could be further analysed using homology modelling. This prediction method could be applied to other protein families sharing conserved interaction types, in order to determine in a fast way large scale cellular protein interaction networks. Thus, it could have an important impact on future in silico structural genomics approaches, in particular with regard to increasing structural proteomics efforts, aiming to determine all possible domain folds and interaction types. All matrices are deposited in the ADAN database (http://adan-embl.ibmc.umh.es/). Supplementary data are available at Bioinformatics online.

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: insight into proteins at intracellular and extracellular spaces.

PubMed

Kim, Jin Yeong; Wu, Jingni; Kwon, Soon Jae; Oh, Haram; Lee, So Eui; Kim, Sang Gon; Wang, Yiming; Agrawal, Ganesh Kumar; Rakwal, Randeep; Kang, Kyu Young; Ahn, Il-Pyung; Kim, Beom-Gi; Kim, Sun Tae

2014-10-01

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coevolution at protein complex interfaces can be detected by the complementarity trace with important impact for predictive docking

PubMed Central

Madaoui, Hocine; Guerois, Raphaël

2008-01-01

Protein surfaces are under significant selection pressure to maintain interactions with their partners throughout evolution. Capturing how selection pressure acts at the interfaces of protein–protein complexes is a fundamental issue with high interest for the structural prediction of macromolecular assemblies. We tackled this issue under the assumption that, throughout evolution, mutations should minimally disrupt the physicochemical compatibility between specific clusters of interacting residues. This constraint drove the development of the so-called Surface COmplementarity Trace in Complex History score (SCOTCH), which was found to discriminate with high efficiency the structure of biological complexes. SCOTCH performances were assessed not only with respect to other evolution-based approaches, such as conservation and coevolution analyses, but also with respect to statistically based scoring methods. Validated on a set of 129 complexes of known structure exhibiting both permanent and transient intermolecular interactions, SCOTCH appears as a robust strategy to guide the prediction of protein–protein complex structures. Of particular interest, it also provides a basic framework to efficiently track how protein surfaces could evolve while keeping their partners in contact. PMID:18511568

Towards a map of the Populus biomass protein-protein interaction network

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

Beers, Eric; Brunner, Amy; Helm, Richard

Biofuels can be produced from a variety of plant feedstocks. The value of a particular feedstock for biofuels production depends in part on the degree of difficulty associated with the extraction of fermentable sugars from the plant biomass. The wood of trees is potentially a rich source fermentable sugars. However, the sugars in wood exist in a tightly cross-linked matrix of cellulose, hemicellulose, and lignin, making them largely recalcitrant to release and fermentation for biofuels production. Before breeders and genetic engineers can effectively develop plants with reduced recalcitrance to fermentation, it is necessary to gain a better understanding of themore » fundamental biology of the mechanisms responsible for wood formation. Regulatory, structural, and enzymatic proteins are required for the complicated process of wood formation. To function properly, proteins must interact with other proteins. Yet, very few of the protein-protein interactions necessary for wood formation are known. The main objectives of this project were to 1) identify new protein-protein interactions relevant to wood formation, and 2) perform in-depth characterizations of selected protein-protein interactions. To identify relevant protein-protein interactions, we cloned a set of approximately 400 genes that were highly expressed in the wood-forming tissue (known as secondary xylem) of poplar (Populus trichocarpa). We tested whether the proteins encoded by these biomass genes interacted with each other in a binary matrix design using the yeast two-hybrid (Y2H) method for protein-protein interaction discovery. We also tested a subset of the 400 biomass proteins for interactions with all proteins present in wood-forming tissue of poplar in a biomass library screen design using Y2H. Together, these two Y2H screens yielded over 270 interactions involving over 75 biomass proteins. For the second main objective we selected several interacting pairs or groups of interacting proteins for in-depth characterizations. Characterizations involved both in vivo and in vitro independent methods to confirm protein-protein interactions and the evaluation of novel phenotypes resulting from creation of transgenic poplar and Arabidopsis plants engineered for increased or decreased expression of the selected genes. Transgenic poplar trees were studied in growth chamber, greenhouse, and two separate replicated field trials involving over 25 distinct wood-associated proteins. In-depth characterizations yielding positive results include the following. First, a NAC domain transcription factor (NAC154) that is a promoter of stress response and dormancy in trees was discovered. Increasing expression of NAC154 caused stunted growth and premature senescence, while decreasing expression led to both delayed bud and leaf expansion in spring and delayed leaf drop (i.e., prolonged leaf retention) in fall. Second, we discovered and characterized a new connection between a negative regulator of wood formation, the NAC domain transcription factor XND1, and an important regulator of cell division and cell differentiation, RBR. Third, we identified a new network of interacting wood-associated transcription factors belonging to the MYB and HD families. One of the HD family proteins, WOX13, was used to prepare transgenic poplar for high-level expression, resulting in significantly increased lateral branch growth. Finally, we modeled and performed in vitro analyses of the insect protein rubber resilin and we prepared transgenic Arabidopsis plants for expression of resilin to test the feasibility of using resilin to modify lignin cross-linking in wood and reduce recalcitrance and improve yield of fermentable sugars for biofuels production. Analysis of these and additional transgenics created with this support is continuing.« less

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

PubMed

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Structural Basis for Interactions Between Contactin Family Members and Protein-tyrosine Phosphatase Receptor Type G in Neural Tissues.

PubMed

Nikolaienko, Roman M; Hammel, Michal; Dubreuil, Véronique; Zalmai, Rana; Hall, David R; Mehzabeen, Nurjahan; Karuppan, Sebastian J; Harroch, Sheila; Stella, Salvatore L; Bouyain, Samuel

2016-10-07

Protein-tyrosine phosphatase receptor type G (RPTPγ/PTPRG) interacts in vitro with contactin-3-6 (CNTN3-6), a group of glycophosphatidylinositol-anchored cell adhesion molecules involved in the wiring of the nervous system. In addition to PTPRG, CNTNs associate with multiple transmembrane proteins and signal inside the cell via cis-binding partners to alleviate the absence of an intracellular region. Here, we use comprehensive biochemical and structural analyses to demonstrate that PTPRG·CNTN3-6 complexes share similar binding affinities and a conserved arrangement. Furthermore, as a first step to identifying PTPRG·CNTN complexes in vivo, we found that PTPRG and CNTN3 associate in the outer segments of mouse rod photoreceptor cells. In particular, PTPRG and CNTN3 form cis-complexes at the surface of photoreceptors yet interact in trans when expressed on the surfaces of apposing cells. Further structural analyses suggest that all CNTN ectodomains adopt a bent conformation and might lie parallel to the cell surface to accommodate these cis and trans binding modes. Taken together, these studies identify a PTPRG·CNTN complex in vivo and provide novel insights into PTPRG- and CNTN-mediated signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tangled web of interactions among proteins involved in iron–sulfur cluster assembly as unraveled by NMR, SAXS, chemical crosslinking, and functional studies☆

PubMed Central

Kim, Jin Hae; Bothe, Jameson R.; Alderson, T. Reid; Markley, John L.

2014-01-01

Proteins containing iron–sulfur (Fe–S) clusters arose early in evolution and are essential to life. Organisms have evolved machinery consisting of specialized proteins that operate together to assemble Fe–S clusters efficiently so as to minimize cellular exposure to their toxic constituents: iron and sulfide ions. To date, the best studied system is the iron sulfur cluster (isc) operon of Escherichia coli, and the eight ISC proteins it encodes. Our investigations over the past five years have identified two functional conformational states for the scaffold protein (IscU) and have shown that the other ISC proteins that interact with IscU prefer to bind one conformational state or the other. From analyses of the NMR spectroscopy-derived network of interactions of ISC proteins and small-angle X-ray scattering (SAXS), chemical crosslinking experiments, and functional assays, we have constructed working models for Fe–S cluster assembly and delivery. Future work is needed to validate and refine what has been learned about the E. coli system and to extend these findings to the homologous Fe–S cluster biosynthetic machinery of yeast and human mitochondria. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases. PMID:25450980

A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics simulations.

PubMed

Michetti, Davide; Brandsdal, Bjørn Olav; Bon, Davide; Isaksen, Geir Villy; Tiberti, Matteo; Papaleo, Elena

2017-01-01

The psychrophilic and mesophilic endonucleases A (EndA) from Aliivibrio salmonicida (VsEndA) and Vibrio cholera (VcEndA) have been studied experimentally in terms of the biophysical properties related to thermal adaptation. The analyses of their static X-ray structures was no sufficient to rationalize the determinants of their adaptive traits at the molecular level. Thus, we used Molecular Dynamics (MD) simulations to compare the two proteins and unveil their structural and dynamical differences. Our simulations did not show a substantial increase in flexibility in the cold-adapted variant on the nanosecond time scale. The only exception is a more rigid C-terminal region in VcEndA, which is ascribable to a cluster of electrostatic interactions and hydrogen bonds, as also supported by MD simulations of the VsEndA mutant variant where the cluster of interactions was introduced. Moreover, we identified three additional amino acidic substitutions through multiple sequence alignment and the analyses of MD-based protein structure networks. In particular, T120V occurs in the proximity of the catalytic residue H80 and alters the interaction with the residue Y43, which belongs to the second coordination sphere of the Mg2+ ion. This makes T120V an amenable candidate for future experimental mutagenesis.

A Protein Domain and Family Based Approach to Rare Variant Association Analysis.

PubMed

Richardson, Tom G; Shihab, Hashem A; Rivas, Manuel A; McCarthy, Mark I; Campbell, Colin; Timpson, Nicholas J; Gaunt, Tom R

2016-01-01

It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals.

Interaction specificity and coexpression of rice NPR1 homologs 1 and 3 (NH1 and NH3), TGA transcription factors and Negative Regulator of Resistance (NRR) proteins.

PubMed

Chern, Mawsheng; Bai, Wei; Ruan, Deling; Oh, Taeyun; Chen, Xuewei; Ronald, Pamela C

2014-06-11

The nonexpressor of pathogenesis-related genes 1, NPR1 (also known as NIM1 and SAI1), is a key regulator of SA-mediated systemic acquired resistance (SAR) in Arabidopsis. In rice, the NPR1 homolog 1 (NH1) interacts with TGA transcriptional regulators and the Negative Regulator of Resistance (NRR) protein to modulate the SAR response. Though five NPR1 homologs (NHs) have been identified in rice, only NH1 and NH3 enhance immunity when overexpressed. To understand why NH1 and NH3, but not NH2, NH4, or NH5, contribute to the rice immune response, we screened TGA transcription factors and NRR-like proteins for interactions specific to NH1 and NH3. We also examined their co-expression patterns using publicly available microarray data. We tested five NHs, four NRR homologs (RHs), and 13 rice TGA proteins for pair-wise protein interactions using yeast two-hybrid (Y2H) and split YFP assays. A survey of 331 inter-family interactions revealed a broad, complex protein interaction network. To investigate preferred interaction partners when all three families of proteins were present, we performed a bridged split YFP assay employing YFPN-fused TGA, YFPC-fused RH, and NH proteins without YFP fusions. We found 64 tertiary interactions mediated by NH family members among the 120 sets we examined. In the yeast two-hybrid assay, each NH protein was capable of interacting with most TGA and RH proteins. In the split YFP assay, NH1 was the most prevalent interactor of TGA and RH proteins, NH3 ranked the second, and NH4 ranked the third. Based on their interaction with TGA proteins, NH proteins can be divided into two subfamilies: NH1, NH2, and NH3 in one family and NH4 and NH5 in the other.In addition to evidence of overlap in interaction partners, co-expression analyses of microarray data suggest a correlation between NH1 and NH3 expression patterns, supporting their common role in rice immunity. However, NH3 is very tightly co-expressed with RH1 and RH2, while NH1 is strongly, inversely co-expressed with RH proteins, representing a difference between NH1 and NH3 expression patterns. Our genome-wide surveys reveal that each rice NH protein can partner with many rice TGA and RH proteins and that each NH protein prefers specific interaction partners. NH1 and NH3 are capable of interacting strongly with most rice TGA and RH proteins, whereas NH2, NH4, and NH5 have weaker, limited interaction with TGA and RH proteins in rice cells. We have identified rTGA2.1, rTGA2.2, rTGA2.3, rLG2, TGAL2 and TGAL4 proteins as the preferred partners of NH1 and NH3, but not NH2, NH4, or NH5. These TGA proteins may play an important role in NH1- and NH3-mediated immune responses. In contrast, NH4 and NH5 preferentially interact with TGAL5, TGAL7, TGAL8 and TGAL9, which are predicted to be involved in plant development.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

PubMed

Mitsui, Shinichi; Hidaka, Chiharu; Furihata, Mutsuo; Osako, Yoji; Yuri, Kazunari

2013-07-12

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology. Copyright © 2013 Elsevier Inc. All rights reserved.

Protein-Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy.

PubMed

Diniz, Ana; Dias, Jorge S; Jiménez-Barbero, Jesús; Marcelo, Filipa; Cabrita, Eurico J

2017-09-21

Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H 15 N-HSQC signals. The intensity of the Gal-3 1 H 15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H 15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Binding of carbonyl flavours to canola, pea and wheat proteins using GC/MS approach.

PubMed

Wang, Kun; Arntfield, Susan D

2014-08-15

Interactions of homologous aldehydes (hexanal, heptanal, and octanal) and ketones (2-hexanone, 2-heptanone, and 2-octanone) to salt and alkaline-extracted canola and pea proteins and commercial wheat gluten were studied using GC/MS. Long-chain aldehyde flavours exhibited higher binding affinity, regardless of protein type and isolation method. Salt-extracted canola protein isolates (CPIs) revealed the highest binding capacity to all aldehydes followed by wheat gluten and salt-extracted pea protein isolates (PPIs), while binding of ketone flavours decreased in the order: PPIs>wheat gluten>CPIs. Two aldolisation products, 2-butyl-2-octenal and 2-pentyl-2-nonenal, were detected from the interactions between CPIs with hexanal and heptanal, respectively. Protein thermal behaviour in the presence of these compounds was analysed by differential scanning calorimeter, where decreased ΔH inferred potential conformational changes due to partial denaturation of PPIs. Compared to ketones, aldehyde flavours possessed much higher "unfolding capacity" (lower ΔH), which accounted for their higher binding affinities. Copyright © 2014 Elsevier Ltd. All rights reserved.

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

PubMed

Nolting, Nicole; Pöggeler, Stefanie

2006-07-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

PubMed Central

König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

2008-01-01

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

PubMed

Lumba, Shelley; Cutler, Sean; McCourt, Peter

2010-01-01

Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

PubMed

Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

2015-02-01

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

HTS-Net: An integrated regulome-interactome approach for establishing network regulation models in high-throughput screenings

PubMed Central

Rioualen, Claire; Da Costa, Quentin; Chetrit, Bernard; Charafe-Jauffret, Emmanuelle; Ginestier, Christophe

2017-01-01

High-throughput RNAi screenings (HTS) allow quantifying the impact of the deletion of each gene in any particular function, from virus-host interactions to cell differentiation. However, there has been less development for functional analysis tools dedicated to RNAi analyses. HTS-Net, a network-based analysis program, was developed to identify gene regulatory modules impacted in high-throughput screenings, by integrating transcription factors-target genes interaction data (regulome) and protein-protein interaction networks (interactome) on top of screening z-scores. HTS-Net produces exhaustive HTML reports for results navigation and exploration. HTS-Net is a new pipeline for RNA interference screening analyses that proves better performance than simple gene rankings by z-scores, by re-prioritizing genes and replacing them in their biological context, as shown by the three studies that we reanalyzed. Formatted input data for the three studied datasets, source code and web site for testing the system are available from the companion web site at http://htsnet.marseille.inserm.fr/. We also compared our program with existing algorithms (CARD and hotnet2). PMID:28949986

Annotation of Alternatively Spliced Proteins and Transcripts with Protein-Folding Algorithms and Isoform-Level Functional Networks.

PubMed

Li, Hongdong; Zhang, Yang; Guan, Yuanfang; Menon, Rajasree; Omenn, Gilbert S

2017-01-01

Tens of thousands of splice isoforms of proteins have been catalogued as predicted sequences from transcripts in humans and other species. Relatively few have been characterized biochemically or structurally. With the extensive development of protein bioinformatics, the characterization and modeling of isoform features, isoform functions, and isoform-level networks have advanced notably. Here we present applications of the I-TASSER family of algorithms for folding and functional predictions and the IsoFunc, MIsoMine, and Hisonet data resources for isoform-level analyses of network and pathway-based functional predictions and protein-protein interactions. Hopefully, predictions and insights from protein bioinformatics will stimulate many experimental validation studies.

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions

PubMed Central

Arjunan, Selvam; Sastri, Narayan P.; Chandra, Nagasuma

2016-01-01

Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue–human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue–human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (http://proline.biochem.iisc.ernet.in/DenHunt/). PMID:27618709

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions.

PubMed

Karyala, Prashanthi; Metri, Rahul; Bathula, Christopher; Yelamanchi, Syam K; Sahoo, Lipika; Arjunan, Selvam; Sastri, Narayan P; Chandra, Nagasuma

2016-09-01

Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue-human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue-human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (http://proline.biochem.iisc.ernet.in/DenHunt/).

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Characterization of the zinc-induced Shank3 interactome of mouse synaptosome.

PubMed

Lee, Yeunkum; Ryu, Jae Ryun; Kang, Hyojin; Kim, Yoonhee; Kim, Shinhyun; Zhang, Yinhua; Jin, Chunmei; Cho, Hyo Min; Kim, Won-Ki; Sun, Woong; Han, Kihoon

2017-12-16

Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl 2 , and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl 2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

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

Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto

2009-12-18

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins aremore » known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.« less

How PEGylation enhances the stability and potency of insulin: a molecular dynamics simulation.

PubMed

Yang, Cheng; Lu, Diannan; Liu, Zheng

2011-04-05

While the effectiveness of PEGylation in enhancing the stability and potency of protein pharmaceuticals has been validated for years, the underlying mechanism remains poorly understood, particularly at the molecular level. A molecular dynamics simulation was developed using an annealing procedure that allowed an all-atom level examination of the interaction between PEG polymers of different chain lengths and a conjugated protein represented by insulin. It was shown that PEG became entangled around the protein surface through hydrophobic interaction and concurrently formed hydrogen bonds with the surrounding water molecules. In addition to enhancing its structural stability, as indicated by the root-mean-square difference (rmsd) and secondary structure analyses, conjugation increased the size of the protein drug while decreasing the solvent accessible surface area of the protein. All these thus led to prolonged circulation life despite kidney filtration, proteolysis, and immunogenic side effects, as experimentally demonstrated elsewhere. Moreover, the simulation results indicated that an optimal chain length exists that would maximize drug potency underpinned by the parameters mentioned above. The simulation provided molecular insight into the interaction between PEG and the conjugated protein at the all-atom level and offered a tool that would allow for the design of PEGylated protein pharmaceuticals for given applications.

Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

PubMed

Smith, Colin A; Kortemme, Tanja

2011-01-01

Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s) are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface), interactions between and within parts of the structure (e.g. domains) can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

The interactome of CCT complex - A computational analysis.

PubMed

Narayanan, Aswathy; Pullepu, Dileep; Kabir, M Anaul

2016-10-01

The eukaryotic chaperonin, CCT (Chaperonin Containing TCP1 or TriC-TCP-1 Ring Complex) has been subjected to physical and genetic analyses in S. cerevisiae which can be extrapolated to human CCT (hCCT), owing to its structural and functional similarities with yeast CCT (yCCT). Studies on hCCT and its interactome acquire an additional dimension, as it has been implicated in several disease conditions like neurodegeneration and cancer. We attempt to study its stress response role in general, which will be reflected in the aspects of human diseases and yeast physiology, through computational analysis of the interactome. Towards consolidating and analysing the interactome data, we prepared and compared the unique CCT-interacting protein lists for S. cerevisiae and H. sapiens, performed GO term classification and enrichment studies which provide information on the diversity in CCT interactome, in terms of protein classes in the data set. Enrichment with disease-associated proteins and pathways highlight the medical importance of CCT. Different analyses converge, suggesting the significance of WD-repeat proteins, protein kinases and cytoskeletal proteins in the interactome. The prevalence of proteasomal subunits and ribosomal proteins suggest a possible cross-talk between protein-synthesis, folding and degradation machinery. A network of chaperones and chaperonins that function in combination can also be envisaged from the CCT interactome-Hsp70 interactome analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural insight into GRIP1-PDZ6 in Alzheimer's disease: study from protein expression data to molecular dynamics simulations.

PubMed

Chatterjee, Paulami; Roy, Debjani

2017-08-01

Protein-protein interaction domain, PDZ, plays a critical role in efficient synaptic transmission in brain. Dysfunction of synaptic transmission is thought to be the underlying basis of many neuropsychiatric and neurodegenerative disorders including Alzheimer's disease (AD). In this study, Glutamate Receptor Interacting Protein1 (GRIP1) was identified as one of the most important differentially expressed, topologically significant proteins in the protein-protein interaction network. To date, very few studies have analyzed the detailed structural basis of PDZ-mediated protein interaction of GRIP1. In order to gain better understanding of structural and dynamic basis of these interactions, we employed molecular dynamics (MD) simulations of GRIP1-PDZ6 dimer bound with Liprin-alpha and GRIP1-PDZ6 dimer alone each with 100 ns simulations. The analyses of MD simulations of Liprin-alpha bound GRIP1-PDZ6 dimer show considerable conformational differences than that of peptide-free dimer in terms of SASA, hydrogen bonding patterns, and along principal component 1 (PC1). Our study also furnishes insight into the structural attunement of the PDZ6 domains of Liprin-alpha bound GRIP1 that is attributed by significant shift of the Liprin-alpha recognition helix in the simulated peptide-bound dimer compared to the crystal structure and simulated peptide-free dimer. It is evident that PDZ6 domains of peptide-bound dimer show differential movements along PC1 than that of peptide-free dimers. Thus, Liprin-alpha also serves an important role in conferring conformational changes along the dimeric interface of the peptide-bound dimer. Results reported here provide information that may lead to novel therapeutic approaches in AD.

The Endoplasmic Reticulum Exit of Glutamate Transporter Is Regulated by the Inducible Mammalian Yip6b/GTRAP3-18 Protein*Ⓢ

PubMed Central

Ruggiero, Alicia M.; Liu, Yiting; Vidensky, Svetlana; Maier, Susanne; Jung, Elizabeth; Farhan, Hesso; Robinson, Michael B.; Sitte, Harald H.; Rothstein, Jeffrey D.

2015-01-01

GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na+/K+ glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively. PMID:18167356

Deconstructing thermodynamic parameters of a coupled system from site-specific observables.

PubMed

Chowdhury, Sandipan; Chanda, Baron

2010-11-02

Cooperative interactions mediate information transfer between structural domains of a protein molecule and are major determinants of protein function and modulation. The prevalent theories to understand the thermodynamic origins of cooperativity have been developed to reproduce the complex behavior of a global thermodynamic observable such as ligand binding or enzyme activity. However, in most cases the measurement of a single global observable cannot uniquely define all the terms that fully describe the energetics of the system. Here we establish a theoretical groundwork for analyzing protein thermodynamics using site-specific information. Our treatment involves extracting a site-specific parameter (defined as χ value) associated with a structural unit. We demonstrate that, under limiting conditions, the χ value is related to the direct interaction terms associated with the structural unit under observation and its intrinsic activation energy. We also introduce a site-specific interaction energy term (χ(diff)) that is a function of the direct interaction energy of that site with every other site in the system. When combined with site-directed mutagenesis and other molecular level perturbations, analyses of χ values of site-specific observables may provide valuable insights into protein thermodynamics and structure.

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

PubMed

Baumann, P; Jackson, S P

1996-06-25

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

Discrete structural features among interface residue-level classes.

PubMed

Sowmya, Gopichandran; Ranganathan, Shoba

2015-01-01

Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs.

Discrete structural features among interface residue-level classes

PubMed Central

2015-01-01

Background Protein-protein interaction (PPI) is essential for molecular functions in biological cells. Investigation on protein interfaces of known complexes is an important step towards deciphering the driving forces of PPIs. Each PPI complex is specific, sensitive and selective to binding. Therefore, we have estimated the relative difference in percentage of polar residues between surface and the interface for each complex in a non-redundant heterodimer dataset of 278 complexes to understand the predominant forces driving binding. Results Our analysis showed ~60% of protein complexes with surface polarity greater than interface polarity (designated as class A). However, a considerable number of complexes (~40%) have interface polarity greater than surface polarity, (designated as class B), with a significantly different p-value of 1.66E-45 from class A. Comprehensive analyses of protein complexes show that interface features such as interface area, interface polarity abundance, solvation free energy gain upon interface formation, binding energy and the percentage of interface charged residue abundance distinguish among class A and class B complexes, while electrostatic visualization maps also help differentiate interface classes among complexes. Conclusions Class A complexes are classical with abundant non-polar interactions at the interface; however class B complexes have abundant polar interactions at the interface, similar to protein surface characteristics. Five physicochemical interface features analyzed from the protein heterodimer dataset are discriminatory among the interface residue-level classes. These novel observations find application in developing residue-level models for protein-protein binding prediction, protein-protein docking studies and interface inhibitor design as drugs. PMID:26679043

A Modular Approach To Study Protein Adsorption on Surface Modified Hydroxyapatite.

PubMed

Ozhukil Kollath, Vinayaraj; Van den Broeck, Freya; Fehér, Krisztina; Martins, José C; Luyten, Jan; Traina, Karl; Mullens, Steven; Cloots, Rudi

2015-07-13

Biocompatible inorganic nano- and microcarriers can be suitable candidates for protein delivery. This study demonstrates facile methods of functionalization by using nanoscale linker molecules to change the protein adsorption capacity of hydroxyapatite (HA) powder. The adsorption capacity of bovine serum albumin as a model protein has been studied with respect to the surface modifications. The selected linker molecules (lysine, arginine, and phosphoserine) can influence the adsorption capacity by changing the electrostatic nature of the HA surface. Qualitative and quantitative analyses of linker-molecule interactions with the HA surface have been performed by using NMR spectroscopy, zeta-potential measurements, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Additionally, correlations to theoretical isotherm models have been calculated with respect to Langmuir and Freundlich isotherms. Lysine and arginine increased the protein adsorption, whereas phosphoserine reduced the protein adsorption. The results show that the adsorption capacity can be controlled with different functionalization, depending on the protein-carrier selections under consideration. The scientific knowledge acquired from this study can be applied in various biotechnological applications that involve biomolecule-inorganic material interfaces. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

PubMed Central

Powell, Amy J; Peabody, David S

2001-01-01

Background The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets. Results We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half. Conclusions These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4. PMID:11504563

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

DOE PAGES

Kemege, Kyle E.; Hickey, John M.; Barta, Michael L.; ...

2014-11-10

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies in this paper expand on those observations through protein structure, mutagenesis andmore » cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. Finally, MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia.« less

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB

PubMed Central

Kemege, Kyle E.; Hickey, John M.; Barta, Michael L.; Wickstrum, Jason; Balwalli, Namita; Lovell, Scott; Battaile, Kevin P.; Hefty, P. Scott

2015-01-01

Summary Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis, and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient E. coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia. PMID:25382739

Chlamydia trachomatis protein CT009 is a structural and functional homolog to the key morphogenesis component RodZ and interacts with division septal plane localized MreB.

PubMed

Kemege, Kyle E; Hickey, John M; Barta, Michael L; Wickstrum, Jason; Balwalli, Namita; Lovell, Scott; Battaile, Kevin P; Hefty, P Scott

2015-02-01

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia. © 2014 John Wiley & Sons Ltd.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

PubMed

Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

Domain Interaction Studies of Herpes Simplex Virus 1 Tegument Protein UL16 Reveal Its Interaction with Mitochondria

PubMed Central

Chadha, Pooja; Sarfo, Akua; Zhang, Dan; Abraham, Thomas; Carmichael, Jillian

2016-01-01

ABSTRACT The UL16 tegument protein of herpes simplex virus 1 (HSV-1) is conserved among all herpesviruses and plays many roles during replication. This protein has an N-terminal domain (NTD) that has been shown to bind to several viral proteins, including UL11, VP22, and glycoprotein E, and these interactions are negatively regulated by a C-terminal domain (CTD). Thus, in pairwise transfections, UL16 binding is enabled only when the CTD is absent or altered. Based on these results, we hypothesized that direct interactions occur between the NTD and the CTD. Here we report that the separated and coexpressed functional domains of UL16 are mutually responsive to each other in transfected cells and form complexes that are stable enough to be captured in coimmunoprecipitation assays. Moreover, we found that the CTD can associate with itself. To our surprise, the CTD was also found to contain a novel and intrinsic ability to localize to specific spots on mitochondria in transfected cells. Subsequent analyses of HSV-infected cells by immunogold electron microscopy and live-cell confocal imaging revealed a population of UL16 that does not merely accumulate on mitochondria but in fact makes dynamic contacts with these organelles in a time-dependent manner. These findings suggest that the domain interactions of UL16 serve to regulate not just the interaction of this tegument protein with its viral binding partners but also its interactions with mitochondria. The purpose of this novel interaction remains to be determined. IMPORTANCE The HSV-1-encoded tegument protein UL16 is involved in multiple events of the virus replication cycle, ranging from virus assembly to cell-cell spread of the virus, and hence it can serve as an important drug target. Unfortunately, a lack of both structural and functional information limits our understanding of this protein. The discovery of domain interactions within UL16 and the novel ability of UL16 to interact with mitochondria in HSV-infected cells lays a foundational framework for future investigations aimed at deciphering the structure and function of not just UL16 of HSV-1 but also its homologs in other herpesviruses. PMID:27847362

Reconstituting protein interaction networks using parameter-dependent domain-domain interactions

PubMed Central

2013-01-01

Background We can describe protein-protein interactions (PPIs) as sets of distinct domain-domain interactions (DDIs) that mediate the physical interactions between proteins. Experimental data confirm that DDIs are more consistent than their corresponding PPIs, lending support to the notion that analyses of DDIs may improve our understanding of PPIs and lead to further insights into cellular function, disease, and evolution. However, currently available experimental DDI data cover only a small fraction of all existing PPIs and, in the absence of structural data, determining which particular DDI mediates any given PPI is a challenge. Results We present two contributions to the field of domain interaction analysis. First, we introduce a novel computational strategy to merge domain annotation data from multiple databases. We show that when we merged yeast domain annotations from six annotation databases we increased the average number of domains per protein from 1.05 to 2.44, bringing it closer to the estimated average value of 3. Second, we introduce a novel computational method, parameter-dependent DDI selection (PADDS), which, given a set of PPIs, extracts a small set of domain pairs that can reconstruct the original set of protein interactions, while attempting to minimize false positives. Based on a set of PPIs from multiple organisms, our method extracted 27% more experimentally detected DDIs than existing computational approaches. Conclusions We have provided a method to merge domain annotation data from multiple sources, ensuring large and consistent domain annotation for any given organism. Moreover, we provided a method to extract a small set of DDIs from the underlying set of PPIs and we showed that, in contrast to existing approaches, our method was not biased towards DDIs with low or high occurrence counts. Finally, we used these two methods to highlight the influence of the underlying annotation density on the characteristics of extracted DDIs. Although increased annotations greatly expanded the possible DDIs, the lack of knowledge of the true biological false positive interactions still prevents an unambiguous assignment of domain interactions responsible for all protein network interactions. Executable files and examples are given at: http://www.bhsai.org/downloads/padds/ PMID:23651452

Plant Cell Wall Dynamics in Compatible and Incompatible Potato Response to Infection Caused by Potato Virus Y (PVYNTN)

PubMed Central

Lockhart, Benham E. L.

2018-01-01

The cell wall provides the structure of the plant, and also acts as a barier against biotic stress. The vein necrosis strain of Potato virus Y (PVYNTN) induces necrotic disease symptoms that affect both plant growth and yield. Virus infection triggers a number of inducible basal defense responses, including defense proteins, especially those involved in cell wall metabolism. This study investigates the comparison of cell wall host dynamics induced in a compatible (potato cv. Irys) and incompatible (potato cv. Sárpo Mira with hypersensitive reaction gene Ny-Smira) PVYNTN–host–plant interaction. Ultrastructural analyses revealed numerous cell wall changes induced by virus infection. Furthermore, the localization of essential defensive wall-associated proteins in susceptible and resistant potato host to PVYNTN infection were investigated. The data revealed a higher level of detection of pathogenesis-related protein 2 (PR-2) in a compatible compared to an incompatible (HR) interaction. Immunofluorescence analyses indicated that hydroxyproline-rich glycoproteins (HRGP) (extensin) synthesis was induced, whereas that of cellulose synthase catalytic subunits (CesA4) decreased as a result of PVYNTN infection. The highest level of extensin localization was found in HR potato plants. Proteins involved in cell wall metabolism play a crucial role in the interaction because they affect the spread of the virus. Analysis of CesA4, PR-2 and HRGP deposition within the apoplast and symplast confirmed the active trafficking of these proteins as a step-in potato cell wall remodeling in response to PVYNTN infection. Therefore, cell wall reorganization may be regarded as an element of “signWALLing”—involving apoplast and symplast activation as a specific response to viruses. PMID:29543714

The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin.

PubMed

Linnemann, Anja; van der Ven, Peter F M; Vakeel, Padmanabhan; Albinus, Britta; Simonis, Dirk; Bendas, Gerd; Schenk, Jörg A; Micheel, Burkhard; Kley, Rudolf A; Fürst, Dieter O

2010-09-01

Here we introduce myopodin as a novel filamin C binding partner. Corroborative yeast two-hybrid and biochemical analyses indicate that the central part of myopodin that shows high homology to the closely related protein synaptopodin and that is common to all its currently known or predicted variants interacts with filamin C immunoglobulin-like domains 20-21. A detailed characterization of the previously described interaction between myopodin and alpha-actinin demonstrates for the first time that myopodin contains three independent alpha-actinin-binding sites. Newly developed myopodin-specific antibodies reveal expression at the earliest stages of in vitro differentiation of human skeletal muscle cells preceding the expression of sarcomeric alpha-actinin. Myopodin colocalizes with filamin and alpha-actinin during all stages of muscle development. By contrast, colocalization with its previously identified binding partner zyxin is restricted to early developmental stages. Genetic and cellular analyses of skeletal muscle provided direct evidence for an alternative transcriptional start site in exon three, corroborating the expression of a myopodin variant lacking the PDZ domain encoded by exons 1 and 2 in skeletal muscle. We conclude that myopodin is a multiadapter protein of the sarcomeric Z-disc that links nascent myofibrils to the sarcolemma via zyxin, and might play a role in early assembly and stabilization of the Z-disc. Mutations in FLNC, ACTN2 and several other genes encoding Z-disc-related proteins cause myopathy and cardiomyopathy. Its localization and its association with the myopathy-associated proteins filamin C and alpha-actinin make myopodin an interesting candidate for a muscle disease gene. Copyright 2010 Elsevier GmbH. All rights reserved.

First large scale chemical synthesis of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active form.

PubMed

de Rocquigny, H; Ficheux, D; Gabus, C; Fournié-Zaluski, M C; Darlix, J L; Roques, B P

1991-10-31

The nucleocapsid protein (NC) of the human immunodeficiency virus type 1 plays a crucial role in the formation of infectious viral particles and therefore should be a major target for the development of antiviral agents. This requires an investigation of NC protein structure and of its interactions with both primer tRNA(Lys,3) and genomic RNA. Nucleocapsid protein NCp7, which results from the maturation of NCp15, contains two zinc fingers flanked by sequences rich in basic and proline residues. Here we report the first synthesis of large quantities of NCp7 able to activate HIV-1 RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In addition UV spectroscopic analyses performed to characterize the Co2+ binding properties of each zinc finger suggest that the two fingers probably interact in NCp7.

Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

Investigation of serum biomarkers in primary gout patients using iTRAQ-based screening.

PubMed

Ying, Ying; Chen, Yong; Zhang, Shun; Huang, Haiyan; Zou, Rouxin; Li, Xiaoke; Chu, Zanbo; Huang, Xianqian; Peng, Yong; Gan, Minzhi; Geng, Baoqing; Zhu, Mengya; Ying, Yinyan; Huang, Zuoan

2018-03-21

Primary gout is a major disease that affects human health; however, its pathogenesis is not well known. The purpose of this study was to identify biomarkers to explore the underlying mechanisms of primary gout. We used the isobaric tags for relative and absolute quantitation (iTRAQ) technique combined with liquid chromatography-tandem mass spectrometry to screen differentially expressed proteins between gout patients and controls. We also identified proteins potentially involved in gout pathogenesis by analysing biological processes, cellular components, molecular functions, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and protein-protein interactions. We further verified some samples using enzyme-linked immunosorbent assay (ELISA). Statistical analyses were carried out using SPSS v. 20.0 and ROC (receiver operating characterstic) curve analyses were carried out using Medcalc software. Two-sided p-values <0.05 were deemed to be statistically significant for all analyses. We identified 95 differentially expressed proteins (50 up-regulated and 45 down-regulated), and selected nine proteins (α-enolase (ENOA), glyceraldehyde-3-phosphate dehydrogenase (G3P), complement component C9 (CO9), profilin-1 (PROF1), lipopolysaccharide-binding protein (LBP), tubulin beta-4A chain (TBB4A), phosphoglycerate kinase (PGK1), glucose-6-phosphate isomerase (G6PI), and transketolase (TKT)) for verification. This showed that the level of TBB4A was significantly higher in primary gout than in controls (p=0.023). iTRAQ technology was useful in the selection of differentially expressed proteins from proteomes, and provides a strong theoretical basis for the study of biomarkers and mechanisms in primary gout. In addition, TBB4A protein may be associated with primary gout.

Three-dimensional (3D) structure prediction and function analysis of the chitin-binding domain 3 protein HD73_3189 from Bacillus thuringiensis HD73.

PubMed

Zhan, Yiling; Guo, Shuyuan

2015-01-01

Bacillus thuringiensis (Bt) is capable of producing a chitin-binding protein believed to be functionally important to bacteria during the stationary phase of its growth cycle. In this paper, the chitin-binding domain 3 protein HD73_3189 from B. thuringiensis has been analyzed by computer technology. Primary and secondary structural analyses demonstrated that HD73_3189 is negatively charged and contains several α-helices, aperiodical coils and β-strands. Domain and motif analyses revealed that HD73_3189 contains a signal peptide, an N-terminal chitin binding 3 domains, two copies of a fibronectin-like domain 3 and a C-terminal carbohydrate binding domain classified as CBM_5_12. Moreover, analysis predicted the protein's associated localization site to be the cell wall. Ligand site prediction determined that amino acid residues GLU-312, TRP-334, ILE-341 and VAL-382 exposed on the surface of the target protein exhibit polar interactions with the substrate.

Protein Corona Analysis of Silver Nanoparticles Links to Their Cellular Effects.

PubMed

Juling, Sabine; Niedzwiecka, Alicia; Böhmert, Linda; Lichtenstein, Dajana; Selve, Sören; Braeuning, Albert; Thünemann, Andreas F; Krause, Eberhard; Lampen, Alfonso

2017-11-03

The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Elucidating Protein Involvement in the Stabilization of the Biogenic Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Ballottin, Daniela; Fulaz, Stephanie; Souza, Michele L.; Corio, Paola; Rodrigues, Alexandre G.; Souza, Ana O.; Gaspari, Priscyla M.; Gomes, Alexandre F.; Gozzo, Fábio; Tasic, Ljubica

2016-06-01

Silver nanoparticles (AgNPs) have been broadly used as antibacterial and antiviral agents. Further, interests for green AgNP synthesis have increased in recent years and several results for AgNP biological synthesis have been reported using bacteria, fungi and plant extracts. The understanding of the role and nature of fungal proteins, their interaction with AgNPs and the subsequent stabilization of nanosilver is yet to be deeply investigated. Therefore, in an attempt to better understand biogenic AgNP stabilization with the extracellular fungal proteins and to describe these supramolecular interactions between proteins and silver nanoparticles, AgNPs, produced extracellularly by Aspergillus tubingensis—isolated as an endophytic fungus from Rizophora mangle—were characterized in order to study their physical characteristics, identify the involved proteins, and shed light into the interactions among protein-NPs by several techniques. AgNPs of around 35 nm in diameter as measured by TEM and a positive zeta potential of +8.48 mV were obtained. These AgNPs exhibited a surface plasmon resonance (SPR) band at 440 nm, indicating the nanoparticles formation, and another band at 280 nm, attributed to the electronic excitations in tryptophan, tyrosine, and/or phenylalanine residues in fungal proteins. Fungal proteins were covalently bounded to the AgNPs, mainly through S-Ag bonds due to cysteine residues (HS-) and with few N-Ag bonds from H2N- groups, as verified by Raman spectroscopy. Observed supramolecular interactions also occur by electrostatic and other protein-protein interactions. Furthermore, proteins that remain free on AgNP surface may perform hydrogen bonds with other proteins or water increasing thus the capping layer around the AgNPs and consequently expanding the hydrodynamic diameter of the particles (~264 nm, measured by DLS). FTIR results enabled us to state that proteins adsorbed to the AgNPs did not suffer relevant secondary structure alteration upon their physical interaction with the AgNPs or when covalently bonded to them. Eight proteins in the AgNP dispersion were identified by mass spectrometry analyses. All these proteins are involved in metabolic pathways of the fungus and are important for carbon, phosphorous and nitrogen uptake, and for the fungal growth. Thereby, important proteins for fungi are also involved in the formation and stabilization of the biogenic AgNPs.

Structural and functional analyses of genes encoding VQ proteins in apple.

PubMed

Dong, Qinglong; Zhao, Shuang; Duan, Dingyue; Tian, Yi; Wang, Yanpeng; Mao, Ke; Zhou, Zongshan; Ma, Fengwang

2018-07-01

Recent studies with Arabidopsis and soybean have shown that a class of valine-glutamine (VQ) motif-containing proteins interacts with some WRKY transcription factors. However, little is known about the evolution, structures, and functions of those proteins in apple. Here, we examined their features and identified 49 apple VQ genes. Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species. Historically, the main characteristics of proteins in Groups I, V, VI, VII, IX, and X were thought to have been generated before the monocot-dicot split, whereas those in Groups II, III + IV, and VIII were generated after that split. In the structural analysis, apple MdVQ proteins appeared to bind only with Group I and IIc MdWRKY proteins. Meanwhile, MdVQ1, MdVQ10, MdVQ15, and MdVQ36 interacted with multiple MdVQ proteins to form heterodimers but MdVQ15 formed a homodimer. The functional analysis indicated that overexpression of some apple MdVQs in Arabidopsis and tobacco plants effected their vegetative and reproductive growth. These results provide important information about the characteristics of apple MdVQ genes and can serve as a solid foundation for further studies about the role of WRKY-VQ interactions in regulating apple developmental and defense mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome-Wide Analyses of Calcium Sensors Reveal Their Involvement in Drought Stress Response and Storage Roots Deterioration after Harvest in Cassava.

PubMed

Hu, Wei; Yan, Yan; Tie, Weiwei; Ding, Zehong; Wu, Chunlai; Ding, Xupo; Wang, Wenquan; Xia, Zhiqiang; Guo, Jianchun; Peng, Ming

2018-04-19

Calcium (Ca 2+ ) plays a crucial role in plant development and responses to environmental stimuli. Currently, calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), such as Ca 2+ sensors, are not well understood in cassava ( Manihot esculenta Crantz), an important tropical crop. In the present study, 8 CaMs, 48 CMLs, and 9 CBLs were genome-wide identified in cassava, which were divided into two, four, and four groups, respectively, based on evolutionary relationship, protein motif, and gene structure analyses. Transcriptomic analysis revealed the expression diversity of cassava CaMs-CMLs-CBLs in distinct tissues and in response to drought stress in different genotypes. Generally, cassava CaMs-CMLs-CBLs showed different expression profiles between cultivated varieties (Arg7 and SC124) and wild ancestor (W14) after drought treatment. In addition, numerous CaMs-CMLs-CBLs were significantly upregulated at 6 h, 12 h, and 48 h after harvest, suggesting their possible role during storage roots (SR) deterioration. Further interaction network and co-expression analyses suggested that a CBL-mediated interaction network was widely involved in SR deterioration. Taken together, this study provides new insights into CaMs-CMLs-CBLs-mediated drought adaption and SR deterioration at the transcription level in cassava, and identifies some candidates for the genetic improvement of cassava.

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Anbazhagan, V; Sankhala, Rajeshwer S; Singh, Bhanu Pratap; Swamy, Musti J

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

PubMed Central

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process. PMID:22022488

Modulation of follistatin and myostatin propeptide by anabolic steroids and gender.

PubMed

Mosler, S; Geisler, S; Hengevoss, J; Schiffer, T; Piechotta, M; Adler, M; Diel, P

2013-07-01

The purpose of this pilot study was to investigate the impact of training, anabolic steroids and endogenous hormones on myostatin-interacting proteins in order to identify manipulations of myostatin signalling. To identify whether analysis of the myostatin interacting proteins follistatin and myostatin propeptide is suitable to detect the abuse of anabolic steroids, their serum concentrations were monitored in untrained males, bodybuilders using anabolic steroids and natural bodybuilders. In addition, we analysed follistatin and myostatin propeptide serum proteins in females during menstrual cycle. Our results showed increased follistatin concentrations in response to anabolic steroids. Furthermore, variations of sex steroid levels during the menstrual cycle had no impact on the expression of follistatin and myostatin propetide. In addition, we identified gender differences in the basal expression of the investigated proteins. In general, follistatin and myostatin propeptide concentrations were relatively stable within the same individual both in males and females. In conclusion, the current findings provide an insight into gender differences in myostatin-interacting proteins and their regulation in response to anabolic steroids and endogenous hormones. Therefore our data provide new aspects for the development of doping prevention strategies. © Georg Thieme Verlag KG Stuttgart · New York.

IFPTarget: A Customized Virtual Target Identification Method Based on Protein-Ligand Interaction Fingerprinting Analyses.

PubMed

Li, Guo-Bo; Yu, Zhu-Jun; Liu, Sha; Huang, Lu-Yi; Yang, Ling-Ling; Lohans, Christopher T; Yang, Sheng-Yong

2017-07-24

Small-molecule target identification is an important and challenging task for chemical biology and drug discovery. Structure-based virtual target identification has been widely used, which infers and prioritizes potential protein targets for the molecule of interest (MOI) principally via a scoring function. However, current "universal" scoring functions may not always accurately identify targets to which the MOI binds from the retrieved target database, in part due to a lack of consideration of the important binding features for an individual target. Here, we present IFPTarget, a customized virtual target identification method, which uses an interaction fingerprinting (IFP) method for target-specific interaction analyses and a comprehensive index (Cvalue) for target ranking. Evaluation results indicate that the IFP method enables substantially improved binding pose prediction, and Cvalue has an excellent performance in target ranking for the test set. When applied to screen against our established target library that contains 11,863 protein structures covering 2842 unique targets, IFPTarget could retrieve known targets within the top-ranked list and identified new potential targets for chemically diverse drugs. IFPTarget prediction led to the identification of the metallo-β-lactamase VIM-2 as a target for quercetin as validated by enzymatic inhibition assays. This study provides a new in silico target identification tool and will aid future efforts to develop new target-customized methods for target identification.

Predicting helix–helix interactions from residue contacts in membrane proteins

PubMed Central

Lo, Allan; Chiu, Yi-Yuan; Rødland, Einar Andreas; Lyu, Ping-Chiang; Sung, Ting-Yi; Hsu, Wen-Lian

2009-01-01

Motivation: Helix–helix interactions play a critical role in the structure assembly, stability and function of membrane proteins. On the molecular level, the interactions are mediated by one or more residue contacts. Although previous studies focused on helix-packing patterns and sequence motifs, few of them developed methods specifically for contact prediction. Results: We present a new hierarchical framework for contact prediction, with an application in membrane proteins. The hierarchical scheme consists of two levels: in the first level, contact residues are predicted from the sequence and their pairing relationships are further predicted in the second level. Statistical analyses on contact propensities are combined with other sequence and structural information for training the support vector machine classifiers. Evaluated on 52 protein chains using leave-one-out cross validation (LOOCV) and an independent test set of 14 protein chains, the two-level approach consistently improves the conventional direct approach in prediction accuracy, with 80% reduction of input for prediction. Furthermore, the predicted contacts are then used to infer interactions between pairs of helices. When at least three predicted contacts are required for an inferred interaction, the accuracy, sensitivity and specificity are 56%, 40% and 89%, respectively. Our results demonstrate that a hierarchical framework can be applied to eliminate false positives (FP) while reducing computational complexity in predicting contacts. Together with the estimated contact propensities, this method can be used to gain insights into helix-packing in membrane proteins. Availability: http://bio-cluster.iis.sinica.edu.tw/TMhit/ Contact: tsung@iis.sinica.edu.tw Supplementary information:Supplementary data are available at Bioinformatics online. PMID:19244388

Network biology discovers pathogen contact points in host protein-protein interactomes.

PubMed

Ahmed, Hadia; Howton, T C; Sun, Yali; Weinberger, Natascha; Belkhadir, Youssef; Mukhtar, M Shahid

2018-06-13

In all organisms, major biological processes are controlled by complex protein-protein interactions networks (interactomes), yet their structural complexity presents major analytical challenges. Here, we integrate a compendium of over 4300 phenotypes with Arabidopsis interactome (AI-1 MAIN ). We show that nodes with high connectivity and betweenness are enriched and depleted in conditional and essential phenotypes, respectively. Such nodes are located in the innermost layers of AI-1 MAIN and are preferential targets of pathogen effectors. We extend these network-centric analyses to Cell Surface Interactome (CSI LRR ) and predict its 35 most influential nodes. To determine their biological relevance, we show that these proteins physically interact with pathogen effectors and modulate plant immunity. Overall, our findings contrast with centrality-lethality rule, discover fast information spreading nodes, and highlight the structural properties of pathogen targets in two different interactomes. Finally, this theoretical framework could possibly be applicable to other inter-species interactomes to reveal pathogen contact points.

Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

PubMed

Kim, Sung Bae; Nishihara, Ryo; Citterio, Daniel; Suzuki, Koji

2016-02-17

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

Lead discovery and in silico 3D structure modeling of tumorigenic FAM72A (p17).

PubMed

Pramanik, Subrata; Kutzner, Arne; Heese, Klaus

2015-01-01

FAM72A (p17) is a novel neuronal protein that has been linked to tumorigenic effects in non-neuronal tissue. Using state of the art in silico physicochemical analyses (e.g., I-TASSER, RaptorX, and Modeller), we determined the three-dimensional (3D) protein structure of FAM72A and further identified potential ligand-protein interactions. Our data indicate a Zn(2+)/Fe(3+)-containing 3D protein structure, based on a 3GA3_A model template, which potentially interacts with the organic molecule RSM ((2s)-2-(acetylamino)-N-methyl-4-[(R)-methylsulfinyl] butanamide). The discovery of RSM may serve as potential lead for further anti-FAM72A drug screening tests in the pharmaceutical industry because interference with FAM72A's activities via RSM-related molecules might be a novel option to influence the tumor suppressor protein p53 signaling pathways for the treatment of various types of cancers.

Identification of Staphylococcal Nuclease Domain-containing 1 (SND1) as a Metadherin-interacting Protein with Metastasis-promoting Functions*

PubMed Central

Blanco, Mario Andres; Alečković, Maša; Hua, Yuling; Li, Tuo; Wei, Yong; Xu, Zhen; Cristea, Ileana M.; Kang, Yibin

2011-01-01

Metastasis is the deadliest and most poorly understood feature of malignant diseases. Recent work has shown that Metadherin (MTDH) is overexpressed in over 40% of breast cancer patients and promotes metastasis and chemoresistance in experimental models of breast cancer progression. Here we applied mass spectrometry-based screen to identify staphylococcal nuclease domain-containing 1 (SND1) as a candidate MTDH-interacting protein. After confirming the interaction between SND1 and MTDH, we tested the role of SND1 in breast cancer and found that it strongly promotes lung metastasis. SND1 was further shown to promote resistance to apoptosis and to regulate the expression of genes associated with metastasis and chemoresistance. Analyses of breast cancer clinical microarray data indicated that high expression of SND1 in primary tumors is strongly associated with reduced metastasis-free survival in multiple large scale data sets. Thus, we have uncovered SND1 as a novel MTDH-interacting protein and shown that it is a functionally and clinically significant mediator of metastasis. PMID:21478147

The Identification and Functional Characterization of WxL Proteins from Enterococcus faecium Reveal Surface Proteins Involved in Extracellular Matrix Interactions

PubMed Central

Galloway-Peña, Jessica R.; Liang, Xiaowen; Singh, Kavindra V.; Yadav, Puja; Chang, Chungyu; La Rosa, Sabina Leanti; Shelburne, Samuel; Ton-That, Hung; Höök, Magnus

2014-01-01

The WxL domain recently has been identified as a novel cell wall binding domain found in numerous predicted proteins within multiple Gram-positive bacterial species. However, little is known about the function of proteins containing this novel domain. Here, we identify and characterize 6 Enterococcus faecium proteins containing the WxL domain which, by reverse transcription-PCR (RT-PCR) and genomic analyses, are located in three similarly organized operons, deemed WxL loci A, B, and C. Western blotting, electron microscopy, and enzyme-linked immunosorbent assays (ELISAs) determined that genes of WxL loci A and C encode antigenic, cell surface proteins exposed at higher levels in clinical isolates than in commensal isolates. Secondary structural analyses of locus A recombinant WxL domain-containing proteins found they are rich in β-sheet structure and disordered segments. Using Biacore analyses, we discovered that recombinant WxL proteins from locus A bind human extracellular matrix proteins, specifically type I collagen and fibronectin. Proteins encoded by locus A also were found to bind to each other, suggesting a novel cell surface complex. Furthermore, bile salt survival assays and animal models using a mutant from which all three WxL loci were deleted revealed the involvement of WxL operons in bile salt stress and endocarditis pathogenesis. In summary, these studies extend our understanding of proteins containing the WxL domain and their potential impact on colonization and virulence in E. faecium and possibly other Gram-positive bacterial species. PMID:25512313

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

PubMed

Chen, Hsin-Yi; Wu, Chien-Ting; Tang, Chieh-Ju C; Lin, Yi-Nan; Wang, Won-Jing; Tang, Tang K

2017-08-15

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2015-01-01

Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD) of heat shock 70 kDa protein (PDB: 1HJO) with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD) simulation. Human DNA binding domain of p53 motif (SCMGGMNR) retrieved from UniProt (UniProtKB: P04637) was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were −0.44 Kcal/mol and −9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy. PMID:26098630

A Novel Protein Interaction between Nucleotide Binding Domain of Hsp70 and p53 Motif.

PubMed

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2015-01-01

Currently, protein interaction of Homo sapiens nucleotide binding domain (NBD) of heat shock 70 kDa protein (PDB: 1HJO) with p53 motif remains to be elucidated. The NBD-p53 motif complex enhances the p53 stabilization, thereby increasing the tumor suppression activity in cancer treatment. Therefore, we identified the interaction between NBD and p53 using STRING version 9.1 program. Then, we modeled the three-dimensional structure of p53 motif through homology modeling and determined the binding affinity and stability of NBD-p53 motif complex structure via molecular docking and dynamics (MD) simulation. Human DNA binding domain of p53 motif (SCMGGMNR) retrieved from UniProt (UniProtKB: P04637) was docked with the NBD protein, using the Autodock version 4.2 program. The binding energy and intermolecular energy for the NBD-p53 motif complex were -0.44 Kcal/mol and -9.90 Kcal/mol, respectively. Moreover, RMSD, RMSF, hydrogen bonds, salt bridge, and secondary structure analyses revealed that the NBD protein had a strong bond with p53 motif and the protein-ligand complex was stable. Thus, the current data would be highly encouraging for designing Hsp70 structure based drug in cancer therapy.

Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

PubMed

White, Courtney L; Kitich, Aleksandar; Gober, James W

2010-05-01

In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

Proteomic analyses of signalling complexes associated with receptor tyrosine kinase identify novel members of fibroblast growth factor receptor 3 interactome.

PubMed

Balek, Lukas; Nemec, Pavel; Konik, Peter; Kunova Bosakova, Michaela; Varecha, Miroslav; Gudernova, Iva; Medalova, Jirina; Krakow, Deborah; Krejci, Pavel

2018-01-01

Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs. Copyright © 2017 Elsevier Inc. All rights reserved.

Quaternary re-arrangement analysed by spectral enhancement: the interaction of a sporulation repressor with its antagonist.

PubMed

Scott, D J; Leejeerajumnean, S; Brannigan, J A; Lewis, R J; Wilkinson, A J; Hoggett, J G

1999-11-12

The protein/protein interaction between SinI and SinR has been studied by analytical ultracentrifugation and gel electrophoresis in an attempt to understand how these proteins contribute to developmental control of sporulation in Bacillus subtilis. SinR was found to be tetrameric, while SinI was found to exist as monomers and dimers in a rapidly reversible equilibrium. Labelling of SinR by incorporating the tryptophan analogue 7-azatryptophan (7AW) into the protein in place of tryptophan shifts the UV absorbance spectrum, thus allowing selective monitoring of 7AWSinR at 315 nm using the UV absorption optics of the analytical ultracentrifuge. Selective monitoring of SinR in mixtures of SinR and SinI enables the binding and stoichiometry of the interaction to be investigated quantitatively and unambiguously. We demonstrate that the oligomeric forms of SinR and SinI re-arrange to form a tight 1:1 SinR:SinI complex, with no stable intermediate species. A fragment of SinR, SinR(1-69), which contains only the DNA-binding domain, was found to be monomeric, showing that the protein appears not to oligomerise in a similar manner to the Cro repressor, a protein with which it shares a marked structural similarity. Copyright 1999 Academic Press.

Root Secreted Metabolites and Proteins Are Involved in the Early Events of Plant-Plant Recognition Prior to Competition

PubMed Central

Badri, Dayakar V.; De-la-Peña, Clelia; Lei, Zhentian; Manter, Daniel K.; Chaparro, Jacqueline M.; Guimarães, Rejane L.; Sumner, Lloyd W.; Vivanco, Jorge M.

2012-01-01

The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually (Col-0, Ler and Cap grown alone) and the plants co-cultured with two homozygous individuals (Col-Col, Ler-Ler and Cap-Cap) or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress- related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions. PMID:23056382

NaviGO: interactive tool for visualization and functional similarity and coherence analysis with gene ontology.

PubMed

Wei, Qing; Khan, Ishita K; Ding, Ziyun; Yerneni, Satwica; Kihara, Daisuke

2017-03-20

The number of genomics and proteomics experiments is growing rapidly, producing an ever-increasing amount of data that are awaiting functional interpretation. A number of function prediction algorithms were developed and improved to enable fast and automatic function annotation. With the well-defined structure and manual curation, Gene Ontology (GO) is the most frequently used vocabulary for representing gene functions. To understand relationship and similarity between GO annotations of genes, it is important to have a convenient pipeline that quantifies and visualizes the GO function analyses in a systematic fashion. NaviGO is a web-based tool for interactive visualization, retrieval, and computation of functional similarity and associations of GO terms and genes. Similarity of GO terms and gene functions is quantified with six different scores including protein-protein interaction and context based association scores we have developed in our previous works. Interactive navigation of the GO function space provides intuitive and effective real-time visualization of functional groupings of GO terms and genes as well as statistical analysis of enriched functions. We developed NaviGO, which visualizes and analyses functional similarity and associations of GO terms and genes. The NaviGO webserver is freely available at: http://kiharalab.org/web/navigo .

3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

PubMed

Nagae, Masamichi; Hirata, Tetsuya; Morita-Matsumoto, Kana; Theiler, Romina; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

2016-10-09

The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (K d = ~10 -4 M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective.

PubMed

Pechanova, Olga; Pechan, Tibor

2015-11-30

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus.

Maize-Pathogen Interactions: An Ongoing Combat from a Proteomics Perspective

PubMed Central

Pechanova, Olga; Pechan, Tibor

2015-01-01

Maize (Zea mays L.) is a host to numerous pathogenic species that impose serious diseases to its ear and foliage, negatively affecting the yield and the quality of the maize crop. A considerable amount of research has been carried out to elucidate mechanisms of maize-pathogen interactions with a major goal to identify defense-associated proteins. In this review, we summarize interactions of maize with its agriculturally important pathogens that were assessed at the proteome level. Employing differential analyses, such as the comparison of pathogen-resistant and susceptible maize varieties, as well as changes in maize proteomes after pathogen challenge, numerous proteins were identified as possible candidates in maize resistance. We describe findings of various research groups that used mainly mass spectrometry-based, high through-put proteomic tools to investigate maize interactions with fungal pathogens Aspergillus flavus, Fusarium spp., and Curvularia lunata, and viral agents Rice Black-streaked Dwarf Virus and Sugarcane Mosaic Virus. PMID:26633370

Norovirus translation requires an interaction between the C Terminus of the genome-linked viral protein VPg and eukaryotic translation initiation factor 4G.

PubMed

Chung, Liliane; Bailey, Dalan; Leen, Eoin N; Emmott, Edward P; Chaudhry, Yasmin; Roberts, Lisa O; Curry, Stephen; Locker, Nicolas; Goodfellow, Ian G

2014-08-01

Viruses have evolved a variety of mechanisms to usurp the host cell translation machinery to enable translation of the viral genome in the presence of high levels of cellular mRNAs. Noroviruses, a major cause of gastroenteritis in man, have evolved a mechanism that relies on the interaction of translation initiation factors with the virus-encoded VPg protein covalently linked to the 5' end of the viral RNA. To further characterize this novel mechanism of translation initiation, we have used proteomics to identify the components of the norovirus translation initiation factor complex. This approach revealed that VPg binds directly to the eIF4F complex, with a high affinity interaction occurring between VPg and eIF4G. Mutational analyses indicated that the C-terminal region of VPg is important for the VPg-eIF4G interaction; viruses with mutations that alter or disrupt this interaction are debilitated or non-viable. Our results shed new light on the unusual mechanisms of protein-directed translation initiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding free energy predictions of farnesoid X receptor (FXR) agonists using a linear interaction energy (LIE) approach with reliability estimation: application to the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Rifai, Eko Aditya; van Dijk, Marc; Vermeulen, Nico P. E.; Geerke, Daan P.

2018-01-01

Computational protein binding affinity prediction can play an important role in drug research but performing efficient and accurate binding free energy calculations is still challenging. In the context of phase 2 of the Drug Design Data Resource (D3R) Grand Challenge 2 we used our automated eTOX ALLIES approach to apply the (iterative) linear interaction energy (LIE) method and we evaluated its performance in predicting binding affinities for farnesoid X receptor (FXR) agonists. Efficiency was obtained by our pre-calibrated LIE models and molecular dynamics (MD) simulations at the nanosecond scale, while predictive accuracy was obtained for a small subset of compounds. Using our recently introduced reliability estimation metrics, we could classify predictions with higher confidence by featuring an applicability domain (AD) analysis in combination with protein-ligand interaction profiling. The outcomes of and agreement between our AD and interaction-profile analyses to distinguish and rationalize the performance of our predictions highlighted the relevance of sufficiently exploring protein-ligand interactions during training and it demonstrated the possibility to quantitatively and efficiently evaluate if this is achieved by using simulation data only.

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins.

PubMed

Tomczynska, Iga; Stumpe, Michael; Mauch, Felix

2018-04-19

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co-immunoprecipitation (Co-IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co-IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co-localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH-sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR-1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase-mediated vesicular secretion of antimicrobial PR-1, PDF1.2 and possibly other defence-related compounds. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

A protein relational database and protein family knowledge bases to facilitate structure-based design analyses.

PubMed

Mobilio, Dominick; Walker, Gary; Brooijmans, Natasja; Nilakantan, Ramaswamy; Denny, R Aldrin; Dejoannis, Jason; Feyfant, Eric; Kowticwar, Rupesh K; Mankala, Jyoti; Palli, Satish; Punyamantula, Sairam; Tatipally, Maneesh; John, Reji K; Humblet, Christine

2010-08-01

The Protein Data Bank is the most comprehensive source of experimental macromolecular structures. It can, however, be difficult at times to locate relevant structures with the Protein Data Bank search interface. This is particularly true when searching for complexes containing specific interactions between protein and ligand atoms. Moreover, searching within a family of proteins can be tedious. For example, one cannot search for some conserved residue as residue numbers vary across structures. We describe herein three databases, Protein Relational Database, Kinase Knowledge Base, and Matrix Metalloproteinase Knowledge Base, containing protein structures from the Protein Data Bank. In Protein Relational Database, atom-atom distances between protein and ligand have been precalculated allowing for millisecond retrieval based on atom identity and distance constraints. Ring centroids, centroid-centroid and centroid-atom distances and angles have also been included permitting queries for pi-stacking interactions and other structural motifs involving rings. Other geometric features can be searched through the inclusion of residue pair and triplet distances. In Kinase Knowledge Base and Matrix Metalloproteinase Knowledge Base, the catalytic domains have been aligned into common residue numbering schemes. Thus, by searching across Protein Relational Database and Kinase Knowledge Base, one can easily retrieve structures wherein, for example, a ligand of interest is making contact with the gatekeeper residue.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

A comparative approach expands the protein-protein interaction node of the immune receptor XA21 in wheat and rice

PubMed Central

Yang, Baoju; Ruan, Randy; Cantu, Dario; Wang, Xiaodong; Ji, Wanquan; Ronald, Pamela C; Dubcovsky, Jorge

2016-01-01

The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a non-syntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation (BiFC) assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that inter-specific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice. PMID:23957671

Background | Office of Cancer Clinical Proteomics Research

Cancer.gov

The term "proteomics" refers to a large-scale comprehensive study of a specific proteome resulting from its genome, including abundances of proteins, their variations and modifications, and interacting partners and networks in order to understand cellular processes involved.  Similarly, “Cancer proteomics” refers to comprehensive analyses of proteins and their derivatives translated from a specific cancer genome using a human biospecimen or a preclinical model (e.g., cultured cell or animal model).

Exploring the mechanistic insights of Cas scaffolding protein family member 4 with protein tyrosine kinase 2 in Alzheimer's disease by evaluating protein interactions through molecular docking and dynamic simulations.

PubMed

Hassan, Mubashir; Shahzadi, Saba; Alashwal, Hany; Zaki, Nazar; Seo, Sung-Yum; Moustafa, Ahmed A

2018-05-22

Cas scaffolding protein family member 4 and protein tyrosine kinase 2 are signaling proteins, which are involved in neuritic plaques burden, neurofibrillary tangles, and disruption of synaptic connections in Alzheimer's disease. In the current study, a computational approach was employed to explore the active binding sites of Cas scaffolding protein family member 4 and protein tyrosine kinase 2 proteins and their significant role in the activation of downstream signaling pathways. Sequential and structural analyses were performed on Cas scaffolding protein family member 4 and protein tyrosine kinase 2 to identify their core active binding sites. Molecular docking servers were used to predict the common interacting residues in both Cas scaffolding protein family member 4 and protein tyrosine kinase 2 and their involvement in Alzheimer's disease-mediated pathways. Furthermore, the results from molecular dynamic simulation experiment show the stability of targeted proteins. In addition, the generated root mean square deviations and fluctuations, solvent-accessible surface area, and gyration graphs also depict their backbone stability and compactness, respectively. A better understanding of CAS and their interconnected protein signaling cascade may help provide a treatment for Alzheimer's disease. Further, Cas scaffolding protein family member 4 could be used as a novel target for the treatment of Alzheimer's disease by inhibiting the protein tyrosine kinase 2 pathway.

Analysis of functional redundancies within the Arabidopsis TCP transcription factor family

PubMed Central

Danisman, Selahattin; de Folter, Stefan; Immink, Richard G. H.

2013-01-01

Analyses of the functions of TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 (TCP) transcription factors have been hampered by functional redundancy between its individual members. In general, putative functionally redundant genes are predicted based on sequence similarity and confirmed by genetic analysis. In the TCP family, however, identification is impeded by relatively low overall sequence similarity. In a search for functionally redundant TCP pairs that control Arabidopsis leaf development, this work performed an integrative bioinformatics analysis, combining protein sequence similarities, gene expression data, and results of pair-wise protein–protein interaction studies for the 24 members of the Arabidopsis TCP transcription factor family. For this, the work completed any lacking gene expression and protein–protein interaction data experimentally and then performed a comprehensive prediction of potential functional redundant TCP pairs. Subsequently, redundant functions could be confirmed for selected predicted TCP pairs by genetic and molecular analyses. It is demonstrated that the previously uncharacterized class I TCP19 gene plays a role in the control of leaf senescence in a redundant fashion with TCP20. Altogether, this work shows the power of combining classical genetic and molecular approaches with bioinformatics predictions to unravel functional redundancies in the TCP transcription factor family. PMID:24129704

Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

NASA Astrophysics Data System (ADS)

Serena, Michela; Giorgetti, Alejandro; Busato, Mirko; Gasparini, Francesca; Diani, Erica; Romanelli, Maria Grazia; Zipeto, Donato

2016-03-01

HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

A data-mining approach to rank candidate protein-binding partners-The case of biogenesis of lysosome-related organelles complex-1 (BLOC-1).

PubMed

Rodriguez-Fernandez, I A; Dell'Angelica, E C

2009-04-01

The study of protein-protein interactions is a powerful approach to uncovering the molecular function of gene products associated with human disease. Protein-protein interaction data are accumulating at an unprecedented pace owing to interactomics projects, although it has been recognized that a significant fraction of these data likely represents false positives. During our studies of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a protein complex involved in protein trafficking and containing the products of genes mutated in Hermansky-Pudlak syndrome, we faced the problem of having too many candidate binding partners to pursue experimentally. In this work, we have explored ways of efficiently gathering high-quality information about candidate binding partners and presenting the information in a visually friendly manner. We applied the approach to rank 70 candidate binding partners of human BLOC-1 and 102 candidates of its counterpart from Drosophila melanogaster. The top candidate for human BLOC-1 was the small GTPase encoded by the RAB11A gene, which is a paralogue of the Rab38 and Rab32 proteins in mammals and the lightoid gene product in flies. Interestingly, genetic analyses in D. melanogaster uncovered a synthetic sick/lethal interaction between Rab11 and lightoid. The data-mining approach described herein can be customized to study candidate binding partners for other proteins or possibly candidates derived from other types of 'omics' data.

Evolution and function of eukaryotic-like proteins from sponge symbionts.

PubMed

Reynolds, David; Thomas, Torsten

2016-10-01

Sponges (Porifera) are ancient metazoans that harbour diverse microorganisms, whose symbiotic interactions are essential for the host's health and function. Although symbiosis between bacteria and sponges are ubiquitous, the molecular mechanisms that control these associations are largely unknown. Recent (meta-) genomic analyses discovered an abundance of genes encoding for eukaryotic-like proteins (ELPs) in bacterial symbionts from different sponge species. ELPs belonging to the ankyrin repeat (AR) class from a bacterial symbiont of the sponge Cymbastela concentrica were subsequently found to modulate amoebal phagocytosis. This might be a molecular mechanism, by which symbionts can control their interaction with the sponge. In this study, we investigated the evolution and function of ELPs from other classes and from symbionts found in other sponges to better understand the importance of ELPs for bacteria-eukaryote interactions. Phylogenetic analyses showed that all of the nine ELPs investigated were most closely related to proteins found either in eukaryotes or in bacteria that can live in association with eukaryotes. ELPs were then recombinantly expressed in Escherichia coli and exposed to the amoeba Acanthamoeba castellanii, which is functionally analogous to phagocytic cells in sponges. Phagocytosis assays with E. coli containing three ELP classes (AR, TPR-SEL1 and NHL) showed a significantly higher percentage of amoeba containing bacteria and average number of intracellular bacteria per amoeba when compared to negative controls. The result that various classes of ELPs found in symbionts of different sponges can modulate phagocytosis indicates that they have a broader function in mediating bacteria-sponge interactions. © 2016 John Wiley & Sons Ltd.

Integrated data analysis for genome-wide research.

PubMed

Steinfath, Matthias; Repsilber, Dirk; Scholz, Matthias; Walther, Dirk; Selbig, Joachim

2007-01-01

Integrated data analysis is introduced as the intermediate level of a systems biology approach to analyse different 'omics' datasets, i.e., genome-wide measurements of transcripts, protein levels or protein-protein interactions, and metabolite levels aiming at generating a coherent understanding of biological function. In this chapter we focus on different methods of correlation analyses ranging from simple pairwise correlation to kernel canonical correlation which were recently applied in molecular biology. Several examples are presented to illustrate their application. The input data for this analysis frequently originate from different experimental platforms. Therefore, preprocessing steps such as data normalisation and missing value estimation are inherent to this approach. The corresponding procedures, potential pitfalls and biases, and available software solutions are reviewed. The multiplicity of observations obtained in omics-profiling experiments necessitates the application of multiple testing correction techniques.

The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition

PubMed Central

Moldovan, John B.; Moran, John V.

2015-01-01

Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements. PMID:25951186

Evolution and Cellular Function of Monothiol Glutaredoxins: Involvement in Iron-Sulphur Cluster Assembly

PubMed Central

Vilella, Felipe; Alves, Rui; Rodríguez-Manzaneque, María Teresa; Bellí, Gemma; Swaminathan, Swarna; Sunnerhagen, Per

2004-01-01

A number of bacterial species, mostly proteobacteria, possess monothiol glutaredoxins homologous to the Saccharomyces cerevisiae mitochondrial protein Grx5, which is involved in iron–sulphur cluster synthesis. Phylogenetic profiling is used to predict that bacterial monothiol glutaredoxins also participate in the iron–sulphur cluster (ISC) assembly machinery, because their phylogenetic profiles are similar to the profiles of the bacterial homologues of yeast ISC proteins. High evolutionary co-occurrence is observed between the Grx5 homologues and the homologues of the Yah1 ferredoxin, the scaffold proteins Isa1 and Isa2, the frataxin protein Yfh1 and the Nfu1 protein. This suggests that a specific functional interaction exists between these ISC machinery proteins. Physical interaction analyses using low-definition protein docking predict the formation of strong and specific complexes between Grx5 and several components of the yeast ISC machinery. Two-hybrid analysis has confirmed the in vivo interaction between Grx5 and Isa1. Sequence comparison techniques and cladistics indicate that the other two monothiol glutaredoxins of S. cerevisiae, Grx3 and Grx4, have evolved from the fusion of a thioredoxin gene with a monothiol glutaredoxin gene early in the eukaryotic lineage, leading to differential functional specialization. While bacteria do not contain these chimaeric glutaredoxins, in many eukaryotic species Grx5 and Grx3/4-type monothiol glutaredoxins coexist in the cell. PMID:18629168

Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions

PubMed Central

Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; West, Graham M; Griffin, Patrick R

2011-01-01

Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery. PMID:21329427

A dynamic interaction process between KaiA and KaiC is critical to the cyanobacterial circadian oscillator.

PubMed

Dong, Pei; Fan, Ying; Sun, Jianqiang; Lv, Mengting; Yi, Ming; Tan, Xiao; Liu, Sen

2016-04-26

The core circadian oscillator of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. This circadian oscillator could be functionally reconstituted in vitro with these three proteins, and therefore has been a very important model in circadian rhythm research. KaiA can bind to KaiC and then stimulate its phosphorylation, but their interaction mechanism remains elusive. In this study, we followed the "second-site suppressor" strategy to investigate the interaction mechanism of KaiA and KaiC. Using protein sequence analyses, we showed that there exist co-varying residues in the binding interface of KaiA and KaiC. The followed mutagenesis study verified that these residues are important to the functions of KaiA and KaiC, but their roles could not be fully explained by the reported complex structures of KaiA and KaiC derived peptides. Combining our data with previous reports, we suggested a dynamic interaction mechanism in KaiA-KaiC interaction, in which both KaiA and the intrinsically disordered tail of KaiC undergo significant structural changes through conformational selection and induced fit during the binding process. At last, we presented a mathematic model to support this hypothesis and explained the importance of this interaction mechanism for the KaiABC circadian oscillator.

A dynamic interaction process between KaiA and KaiC is critical to the cyanobacterial circadian oscillator

NASA Astrophysics Data System (ADS)

Dong, Pei; Fan, Ying; Sun, Jianqiang; Lv, Mengting; Yi, Ming; Tan, Xiao; Liu, Sen

2016-04-01

The core circadian oscillator of cyanobacteria consists of three proteins, KaiA, KaiB, and KaiC. This circadian oscillator could be functionally reconstituted in vitro with these three proteins, and therefore has been a very important model in circadian rhythm research. KaiA can bind to KaiC and then stimulate its phosphorylation, but their interaction mechanism remains elusive. In this study, we followed the “second-site suppressor” strategy to investigate the interaction mechanism of KaiA and KaiC. Using protein sequence analyses, we showed that there exist co-varying residues in the binding interface of KaiA and KaiC. The followed mutagenesis study verified that these residues are important to the functions of KaiA and KaiC, but their roles could not be fully explained by the reported complex structures of KaiA and KaiC derived peptides. Combining our data with previous reports, we suggested a dynamic interaction mechanism in KaiA-KaiC interaction, in which both KaiA and the intrinsically disordered tail of KaiC undergo significant structural changes through conformational selection and induced fit during the binding process. At last, we presented a mathematic model to support this hypothesis and explained the importance of this interaction mechanism for the KaiABC circadian oscillator.

Mutation of a Short Variable Region in HCpro Protein of Potato virus A Affects Interactions with a Microtubule-Associated Protein and Induces Necrotic Responses in Tobacco.

PubMed

Haikonen, Tuuli; Rajamäki, Minna-Liisa; Tian, Yan-Ping; Valkonen, Jari P T

2013-07-01

Helper component proteinase (HCpro) is a multifunctional protein of potyviruses (genus Potyvirus). HCpro of Potato virus A (PVA) interacts with the microtubule-associated protein HIP2 in host cells, and depletion of HIP2 reduces virus accumulation. This study shows that HCpro of Potato virus Y and Tobacco etch virus also interact with HIP2. The C-proximal portion of PVA HCpro determines the interaction with HIP2 and was found to contain a stretch of six residues comprising a highly variable region (HVR) in potyviruses. Mutations in HVR reduced PVA accumulation in tobacco plants and induced necrotic symptoms novel to PVA. Microarray and quantitative reverse transcription polymerase chain reaction analyses revealed induction of many defense-related genes including ethylene- and jasmonic acid-inducible pathways in systemically infected leaves at necrosis onset. Salicylic acid-mediated signaling was dispensable for the response. Genes related to microtubule functions were down-regulated. Structural modeling of HCpro suggested that all mutations in HVR caused conformational changes in adjacent regions containing functionally important motifs conserved in potyviruses. Those mutations, which also caused conformational changes in HVR, led to the greatest reduction of fitness. Our results implicate HVR in the regulation of HCpro conformation and virus-host interactions and suggest that mutation of HVR induces host defense.

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

Mei, Yang; Glover, Karen; Su, Minfei

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

A MADS Box Protein Interacts with a Mating-Type Protein and Is Required for Fruiting Body Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Nolting, Nicole; Pöggeler, Stefanie

2006-01-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Δmcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development. PMID:16835449

Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome

PubMed Central

Kosová, Klára; Vítámvás, Pavel; Urban, Milan O.; Prášil, Ilja T.; Renaut, Jenny

2018-01-01

HIGHLIGHTS: Major environmental and genetic factors determining stress-related protein abundance are discussed.Major aspects of protein biological function including protein isoforms and PTMs, cellular localization and protein interactions are discussed.Functional diversity of protein isoforms and PTMs is discussed. Abiotic stresses reveal profound impacts on plant proteomes including alterations in protein relative abundance, cellular localization, post-transcriptional and post-translational modifications (PTMs), protein interactions with other protein partners, and, finally, protein biological functions. The main aim of the present review is to discuss the major factors determining stress-related protein accumulation and their final biological functions. A dynamics of stress response including stress acclimation to altered ambient conditions and recovery after the stress treatment is discussed. The results of proteomic studies aimed at a comparison of stress response in plant genotypes differing in stress adaptability reveal constitutively enhanced levels of several stress-related proteins (protective proteins, chaperones, ROS scavenging- and detoxification-related enzymes) in the tolerant genotypes with respect to the susceptible ones. Tolerant genotypes can efficiently adjust energy metabolism to enhanced needs during stress acclimation. Stress tolerance vs. stress susceptibility are relative terms which can reflect different stress-coping strategies depending on the given stress treatment. The role of differential protein isoforms and PTMs with respect to their biological functions in different physiological constraints (cellular compartments and interacting partners) is discussed. The importance of protein functional studies following high-throughput proteome analyses is presented in a broader context of plant biology. In summary, the manuscript tries to provide an overview of the major factors which have to be considered when interpreting data from proteomic studies on stress-treated plants. PMID:29472941

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP).

PubMed

Kamina, Anyango D; Williams, Noreen

2017-01-01

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains' interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

Non-canonical binding interactions of the RNA recognition motif (RRM) domains of P34 protein modulate binding within the 5S ribonucleoprotein particle (5S RNP)

PubMed Central

Kamina, Anyango D.; Williams, Noreen

2017-01-01

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains’ interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP. PMID:28542332

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ.

PubMed

Di Matteo, A; Franceschini, M; Paiardini, A; Grottesi, A; Chiarella, S; Rocchio, S; Di Natale, C; Marasco, D; Vitagliano, L; Travaglini-Allocatelli, C; Federici, L

2017-09-18

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

Binding preference of carbon nanotube over proline-rich motif ligand on SH3-domain: a comparison with different force fields.

PubMed

Shi, Biyun; Zuo, Guanghong; Xiu, Peng; Zhou, Ruhong

2013-04-04

With the widespread applications of nanomaterials such as carbon nanotubes, there is a growing concern on the biosafety of these engineered nanoparticles, in particular their interactions with proteins. In molecular simulations of nanoparticle-protein interactions, the choice of empirical parameters (force fields) plays a decisive role, and thus is of great importance and should be examined carefully before wider applications. Here we compare three commonly used force fields, CHARMM, OPLSAA, and AMBER in study of the competitive binding of a single wall carbon nanotube (SWCNT) with a native proline-rich motif (PRM) ligand on its target protein SH3 domain, a ubiquitous protein-protein interaction mediator involved in signaling and regulatory pathways. We find that the SWCNT displays a general preference over the PRM in binding with SH3 domain in all the three force fields examined, although the degree of preference can be somewhat different, with the AMBER force field showing the highest preference. The SWCNT prevents the ligand from reaching its native binding pocket by (i) occupying the binding pocket directly, and (ii) binding with the ligand itself and then being trapped together onto some off-sites. The π-π stacking interactions between the SWCNT and aromatic residues are found to play a significant role in its binding to the SH3 domain in all the three force fields. Further analyses show that even the SWCNT-ligand binding can also be relatively more stable than the native ligand-protein binding, indicating a serious potential disruption to the protein SH3 function.

Analysis of the interaction of calcitriol with the disulfide isomerase ERp57

NASA Astrophysics Data System (ADS)

Gaucci, Elisa; Raimondo, Domenico; Grillo, Caterina; Cervoni, Laura; Altieri, Fabio; Nittari, Giulio; Eufemi, Margherita; Chichiarelli, Silvia

2016-11-01

Calcitriol, the active form of vitamin D3, can regulate the gene expression through the binding to the nuclear receptor VDR, but it can also display nongenomic actions, acting through a membrane-associated receptor, which has been discovered as the disulfide isomerase ERp57. The aim of our research is to identify the binding sites for calcitriol in ERp57 and to analyze their interaction. We first studied the interaction through bioinformatics and fluorimetric analyses. Subsequently, we focused on two protein mutants containing the predicted interaction domains with calcitriol: abb’-ERp57, containing the first three domains, and a’-ERp57, the fourth domain only. To consolidate the achievements we used the calorimetric approach to the whole protein and its mutants. Our results allow us to hypothesize that the interaction with the a’ domain contributes to a greater extent than the other potential binding sites to the dissociation constant, calculated as a Kd of about 10-9 M.

Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones.

PubMed

Watts, Joel C; Huo, Hairu; Bai, Yu; Ehsani, Sepehr; Jeon, Amy Hye Won; Won, Amy Hye; Shi, Tujin; Daude, Nathalie; Lau, Agnes; Young, Rebecca; Xu, Lei; Carlson, George A; Williams, David; Westaway, David; Schmitt-Ulms, Gerold

2009-10-01

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP(C) interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP(C) paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP(C) and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP(C) with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP(Sc). A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP(C) organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.

[MALDI-TOF mass spectrometry in the investigation of large high-molecular biological compounds].

PubMed

Porubl'ova, L V; Rebriiev, A V; Hromovyĭ, T Iu; Minia, I I; Obolens'ka, M Iu

2009-01-01

MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry has become, in the recent years, a tool of choice for analyses of biological polymers. The wide mass range, high accuracy, informativity and sensitivity make it a superior method for analysis of all kinds of high-molecular biological compounds including proteins, nucleic acids and lipids. MALDI-TOF-MS is particularly suitable for the identification of proteins by mass fingerprint or microsequencing. Therefore it has become an important technique of proteomics. Furthermore, the method allows making a detailed analysis of post-translational protein modifications, protein-protein and protein-nucleic acid interactions. Recently, the method was also successfully applied to nucleic acid sequencing as well as screening for mutations.

A recruiting protein of geranylgeranyl diphosphate synthase controls metabolic flux toward chlorophyll biosynthesis in rice.

PubMed

Zhou, Fei; Wang, Cheng-Yuan; Gutensohn, Michael; Jiang, Ling; Zhang, Peng; Zhang, Dabing; Dudareva, Natalia; Lu, Shan

2017-06-27

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

PubMed Central

Baumann, P; Jackson, S P

1996-01-01

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya. Images Fig. 1 Fig. 2 PMID:8692886

Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy - An Enhanced Method for Examining Protein Conformations and Protein Interactions

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

B Wallace; R Janes

CD (circular dichroism) spectroscopy is a well-established technique in structural biology. SRCD (synchrotron radiation circular dichroism) spectroscopy extends the utility and applications of conventional CD spectroscopy (using laboratory-based instruments) because the high flux of a synchrotron enables collection of data at lower wavelengths (resulting in higher information content), detection of spectra with higher signal-to-noise levels and measurements in the presence of absorbing components (buffers, salts, lipids and detergents). SRCD spectroscopy can provide important static and dynamic structural information on proteins in solution, including secondary structures of intact proteins and their domains, protein stability, the differences between wild-type and mutant proteins,more » the identification of natively disordered regions in proteins, and the dynamic processes of protein folding and membrane insertion and the kinetics of enzyme reactions. It has also been used to effectively study protein interactions, including protein-protein complex formation involving either induced-fit or rigid-body mechanisms, and protein-lipid complexes. A new web-based bioinformatics resource, the Protein Circular Dichroism Data Bank (PCDDB), has been created which enables archiving, access and analyses of CD and SRCD spectra and supporting metadata, now making this information publicly available. To summarize, the developing method of SRCD spectroscopy has the potential for playing an important role in new types of studies of protein conformations and their complexes.« less

A stoichiometry driven universal spatial organization of backbones of folded proteins: are there Chargaff's rules for protein folding?

PubMed

Mittal, A; Jayaram, B; Shenoy, Sandhya; Bawa, Tejdeep Singh

2010-10-01

Protein folding is at least a six decade old problem, since the times of Pauling and Anfinsen. However, rules of protein folding remain elusive till date. In this work, rigorous analyses of several thousand crystal structures of folded proteins reveal a surprisingly simple unifying principle of backbone organization in protein folding. We find that protein folding is a direct consequence of a narrow band of stoichiometric occurrences of amino-acids in primary sequences, regardless of the size and the fold of a protein. We observe that "preferential interactions" between amino-acids do not drive protein folding, contrary to all prevalent views. We dedicate our discovery to the seminal contribution of Chargaff which was one of the major keys to elucidation of the stoichiometry-driven spatially organized double helical structure of DNA.

Atomic decomposition of the protein solvation free energy and its application to amyloid-beta protein in water

NASA Astrophysics Data System (ADS)

Chong, Song-Ho; Ham, Sihyun

2011-07-01

We report the development of an atomic decomposition method of the protein solvation free energy in water, which ascribes global change in the solvation free energy to local changes in protein conformation as well as in hydration structure. So far, empirical decomposition analyses based on simple continuum solvation models have prevailed in the study of protein-protein interactions, protein-ligand interactions, as well as in developing scoring functions for computer-aided drug design. However, the use of continuum solvation model suffers serious drawbacks since it yields the protein free energy landscape which is quite different from that of the explicit solvent model and since it does not properly account for the non-polar hydrophobic effects which play a crucial role in biological processes in water. Herein, we develop an exact and general decomposition method of the solvation free energy that overcomes these hindrances. We then apply this method to elucidate the molecular origin for the solvation free energy change upon the conformational transitions of 42-residue amyloid-beta protein (Aβ42) in water, whose aggregation has been implicated as a primary cause of Alzheimer's disease. We address why Aβ42 protein exhibits a great propensity to aggregate when transferred from organic phase to aqueous phase.

Visualisation and graph-theoretic analysis of a large-scale protein structural interactome

PubMed Central

Bolser, Dan; Dafas, Panos; Harrington, Richard; Park, Jong; Schroeder, Michael

2003-01-01

Background Large-scale protein interaction maps provide a new, global perspective with which to analyse protein function. PSIMAP, the Protein Structural Interactome Map, is a database of all the structurally observed interactions between superfamilies of protein domains with known three-dimensional structure in the PDB. PSIMAP incorporates both functional and evolutionary information into a single network. Results We present a global analysis of PSIMAP using several distinct network measures relating to centrality, interactivity, fault-tolerance, and taxonomic diversity. We found the following results: Centrality: we show that the center and barycenter of PSIMAP do not coincide, and that the superfamilies forming the barycenter relate to very general functions, while those constituting the center relate to enzymatic activity. Interactivity: we identify the P-loop and immunoglobulin superfamilies as the most highly interactive. We successfully use connectivity and cluster index, which characterise the connectivity of a superfamily's neighbourhood, to discover superfamilies of complex I and II. This is particularly significant as the structure of complex I is not yet solved. Taxonomic diversity: we found that highly interactive superfamilies are in general taxonomically very diverse and are thus amongst the oldest. Fault-tolerance: we found that the network is very robust as for the majority of superfamilies removal from the network will not break up the network. Conclusions Overall, we can single out the P-loop containing nucleotide triphosphate hydrolases superfamily as it is the most highly connected and has the highest taxonomic diversity. In addition, this superfamily has the highest interaction rank, is the barycenter of the network (it has the shortest average path to every other superfamily in the network), and is an articulation vertex, whose removal will disconnect the network. More generally, we conclude that the graph-theoretic and taxonomic analysis of PSIMAP is an important step towards the understanding of protein function and could be an important tool for tracing the evolution of life at the molecular level. PMID:14531933

Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA

PubMed Central

Singhal, Anshika; Joshi, Jayadev; Virmani, Richa; Gupta, Meetu; Verma, Nupur; Maji, Abhijit; Misra, Richa; Baronian, Grégory; Pandey, Amit K.; Molle, Virginie; Singh, Yogendra

2014-01-01

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. Methodology/Principal Findings In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Conclusions/Significance Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes. PMID:25412098

Integrative proteomics and biochemical analyses define Ptc6p as the Saccharomyces cerevisiae pyruvate dehydrogenase phosphatase.

PubMed

Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J

2017-07-14

The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differential protein-coding gene and long noncoding RNA expression in smoking-related lung squamous cell carcinoma.

PubMed

Li, Shicheng; Sun, Xiao; Miao, Shuncheng; Liu, Jia; Jiao, Wenjie

2017-11-01

Cigarette smoking is one of the greatest preventable risk factors for developing cancer, and most cases of lung squamous cell carcinoma (lung SCC) are associated with smoking. The pathogenesis mechanism of tumor progress is unclear. This study aimed to identify biomarkers in smoking-related lung cancer, including protein-coding gene, long noncoding RNA, and transcription factors. We selected and obtained messenger RNA microarray datasets and clinical data from the Gene Expression Omnibus database to identify gene expression altered by cigarette smoking. Integrated bioinformatic analysis was used to clarify biological functions of the identified genes, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the construction of a protein-protein interaction network, transcription factor, and statistical analyses. Subsequent quantitative real-time PCR was utilized to verify these bioinformatic analyses. Five hundred and ninety-eight differentially expressed genes and 21 long noncoding RNA were identified in smoking-related lung SCC. GO and KEGG pathway analysis showed that identified genes were enriched in the cancer-related functions and pathways. The protein-protein interaction network revealed seven hub genes identified in lung SCC. Several transcription factors and their binding sites were predicted. The results of real-time quantitative PCR revealed that AURKA and BIRC5 were significantly upregulated and LINC00094 was downregulated in the tumor tissues of smoking patients. Further statistical analysis indicated that dysregulation of AURKA, BIRC5, and LINC00094 indicated poor prognosis in lung SCC. Protein-coding genes AURKA, BIRC5, and LINC00094 could be biomarkers or therapeutic targets for smoking-related lung SCC. © 2017 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Part I---Evaluating Effects of Oligomer Formation on Cytochrome P450 2C9 Electron Transfer and Drug Metabolism, Part II---Utilizing Molecular Modeling Techniques to Study the Src-Interacting Proteins Actin Filament Associated Protein of 110 kDa (AFAP-110) and Cortactin

NASA Astrophysics Data System (ADS)

Jett, John Edward, Jr.

The dissertation has been divided into two parts to accurately reflect the two distinct areas of interest pursued during my matriculation in the School of Pharmacy at West Virginia University. In Part I, I discuss research probing the nature of electron transfer in the Cytochrome P450 family of proteins, a group of proteins well-known for their role in drug metabolism. In Part II, I focus on in silico and in vitro work developed in concert to probe protein structure and protein-protein interactions involved in actin filament reorganization and cellular motility. Part I. Cytochrome P450s (P450s) are an important class of enzymes known to metabolize a variety of endogenous and xenobiotic compounds. P450s are most commonly found in liver and intestinal endothelial cells and are responsible for the metabolism of approximately 75% of pharmaceutical drugs on the market. CYP2C9---one of the six major P450 isoforms---is responsible for ˜20% of drug metabolism. Elucidation of the factors that affect in vitro drug metabolism is crucial to the accurate prediction of in vivo drug metabolism kinetics. Currently, the two major techniques for studying in vitro drug metabolism are solution-based. However, it is known that the results of solution-based studies can vary from in vivo drug metabolism. One reason suggested to account for this variation is the state of P450 oligomer formation in solution compared to the in vivo environment, where P450s are membrane-bound. To understand the details of how oligomer formation affects in vitro drug metabolism, it is imperative that techniques be developed which will allow for the unequivocal control of oligomer formation without altering other experimental parameters. Our long term goal of this research is to develop methods to more accurately predict in vivo drug metabolism from in vitro data. This section of the dissertation will discuss the development of a platform consisting of a doped silicon surface containing a large array of gold nanopillars, the immobilization of CYP2C9 enzymes to those nanopillars, and the utilization of the array to perform conductive probe atomic force microscopy experiments examining the electron transfer process of CYP2C9 in the absence and presence of substrate molecules. Part II. The Src protein has been known to play a role in cancer cell progression for over 30 years. The function of a non-receptor tyrosine kinase such as Src is to relay extracellular signals through intracellular tyrosine phosphorylation. As a tyrosine kinase, Src and the cellular signaling pathways it is involved in play many functional roles in the cell, both in cellular proliferation and in cytoskeletal dynamics, cell adhesion, motility and invasion. Two of the many proteins comprising Src cellular signaling pathways are actin filament associated protein of 110 kDa (AFAP-110) and cortactin. AFAP-110 is a known activator of Src; one mechanism to abrogate the AFAP-110-induced activation of Src is to inhibit their colocalization within the cell. This colocalization is expected to occur when the pleckstrin homology (PH1 and PH2) domains of AFAP-110 are allowed to interact with membrane-bound phospholipids. Cortactin, on the other hand, is a cytosolic protein capable of being phosphorylated on various tyrosine residues, activating it and allowing it to interact with actin. The Src homology 2 (SH2) domain of Src has been shown to be capable of interacting with cortactin, an association which will be probed here. This section of the dissertation will discuss the use of molecular modeling techniques to develop structural models of the AFAP-110 PH1 and PH2 domains and use them to make predictions about how the protein interacts with phospholipids in the plasma membrane and how they might be stabilized to interact with other proteins. Structural models were designed using homology modeling methods, docking programs were used to predict key residues of AFAP-110 involved in binding to phospholipids and mutational analyses was used to test those predictions. This section will also discuss the use of molecular modeling techniques to explore protein-protein interactions between cortactin and Src. These include docking experiments and binding interaction analyses between Src and key areas of cortactin known to be involved in protein-protein interactions with Src. The data point to a cysteine-cysteine interaction between the two proteins, a result which is confirmed through in vitro experiments in collaboration with the lab of Dr. Scott Weed.

Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

PubMed

DeBalsi, Karen L; Wong, Kari E; Koves, Timothy R; Slentz, Dorothy H; Seiler, Sarah E; Wittmann, April H; Ilkayeva, Olga R; Stevens, Robert D; Perry, Christopher G R; Lark, Daniel S; Hui, Simon T; Szweda, Luke; Neufer, P Darrell; Muoio, Deborah M

2014-03-21

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

PubMed

Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

2017-05-01

Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Targeted Metabolomics Connects Thioredoxin-interacting Protein (TXNIP) to Mitochondrial Fuel Selection and Regulation of Specific Oxidoreductase Enzymes in Skeletal Muscle*

PubMed Central

DeBalsi, Karen L.; Wong, Kari E.; Koves, Timothy R.; Slentz, Dorothy H.; Seiler, Sarah E.; Wittmann, April H.; Ilkayeva, Olga R.; Stevens, Robert D.; Perry, Christopher G. R.; Lark, Daniel S.; Hui, Simon T.; Szweda, Luke; Neufer, P. Darrell; Muoio, Deborah M.

2014-01-01

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIPSKM−/−) Txnip deficiency. Compared with littermate controls, both TKO and TXNIPSKM−/− mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability. PMID:24482226

Study of intermolecular contacts in proteins and oligomer interfaces and preliminary investigations into the design and production of nanomaterials from proteins

NASA Astrophysics Data System (ADS)

Iyer, Ganesh Hariharan

The first part of this research involved a study of the nature and extent of nonbonded interactions at crystal and oligomer interfaces. A survey was compiled of several characteristics of intersubunit contacts in 58 different oligomeric proteins, and of the intermolecular contacts in 223 protein crystal structures. Routines written in "S" language were utilized for the generation of the observed and expected contacts. The information in the Protein Data Bank (PDB) was extracted using the database management system, Protein Knowledge Base (PKB). Potentials of mean force for atom-atom contacts and residue-residue contacts were derived by comparison of the number of observed interactions with the number expected by mass action. Preference association matrices and log-linear analyses were applied to determine the different factors that could contribute to the overall interactions at the interfaces of oligomers and crystals. Surface patches at oligomer and crystal interfaces were also studied to further investigate the origin of the differences in their stabilities. Total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acid prominent in oligomer interfaces. Contact potentials indicate that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. The second part involved the development of a new class of biomaterials from two-dimensional arrays of ordered proteins. Point mutations were planned to introduce cysteine residues at appropriate locations to enable cross-linking at the molecular interface within given crystallographic planes. Crystallization and subsequent cross-linking of the modified protein would lead to the formation of arrays on subsequent dissociation of the crystal. Novel protein architectures can be generated from these cross-linked nanostructures. Experiments with model protein, maltose-binding protein (MBP) were performed to develop purification, cross-linking and crystallization techniques. The long-term goal of this project is to apply the experience gained with MBP to the fabrication of nanomaterials from other, application-specific proteins for ultrafiltration and microelectronic devices.

The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer.

PubMed

Hannemann, Frank; Guyot, Arnaud; Zöllner, Andy; Müller, Jürgen J; Heinemann, Udo; Bernhardt, Rita

2009-07-01

Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.

Gene-gene interactions among genetic variants from obesity candidate genes for nonobese and obese populations in type 2 diabetes.

PubMed

Lin, Eugene; Pei, Dee; Huang, Yi-Jen; Hsieh, Chang-Hsun; Wu, Lawrence Shih-Hsin

2009-08-01

Recent studies indicate that obesity may play a key role in modulating genetic predispositions to type 2 diabetes (T2D). This study examines the main effects of both single-locus and multilocus interactions among genetic variants in Taiwanese obese and nonobese individuals to test the hypothesis that obesity-related genes may contribute to the etiology of T2D independently and/or through such complex interactions. We genotyped 11 single nucleotide polymorphisms for 10 obesity candidate genes including adrenergic beta-2-receptor surface, adrenergic beta-3-receptor surface, angiotensinogen, fat mass and obesity associated gene, guanine nucleotide binding protein beta polypeptide 3 (GNB3), interleukin 6 receptor, proprotein convertase subtilisin/kexin type 1 (PCSK1), uncoupling protein 1, uncoupling protein 2, and uncoupling protein 3. There were 389 patients diagnosed with T2D and 186 age- and sex-matched controls. Single-locus analyses showed significant main effects of the GNB3 and PCSK1 genes on the risk of T2D among the nonobese group (p = 0.002 and 0.047, respectively). Further, interactions involving GNB3 and PCSK1 were suggested among the nonobese population using the generalized multifactor dimensionality reduction method (p = 0.001). In addition, interactions among angiotensinogen, fat mass and obesity associated gene, GNB3, and uncoupling protein 3 genes were found in a significant four-locus generalized multifactor dimensionality reduction model among the obese population (p = 0.001). The results suggest that the single nucleotide polymorphisms from the obesity candidate genes may contribute to the risk of T2D independently and/or in an interactive manner according to the presence or absence of obesity.

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed Central

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-01-01

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. Images Fig. 2 Fig. 4 PMID:8265602

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

PubMed

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J

1993-12-15

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27

PubMed Central

Balana, Bartosz; Maslennikov, Innokentiy; Kwiatkowski, Witek; Stern, Kalyn M.; Bahima, Laia; Choe, Senyon; Slesinger, Paul A.

2011-01-01

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases. PMID:21422294

Monothiol CGFS Glutaredoxins and BolA-like Proteins: [2Fe-2S] Binding Partners in Iron Homeostasis

PubMed Central

Li, Haoran; Outten, Caryn E.

2012-01-01

Monothiol glutaredoxins (Grxs) with a signature CGFS active site and BolA-like proteins have recently emerged as novel players in iron homeostasis. Elegant genetic and biochemical studies examining the functional and physical interactions of CGFS Grxs in the fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe have unveiled their essential roles in intracellular iron signaling, iron trafficking, and the maturation of Fe-S cluster proteins. Biophysical and biochemical analyses of the [2Fe-2S]-bridging interaction between CGFS Grxs and a BolA-like protein in S. cerevisiae provided the first molecular-level understanding of the iron regulation mechanism in this model eukaryote, and established the ubiquitous CGFS Grxs and BolA-like proteins as novel Fe-S cluster-binding regulatory partners. Parallel studies focused on E. coli and human homologues for CGFS Grxs and BolA-like proteins have supported the studies in yeast and provided additional clues to their involvement in cellular iron metabolism. Herein we review recent progress in uncovering the cellular and molecular mechanisms by which CGFS Grxs and BolA-like proteins help regulate iron metabolism in both eukaryotic and prokaryotic organisms. PMID:22583368

Binding of perlecan to transthyretin in vitro.

PubMed Central

Smeland, S; Kolset, S O; Lyon, M; Norum, K R; Blomhoff, R

1997-01-01

Transthyretin is one of two specific proteins involved in the transport of thyroid hormones in plasma; it possesses two binding sites for serum retinol-binding protein. In the present study we demonstrate that transthyretin also interacts in vitro with [35S]sulphate-labelled material from the medium of HepG2 cells. By using the same strategy as for purifying serum retinol-binding protein, [35S]sulphate-labelled medium was specifically eluted from a transthyretin-affinity column. Ion-exchange chromatography showed that the material was highly polyanionic, and its size and alkali susceptibility suggested that it was a proteoglycan. Structural analyses with chondroitinase ABC lyase and nitrous acid revealed that approx. 20% was chondroitin sulphate and 80% heparan sulphate. Immunoprecipitation showed that the [35S]sulphate-labelled material contained perlecan. Further analysis by binding studies revealed specific and saturable binding of 125I-transthyretin to perlecan-enriched Matrigel. Because inhibition of sulphation by treating HepG2 cells with sodium chlorate increased the affinity of the perlecan for transthyretin, and [3H]heparin was not retained by the transthyretin affinity column, the binding is probably mediated by the core protein and is not a protein-glycosaminoglycan interaction. Because perlecan is released from transthyretin in water, the binding might be due to hydrophobic interactions. PMID:9307034

Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

PubMed Central

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK. PMID:22540012

Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

PubMed

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

PubMed Central

Nakahashi, Mito; Matsushima, Yoshiaki; Ito, Morihiro; Nishio, Machiko; Kawano, Mitsuo; Komada, Hiroshi; Nosaka, Tetsuya

2013-01-01

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity. PMID:23698295

Synergistic behavior of glycine betaine-urea mixture: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Kumar, Narendra; Kishore, Nand

2013-09-01

Glycine betaine (GB) is one of the most important osmolyte which is known to stabilize proteins as well as counteract the denaturing effect of urea. There have been many studies indicating protein stabilization and counteraction of the effect of urea by GB. However, the exact mechanism of counteraction is still debated and is of important research interest. In this study, distribution functions, hydrogen bonds, and energetics were analysed to understand different interactions between GB and urea, and their solvation properties in presence of each other. The results show that in the GB-urea mixture, GB acted as a stronger osmolyte and urea became a weaker denaturing agent than its individual counterparts. The increase in the solvation of urea and GB in GB-urea mixture and their mutual interactions through hydrogen bonding and coulombic energy resulted in more involvement of GB and urea with solvent as well as with themselves. This might result in the increase of the exclusion of GB from protein surface and decrease in the protein-urea interactions in the mixture. This synergistic behavior might be the prime reason for the counteraction of denaturing effect of urea by GB.

LiGRO: a graphical user interface for protein-ligand molecular dynamics.

PubMed

Kagami, Luciano Porto; das Neves, Gustavo Machado; da Silva, Alan Wilter Sousa; Caceres, Rafael Andrade; Kawano, Daniel Fábio; Eifler-Lima, Vera Lucia

2017-10-04

To speed up the drug-discovery process, molecular dynamics (MD) calculations performed in GROMACS can be coupled to docking simulations for the post-screening analyses of large compound libraries. This requires generating the topology of the ligands in different software, some basic knowledge of Linux command lines, and a certain familiarity in handling the output files. LiGRO-the python-based graphical interface introduced here-was designed to overcome these protein-ligand parameterization challenges by allowing the graphical (non command line-based) control of GROMACS (MD and analysis), ACPYPE (ligand topology builder) and PLIP (protein-binder interactions monitor)-programs that can be used together to fully perform and analyze the outputs of complex MD simulations (including energy minimization and NVT/NPT equilibration). By allowing the calculation of linear interaction energies in a simple and quick fashion, LiGRO can be used in the drug-discovery pipeline to select compounds with a better protein-binding interaction profile. The design of LiGRO allows researchers to freely download and modify the software, with the source code being available under the terms of a GPLv3 license from http://www.ufrgs.br/lasomfarmacia/ligro/ .

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa

PubMed Central

Wulf, Alexander; Campagne, Sébastien; Pei, Xue-Yuan; Forlani, Giada; Prindl, Konstantin; Abdou, Laetitia; Resch, Armin; Allain, Frederic H -T; Luisi, Ben F; Urlaub, Henning

2018-01-01

Abstract In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources. PMID:29244160

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa.

PubMed

Sonnleitner, Elisabeth; Wulf, Alexander; Campagne, Sébastien; Pei, Xue-Yuan; Wolfinger, Michael T; Forlani, Giada; Prindl, Konstantin; Abdou, Laetitia; Resch, Armin; Allain, Frederic H-T; Luisi, Ben F; Urlaub, Henning; Bläsi, Udo

2018-02-16

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

PubMed Central

Müller, G; Zimmermann, R

1987-01-01

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2820722

Conformational dynamics of proanthocyanidins: physical and computational approaches

Treesearch

Fred L. Tobiason; Richard W. Hemingway; T. Hatano

1998-01-01

The interaction of plant polyphenols with proteins accounts for a good part of their commercial (e.g., leather manufacture) and biological (e.g., antimicrobial activity) significance. The interplay between observations of physical data such as crystal structure, NMR analyses, and time-resolved fluorescence with results of computational chemistry approaches has been...

Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

PubMed

Sarkar, Mitul; Koland, John G

2016-01-01

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

Necdin interacts with the Msx2 homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells.

PubMed

Kuwajima, Takaaki; Taniura, Hideo; Nishimura, Isao; Yoshikawa, Kazuaki

2004-09-24

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

Structural basis of light chain amyloidogenicity: comparison of the thermodynamic properties, fibrillogenic potential and tertiary structural features of four vλ6 proteins

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

Wall, J.S.; Gupta, V.; Wilkerson, M.

2004-04-01

Primary (AL) amyloidosis results from the pathologic deposition of monoclonal light chains as amyloid fibrils. Studies of recombinant-derived variable region (V{sub L}) fragments of these proteins have shown an inverse relationship between thermodynamic stability and fibrillogenic potential. Further, ionic interactions within the V{sub L} domain were predicted to influence the kinetics of light chain fibrillogenicity, as evidenced from our analyses of a relatively stable V{sub {lambda}}6 protein (Jto) with a long range electrostatic interaction between Asp and Arg side chains at position 29 and 68, respectively, and an unstable, highly fibrillogenic V{sub {lambda}}6 protein (Wil) that had neutral amino acidsmore » at these locations. To test this hypothesis, we have generated two Jto-related mutants designed to disrupt the interaction between Asp 29 and Arg 68 (JtoD29A and JtoR68S). Although the thermodynamic stabilities of unfolding for these two molecules were identical, they exhibited very different kinetics of fibril formation: the rate of JtoD29A fibrillogenesis was slow and comparable to the parent molecule, whereas that of JtoR68S was significantly faster. High-resolution X-ray diffraction analyses of crystals prepared from the two mutants having the same space group and unit cell dimensions revealed no significant main-chain conformational changes. However, several notable side-chain alterations were observed in JtoR68S, as compared with JtoD29A, that resulted in the solvent exposure of a greater hydrophobic surface and modifications in the electrostatic potential surface. We posit that these differences contributed to the enhanced fibrillogenic potential of the Arg 68 mutant, since both Jto mutants lacked the intrachain ionic interaction and were equivalently unstable. The information gleaned from our studies has provided insight into structural parameters that in addition to overall thermodynamic stability, contribute to the fibril forming propensity of immunoglobulin light chains.« less

RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery

PubMed Central

Fontrodona, Laura; Porta-de-la-Riva, Montserrat; Morán, Tomás; Niu, Wei; Díaz, Mònica; Aristizábal-Corrales, David; Villanueva, Alberto; Schwartz, Simó; Reinke, Valerie; Cerón, Julián

2013-01-01

Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development. PMID:23754964

Integrated Analyses of Gene Expression Profiles Digs out Common Markers for Rheumatic Diseases

PubMed Central

Wang, Lan; Wu, Long-Fei; Lu, Xin; Mo, Xing-Bo; Tang, Zai-Xiang; Lei, Shu-Feng; Deng, Fei-Yan

2015-01-01

Objective Rheumatic diseases have some common symptoms. Extensive gene expression studies, accumulated thus far, have successfully identified signature molecules for each rheumatic disease, individually. However, whether there exist shared factors across rheumatic diseases has yet to be tested. Methods We collected and utilized 6 public microarray datasets covering 4 types of representative rheumatic diseases including rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, and osteoarthritis. Then we detected overlaps of differentially expressed genes across datasets and performed a meta-analysis aiming at identifying common differentially expressed genes that discriminate between pathological cases and normal controls. To further gain insights into the functions of the identified common differentially expressed genes, we conducted gene ontology enrichment analysis and protein-protein interaction analysis. Results We identified a total of eight differentially expressed genes (TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, PRF1), each associated with at least 3 of the 4 studied rheumatic diseases. Meta-analysis warranted the significance of the eight genes and highlighted the general significance of four genes (CX3CR1, LY96, TLR5, and PRF1). Protein-protein interaction and gene ontology enrichment analyses indicated that the eight genes interact with each other to exert functions related to immune response and immune regulation. Conclusion The findings support that there exist common factors underlying rheumatic diseases. For rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and osteoarthritis diseases, those common factors include TNFSF10, CX3CR1, LY96, TLR5, TXN, TIA1, PRKCH, and PRF1. In-depth studies on these common factors may provide keys to understanding the pathogenesis and developing intervention strategies for rheumatic diseases. PMID:26352601

Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis.

PubMed

Liu, Long; Tian, Jiao; Nan, Hao; Tian, Mengmeng; Li, Yuan; Xu, Xiaodong; Huang, Baicheng; Zhou, Enmin; Hiscox, Julian A; Chen, Hongying

2016-06-01

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis. It is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

MinCD cell division proteins form alternating co-polymeric cytomotive filaments

PubMed Central

Ghosal, Debnath; Trambaiolo, Daniel; Amos, Linda A.; Löwe, Jan

2014-01-01

Summary During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell. Here, using X-ray crystallography, electron microscopy and in vivo analyses we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments A non-polymerising mutation in MinD causes aberrant cell division in E. coli. MinCD copolymers bind to membrane, interact with FtsZ, and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers. PMID:25500731

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

NASA Astrophysics Data System (ADS)

Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

2015-07-01

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

Preservation of protein clefts in comparative models.

PubMed

Piedra, David; Lois, Sergi; de la Cruz, Xavier

2008-01-16

Comparative, or homology, modelling of protein structures is the most widely used prediction method when the target protein has homologues of known structure. Given that the quality of a model may vary greatly, several studies have been devoted to identifying the factors that influence modelling results. These studies usually consider the protein as a whole, and only a few provide a separate discussion of the behaviour of biologically relevant features of the protein. Given the value of the latter for many applications, here we extended previous work by analysing the preservation of native protein clefts in homology models. We chose to examine clefts because of their role in protein function/structure, as they are usually the locus of protein-protein interactions, host the enzymes' active site, or, in the case of protein domains, can also be the locus of domain-domain interactions that lead to the structure of the whole protein. We studied how the largest cleft of a protein varies in comparative models. To this end, we analysed a set of 53507 homology models that cover the whole sequence identity range, with a special emphasis on medium and low similarities. More precisely we examined how cleft quality - measured using six complementary parameters related to both global shape and local atomic environment, depends on the sequence identity between target and template proteins. In addition to this general analysis, we also explored the impact of a number of factors on cleft quality, and found that the relationship between quality and sequence identity varies depending on cleft rank amongst the set of protein clefts (when ordered according to size), and number of aligned residues. We have examined cleft quality in homology models at a range of seq.id. levels. Our results provide a detailed view of how quality is affected by distinct parameters and thus may help the user of comparative modelling to determine the final quality and applicability of his/her cleft models. In addition, the large variability in model quality that we observed within each sequence bin, with good models present even at low sequence identities (between 20% and 30%), indicates that properly developed identification methods could be used to recover good cleft models in this sequence range.

A Missense Mutation in the Aggrecan C-type Lectin Domain Disrupts Extracellular Matrix Interactions and Causes Dominant Familial Osteochondritis Dissecans

PubMed Central

Stattin, Eva-Lena; Wiklund, Fredrik; Lindblom, Karin; Önnerfjord, Patrik; Jonsson, Björn-Anders; Tegner, Yelverton; Sasaki, Takako; Struglics, André; Lohmander, Stefan; Dahl, Niklas; Heinegård, Dick; Aspberg, Anders

2010-01-01

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo. PMID:20137779

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

PubMed

Maerker, Tina; van Wijk, Erwin; Overlack, Nora; Kersten, Ferry F J; McGee, Joann; Goldmann, Tobias; Sehn, Elisabeth; Roepman, Ronald; Walsh, Edward J; Kremer, Hannie; Wolfrum, Uwe

2008-01-01

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.

An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B.

PubMed

Rallapalli, P M; Kemball-Cook, G; Tuddenham, E G; Gomez, K; Perkins, S J

2013-07-01

Factor IX (FIX) is important in the coagulation cascade, being activated to FIXa on cleavage. Defects in the human F9 gene frequently lead to hemophilia B. To assess 1113 unique F9 mutations corresponding to 3721 patient entries in a new and up-to-date interactive web database alongside the FIXa protein structure. The mutations database was built using MySQL and structural analyses were based on a homology model for the human FIXa structure based on closely-related crystal structures. Mutations have been found in 336 (73%) out of 461 residues in FIX. There were 812 unique point mutations, 182 deletions, 54 polymorphisms, 39 insertions and 26 others that together comprise a total of 1113 unique variants. The 64 unique mild severity mutations in the mature protein with known circulating protein phenotypes include 15 (23%) quantitative type I mutations and 41 (64%) predominantly qualitative type II mutations. Inhibitors were described in 59 reports (1.6%) corresponding to 25 unique mutations. The interactive database provides insights into mechanisms of hemophilia B. Type II mutations are deduced to disrupt predominantly those structural regions involved with functional interactions. The interactive features of the database will assist in making judgments about patient management. © 2013 International Society on Thrombosis and Haemostasis.

Interaction of lipids with the neurotensin receptor 1.

PubMed

Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

2016-06-01

Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs. Copyright © 2016 Elsevier B.V. All rights reserved.

HomPPI: a class of sequence homology based protein-protein interface prediction methods

PubMed Central

2011-01-01

Background Although homology-based methods are among the most widely used methods for predicting the structure and function of proteins, the question as to whether interface sequence conservation can be effectively exploited in predicting protein-protein interfaces has been a subject of debate. Results We studied more than 300,000 pair-wise alignments of protein sequences from structurally characterized protein complexes, including both obligate and transient complexes. We identified sequence similarity criteria required for accurate homology-based inference of interface residues in a query protein sequence. Based on these analyses, we developed HomPPI, a class of sequence homology-based methods for predicting protein-protein interface residues. We present two variants of HomPPI: (i) NPS-HomPPI (Non partner-specific HomPPI), which can be used to predict interface residues of a query protein in the absence of knowledge of the interaction partner; and (ii) PS-HomPPI (Partner-specific HomPPI), which can be used to predict the interface residues of a query protein with a specific target protein. Our experiments on a benchmark dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably predict protein-protein interface residues in a given protein, with an average correlation coefficient (CC) of 0.76, sensitivity of 0.83, and specificity of 0.78, when sequence homologs of the query protein can be reliably identified. NPS-HomPPI also reliably predicts the interface residues of intrinsically disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with several state-of-the-art interface prediction servers including those that exploit the structure of the query proteins. The partner-specific classifier, PS-HomPPI can, on a large dataset of transient complexes, predict the interface residues of a query protein with a specific target, with a CC of 0.65, sensitivity of 0.69, and specificity of 0.70, when homologs of both the query and the target can be reliably identified. The HomPPI web server is available at http://homppi.cs.iastate.edu/. Conclusions Sequence homology-based methods offer a class of computationally efficient and reliable approaches for predicting the protein-protein interface residues that participate in either obligate or transient interactions. For query proteins involved in transient interactions, the reliability of interface residue prediction can be improved by exploiting knowledge of putative interaction partners. PMID:21682895

Structure and sequence analyses of Bacteroides proteins BVU_4064 and BF1687 reveal presence of two novel predominantly-beta domains, predicted to be involved in lipid and cell surface interactions

DOE PAGES

Natarajan, Padmaja; Punta, Marco; Kumar, Abhinav; ...

2015-01-16

N-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.

Sulphur Atoms from Methionines Interacting with Aromatic Residues Are Less Prone to Oxidation

PubMed Central

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

2015-01-01

Methionine residues exhibit different degrees of susceptibility to oxidation. Although solvent accessibility is a relevant factor, oxidation at particular sites cannot be unequivocally explained by accessibility alone. To explore other possible structural determinants, we assembled different sets of oxidation-sensitive and oxidation-resistant methionines contained in human proteins. Comparisons of the proteins containing oxidized methionines with all proteins in the human proteome led to the conclusion that the former exhibit a significantly higher mean value of methionine content than the latter. Within a given protein, an examination of the sequence surrounding the non-oxidized methionine revealed a preference for neighbouring tyrosine and tryptophan residues, but not for phenylalanine residues. However, because the interaction between sulphur atoms and aromatic residues has been reported to be important for the stabilization of protein structure, we carried out an analysis of the spatial interatomic distances between methionines and aromatic residues, including phenylalanine. The results of these analyses uncovered a new determinant for methionine oxidation: the S-aromatic motif, which decreases the reactivity of the involved sulphur towards oxidants. PMID:26597773

Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev.

PubMed

Banerjee, Atoshi; Benjamin, Ronald; Balakrishnan, Kannan; Ghosh, Payel; Banerjee, Sharmistha

2014-02-13

The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell. In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines. With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

Ethanol Influences on Bax Associations with Mitochondrial Membrane Proteins in Neonatal Rat Cerebellum

PubMed Central

Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

2012-01-01

These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure, and focused on interactions between pro-apoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC), and adenine nucleotide translocator (ANT), of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that following ethanol exposure, Bax pro-apoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least two hours post-exposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment, and found such interactions were altered by ethanol treatment, but only at two-hours post-exposure, and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax, but not by a Bax channel blocker. Therefore, we conclude that at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex, and not channel formation independent of PTP constituents. PMID:22767450

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ

PubMed Central

Di Matteo, A; Franceschini, M; Paiardini, A; Grottesi, A; Chiarella, S; Rocchio, S; Di Natale, C; Marasco, D; Vitagliano, L; Travaglini-Allocatelli, C; Federici, L

2017-01-01

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment. PMID:28920929

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

PubMed

Kachhap, Sangita; Singh, Balvinder

2015-01-01

In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

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

Evolution of hormone signaling in elasmobranchs by exploitation of promiscuous receptors.

PubMed

Carroll, Sean Michael; Bridgham, Jamie T; Thornton, Joseph W

2008-12-01

Specific interactions among proteins, nucleic acids, and metabolites drive virtually all cellular functions and underlie phenotypic complexity and diversity. Despite the fundamental importance of interactions, the mechanisms and dynamics by which they evolve are poorly understood. Here we describe novel interactions between a lineage-specific hormone and its receptors in elasmobranchs, a subclass of cartilaginous fishes, and infer how these associations evolved using phylogenetic and protein structural analyses. The hormone 1alpha-hydroxycorticosterone (1alpha-B) is a physiologically important steroid synthesized only in elasmobranchs. We show that 1alpha-B modulates gene expression in vitro by activating two paralogous intracellular transcription factors, the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the little skate Leucoraja erinacea; MR serves as a high-sensitivity and GR as a low-sensitivity receptor. Using functional analysis of extant and resurrected ancestral proteins, we show that receptor sensitivity to 1alpha-B evolved millions of years before the hormone itself evolved. The 1alpha-B differs from more ancient corticosteroids only by the addition of a hydroxyl group; the three-dimensional structure of the ancestral receptor shows that the ligand pocket contained ample unoccupied space to accommodate this moiety. Our findings indicate that the interactions between 1alpha-B and elasmobranch GR and MR proteins evolved by molecular exploitation: a novel hormone recruited into new functional partnerships two ancient receptors that had previously interacted with other ligands. The ancestral receptor's promiscuous capacity to fortuitously bind compounds that are slight structural variants of its original ligands set the stage for the evolution of this new interaction.

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

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

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions.

PubMed

Glover, Karen; Mei, Yang; Sinha, Sangita C

2016-10-01

Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions. Copyright © 2016 Elsevier B.V. All rights reserved.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

PubMed Central

Guo, B; Odgren, P R; van Wijnen, A J; Last, T J; Nickerson, J; Penman, S; Lian, J B; Stein, J L; Stein, G S

1995-01-01

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479833

Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

PubMed

Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

2017-12-01

Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

Functional regulation of Q by microRNA172 and transcriptional co-repressor TOPLESS in controlling bread wheat spikelet density.

PubMed

Liu, Pan; Liu, Jie; Dong, Huixue; Sun, Jiaqiang

2018-02-01

Bread wheat (Triticum aestivum) spike architecture is an important agronomic trait. The Q gene plays a key role in the domestication of bread wheat spike architecture. However, the regulatory mechanisms of Q expression and transcriptional activity remain largely unknown. In this study, we show that overexpression of bread wheat tae-miR172 caused a speltoid-like spike phenotype, reminiscent of that in wheat plants with the q gene. The reduction in Q transcript levels in the tae-miR172 overexpression transgenic bread wheat lines suggests that the Q expression can be suppressed by tae-miR172 in bread wheat. Indeed, our RACE analyses confirmed that the Q mRNA is targeted by tae-miR172 for cleavage. According to our analyses, the Q protein is localized in nucleus and confers transcriptional repression activity. Meanwhile, the Q protein could physically interact with the bread wheat transcriptional co-repressor TOPLESS (TaTPL). Specifically, the N-terminal ethylene-responsive element binding factor-associated amphiphilic repression (EAR) (LDLNVE) motif but not the C-terminal EAR (LDLDLR) motif of Q protein mediates its interaction with the CTLH motif of TaTPL. Moreover, we show that the N-terminal EAR motif of Q protein is also essentially required for the transcriptional repression activity of Q protein. Taken together, we reveal the functional regulation of Q protein by tae-miR172 and transcriptional co-repressor TaTPL in controlling the bread wheat spike architecture. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

P-MartCancer: A New Online Platform to Access CPTAC Datasets and Enable New Analyses | Office of Cancer Clinical Proteomics Research

Cancer.gov

The November 1, 2017 issue of Cancer Research is dedicated to a collection of computational resource papers in genomics, proteomics, animal models, imaging, and clinical subjects for non-bioinformaticists looking to incorporate computing tools into their work. Scientists at Pacific Northwest National Laboratory have developed P-MartCancer, an open, web-based interactive software tool that enables statistical analyses of peptide or protein data generated from mass-spectrometry (MS)-based global proteomics experiments.

Identification of key genes and pathways associated with neuropathic pain in uninjured dorsal root ganglion by using bioinformatic analysis.

PubMed

Chen, Chao-Jin; Liu, De-Zhao; Yao, Wei-Feng; Gu, Yu; Huang, Fei; Hei, Zi-Qing; Li, Xiang

2017-01-01

Neuropathic pain is a complex chronic condition occurring post-nervous system damage. The transcriptional reprogramming of injured dorsal root ganglia (DRGs) drives neuropathic pain. However, few comparative analyses using high-throughput platforms have investigated uninjured DRG in neuropathic pain, and potential interactions among differentially expressed genes (DEGs) and pathways were not taken into consideration. The aim of this study was to identify changes in genes and pathways associated with neuropathic pain in uninjured L4 DRG after L5 spinal nerve ligation (SNL) by using bioinformatic analysis. The microarray profile GSE24982 was downloaded from the Gene Expression Omnibus database to identify DEGs between DRGs in SNL and sham rats. The prioritization for these DEGs was performed using the Toppgene database followed by gene ontology and pathway enrichment analyses. The relationships among DEGs from the protein interactive perspective were analyzed using protein-protein interaction (PPI) network and module analysis. Real-time polymerase chain reaction (PCR) and Western blotting were used to confirm the expression of DEGs in the rodent neuropathic pain model. A total of 206 DEGs that might play a role in neuropathic pain were identified in L4 DRG, of which 75 were upregulated and 131 were downregulated. The upregulated DEGs were enriched in biological processes related to transcription regulation and molecular functions such as DNA binding, cell cycle, and the FoxO signaling pathway. Ctnnb1 protein had the highest connectivity degrees in the PPI network. The in vivo studies also validated that mRNA and protein levels of Ctnnb1 were upregulated in both L4 and L5 DRGs. This study provides insight into the functional gene sets and pathways associated with neuropathic pain in L4 uninjured DRG after L5 SNL, which might promote our understanding of the molecular mechanisms underlying the development of neuropathic pain.

Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 1 Nef Proteins Show Distinct Patterns and Mechanisms of Src Kinase Activation

PubMed Central

Greenway, Alison L.; Dutartre, Hélène; Allen, Kelly; McPhee, Dale A.; Olive, Daniel; Collette, Yves

1999-01-01

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases. PMID:10364375

Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions

PubMed Central

Morris, John H; Knudsen, Giselle M; Verschueren, Erik; Johnson, Jeffrey R; Cimermancic, Peter; Greninger, Alexander L; Pico, Alexander R

2015-01-01

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification–mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography–MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one’s familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2–4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one’s data and find the right questions. PMID:25275790

Leupaxin stimulates adhesion and migration of prostate cancer cells through modulation of the phosphorylation status of the actin-binding protein caldesmon

PubMed Central

Schmidt, Thomas; Bremmer, Felix; Burfeind, Peter; Kaulfuß, Silke

2015-01-01

The focal adhesion protein leupaxin (LPXN) is overexpressed in a subset of prostate cancers (PCa) and is involved in the progression of PCa. In the present study, we analyzed the LPXN-mediated adhesive and cytoskeletal changes during PCa progression. We identified an interaction between the actin-binding protein caldesmon (CaD) and LPXN and this interaction is increased during PCa cell migration. Furthermore, knockdown of LPXN did not affect CaD expression but reduced CaD phosphorylation. This is known to destabilize the affinity of CaD to F-actin, leading to dynamic cell structures that enable cell motility. Thus, downregulation of CaD increased migration and invasion of PCa cells. To identify the kinase responsible for the LPXN-mediated phosphorylation of CaD, we used data from an antibody array, which showed decreased expression of TGF-beta-activated kinase 1 (TAK1) after LPXN knockdown in PC-3 PCa cells. Subsequent analyses of the downstream kinases revealed the extracellular signal-regulated kinase (ERK) as an interaction partner of LPXN that facilitates CaD phosphorylation during LPXN-mediated PCa cell migration. In conclusion, we demonstrate that LPXN directly influences cytoskeletal dynamics via interaction with the actin-binding protein CaD and regulates CaD phosphorylation by recruiting ERK to highly dynamic structures within PCa cells. PMID:26079947

A Role for USP7 in DNA Replication

PubMed Central

Jagannathan, Madhav; Nguyen, Tin; Gallo, David; Luthra, Niharika; Brown, Grant W.; Saridakis, Vivian

2014-01-01

The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7–MCM-BP interaction showed that the 155PSTS158 MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase. PMID:24190967

Unifying measures of gene function and evolution.

PubMed

Wolf, Yuri I; Carmel, Liran; Koonin, Eugene V

2006-06-22

Recent genome analyses revealed intriguing correlations between variables characterizing the functioning of a gene, such as expression level (EL), connectivity of genetic and protein-protein interaction networks, and knockout effect, and variables describing gene evolution, such as sequence evolution rate (ER) and propensity for gene loss. Typically, variables within each of these classes are positively correlated, e.g. products of highly expressed genes also have a propensity to be involved in many protein-protein interactions, whereas variables between classes are negatively correlated, e.g. highly expressed genes, on average, evolve slower than weakly expressed genes. Here, we describe principal component (PC) analysis of seven genome-related variables and propose biological interpretations for the first three PCs. The first PC reflects a gene's 'importance', or the 'status' of a gene in the genomic community, with positive contributions from knockout lethality, EL, number of protein-protein interaction partners and the number of paralogues, and negative contributions from sequence ER and gene loss propensity. The next two PCs define a plane that seems to reflect the functional and evolutionary plasticity of a gene. Specifically, PC2 can be interpreted as a gene's 'adaptability' whereby genes with high adaptability readily duplicate, have many genetic interaction partners and tend to be non-essential. PC3 also might reflect the role of a gene in organismal adaptation albeit with a negative rather than a positive contribution of genetic interactions; we provisionally designate this PC 'reactivity'. The interpretation of PC2 and PC3 as measures of a gene's plasticity is compatible with the observation that genes with high values of these PCs tend to be expressed in a condition- or tissue-specific manner. Functional classes of genes substantially vary in status, adaptability and reactivity, with the highest status characteristic of the translation system and cytoskeletal proteins, highest adaptability seen in cellular processes and signalling genes, and top reactivity characteristic of metabolic enzymes.

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

PubMed Central

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P.; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis. PMID:28401063

Functional and genomic analyses of alpha-solenoid proteins.

PubMed

Fournier, David; Palidwor, Gareth A; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A

2013-01-01

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.

Evaluation of genetic and metabolic role of SKIP11 in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Hassan, Muhammad Naeem ul; Ismail, Ismanizan

2015-09-01

Most of the regulatory proteins are degraded by 26S proteasome complex, only when they are tagged by Ubiquitin. A complex of four proteins, SKP1-Cullin-Ring box-F box (SCF) catalyses the final step to link the Ubiquitin tag with the target proteins. SCF complex interacts with the target proteins through F-box proteins, which confer the overall substrate specificity to the complex. F-box proteins, one of the largest family of proteins in plants have an N-terminal F-box domain and variable C-terminal domains, like leucine-rich repeat, WD-40 repeat and the kelch-repeat domains. In this study, we analysed the role of SKIP11, a kelch containing F-box protein (KFB) from Arabidopsis thaliana, by using reverse genetics strategy. The results show that SKIP11 is involved in the down-regulation of oxylipin pathway, possibly through the degradation of enzymes or/ and the regulatory factors of the pathway.

Molecular responses of genetically modified maize to abiotic stresses as determined through proteomic and metabolomic analyses

PubMed Central

Benevenuto, Rafael Fonseca; Agapito-Tenfen, Sarah Zanon; Vilperte, Vinicius; Wikmark, Odd-Gunnar; van Rensburg, Peet Jansen; Nodari, Rubens Onofre

2017-01-01

Some genetically modified (GM) plants have transgenes that confer tolerance to abiotic stressors. Meanwhile, other transgenes may interact with abiotic stressors, causing pleiotropic effects that will affect the plant physiology. Thus, physiological alteration might have an impact on the product safety. However, routine risk assessment (RA) analyses do not evaluate the response of GM plants exposed to different environmental conditions. Therefore, we here present a proteome profile of herbicide-tolerant maize, including the levels of phytohormones and related compounds, compared to its near-isogenic non-GM variety under drought and herbicide stresses. Twenty differentially abundant proteins were detected between GM and non-GM hybrids under different water deficiency conditions and herbicide sprays. Pathway enrichment analysis showed that most of these proteins are assigned to energetic/carbohydrate metabolic processes. Among phytohormones and related compounds, different levels of ABA, CA, JA, MeJA and SA were detected in the maize varieties and stress conditions analysed. In pathway and proteome analyses, environment was found to be the major source of variation followed by the genetic transformation factor. Nonetheless, differences were detected in the levels of JA, MeJA and CA and in the abundance of 11 proteins when comparing the GM plant and its non-GM near-isogenic variety under the same environmental conditions. Thus, these findings do support molecular studies in GM plants Risk Assessment analyses. PMID:28245233

Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

NASA Astrophysics Data System (ADS)

Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

PubMed

Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

2016-04-01

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. © 2016 Authors; published by Portland Press Limited.

Competing endogenous RNA and interactome bioinformatic analyses on human telomerase.

PubMed

Arancio, Walter; Pizzolanti, Giuseppe; Genovese, Swonild Ilenia; Baiamonte, Concetta; Giordano, Carla

2014-04-01

We present a classic interactome bioinformatic analysis and a study on competing endogenous (ce) RNAs for hTERT. The hTERT gene codes for the catalytic subunit and limiting component of the human telomerase complex. Human telomerase reverse transcriptase (hTERT) is essential for the integrity of telomeres. Telomere dysfunctions have been widely reported to be involved in aging, cancer, and cellular senescence. The hTERT gene network has been analyzed using the BioGRID interaction database (http://thebiogrid.org/) and related analysis tools such as Osprey (http://biodata.mshri.on.ca/osprey/servlet/Index) and GeneMANIA (http://genemania.org/). The network of interaction of hTERT transcripts has been further analyzed following the competing endogenous (ce) RNA hypotheses (messenger [m] RNAs cross-talk via micro [mi] RNAs) using the miRWalk database and tools (www.ma.uni-heidelberg.de/apps/zmf/mirwalk/). These analyses suggest a role for Akt, nuclear factor-κB (NF-κB), heat shock protein 90 (HSP90), p70/p80 autoantigen, 14-3-3 proteins, and dynein in telomere functions. Roles for histone acetylation/deacetylation and proteoglycan metabolism are also proposed.

ExAtlas: An interactive online tool for meta-analysis of gene expression data.

PubMed

Sharov, Alexei A; Schlessinger, David; Ko, Minoru S H

2015-12-01

We have developed ExAtlas, an on-line software tool for meta-analysis and visualization of gene expression data. In contrast to existing software tools, ExAtlas compares multi-component data sets and generates results for all combinations (e.g. all gene expression profiles versus all Gene Ontology annotations). ExAtlas handles both users' own data and data extracted semi-automatically from the public repository (GEO/NCBI database). ExAtlas provides a variety of tools for meta-analyses: (1) standard meta-analysis (fixed effects, random effects, z-score, and Fisher's methods); (2) analyses of global correlations between gene expression data sets; (3) gene set enrichment; (4) gene set overlap; (5) gene association by expression profile; (6) gene specificity; and (7) statistical analysis (ANOVA, pairwise comparison, and PCA). ExAtlas produces graphical outputs, including heatmaps, scatter-plots, bar-charts, and three-dimensional images. Some of the most widely used public data sets (e.g. GNF/BioGPS, Gene Ontology, KEGG, GAD phenotypes, BrainScan, ENCODE ChIP-seq, and protein-protein interaction) are pre-loaded and can be used for functional annotations.

A network biology approach to understanding the importance of chameleon proteins in human physiology and pathology.

PubMed

Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Marashi, Sayed-Amir

2017-02-01

Chameleon proteins are proteins which include sequences that can adopt α-helix-β-strand (HE-chameleon) or α-helix-coil (HC-chameleon) or β-strand-coil (CE-chameleon) structures to operate their crucial biological functions. In this study, using a network-based approach, we examined the chameleon proteins to give a better knowledge on these proteins. We focused on proteins with identical chameleon sequences with more than or equal to seven residues long in different PDB entries, which adopt HE-chameleon, HC-chameleon, and CE-chameleon structures in the same protein. One hundred and ninety-one human chameleon proteins were identified via our in-house program. Then, protein-protein interaction (PPI) networks, Gene ontology (GO) enrichment, disease network, and pathway enrichment analyses were performed for our derived data set. We discovered that there are chameleon sequences which reside in protein-protein interaction regions between two proteins critical for their dual function. Analysis of the PPI networks for chameleon proteins introduced five hub proteins, namely TP53, EGFR, HSP90AA1, PPARA, and HIF1A, which were presented in four PPI clusters. The outcomes demonstrate that the chameleon regions are in critical domains of these proteins and are important in the development and treatment of human cancers. The present report is the first network-based functional study of chameleon proteins using computational approaches and might provide a new perspective for understanding the mechanisms of diseases helping us in developing new medical therapies along with discovering new proteins with chameleon properties which are highly important in cancer.

The roles of autophagy and hypoxia in human inflammatory periapical lesions.

PubMed

Huang, H Y; Wang, W C; Lin, P Y; Huang, C P; Chen, C Y; Chen, Y K

2018-02-01

To determine the expressions of hypoxia-related [hypoxia-inducible transcription factors (HIF)-1α, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and phospho-adenosine monophosphate activated protein kinase (pAMPK)] and autophagy-related [microtubule-associated protein 1 light chain 3 (LC3), beclin-1 (BECN-1), autophagy-related gene (Atg)5-12, and p62] proteins in human inflammatory periapical lesions. Fifteen samples of radicular cysts (RCs) and 21 periapical granulomas (PGs), combined with 17 healthy dental pulp tissues, were examined. Enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β cytokine; immunohistochemical (IHC) and Western blot (WB) analyses were employed to examine autophagy-related and hypoxia-related proteins. Transmission electron microscopy (TEM) was used to explore the ultrastructural morphology of autophagy in periapical lesions. Nonparametric Kruskal-Wallis tests and Mann-Whitney U-tests were used for statistical analyses. ELISA revealed a significantly higher (P < 0.001) IL-1β expression in periapical lesions than in normal pulp tissue. Immunoscores of IHC expressions of pAMPK, HIF-1α, BNIP3, BECN-1 and Atg5-12 proteins in periapical lesions were significantly higher (P < 0.001) (except BECN-1) than those in normal pulp tissue. The results of IHC studies were largely compatible with those of WB analyses, where significantly higher (P < 0.05) expressions of hypoxia-related and autophagy-related proteins (except BECN-1, p62 and LC3II in WB analyses) in periapical lesions were noted as compared to normal pulp tissue. Upon TEM, ultrastructural double-membrane autophagosomes and autolysosomes were observed in PGs and RCs. Autophagy associated with hypoxia may play a potential causative role in the development and maintenance of inflamed periapical lesions. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Conserved Domains in the Transformer Protein Act Complementary to Regulate Sex-Specific Splicing of Its Own Pre-mRNA.

PubMed

Tanaka, Arisa; Aoki, Fugaku; Suzuki, Masataka G

2018-05-26

The transformer (tra) gene, which is a female-determining master gene in the housefly Musca domestica, acts as a memory device for sex determination via its auto-regulatory function, i.e., through the contribution of the TRA protein to female-specific splicing of its own pre-mRNA. The TRA protein contains 4 small domains that are specifically conserved among TRA proteins (domains 1-4). Domain 2, also named TRA-CAM domain, is the most conserved, but its function remains unknown. To examine whether these domains are involved in the auto-regulatory function, we performed in vitro splicing assays using a tra minigene containing a partial genomic sequence of the M. domestica tra (Mdtra) gene. Co-transfection of the Mdtra minigene and an MdTRA protein expression vector into cultured insect cells strongly induced female-specific splicing of the minigene. A series of deletion mutation analyses demonstrated that these domains act complementarily to induce female-specific splicing. Domain 1 and the TRA-CAM domain were necessary for the female-specific splicing when the MdTRA protein lacked both domains 3 and 4. In this situation, mutation of the well-conserved 3 amino acids (GEG) in the TRA-CAM domain significantly reduced the female-specific splicing activity of MdTRA. GST-pull down analyses demonstrated that the MdTRA protein specifically enriched on the male-specific exonic region (exon 2b), which contains the putative TRA/TRA-2 binding sites, and that the GEG mutation disrupts this enrichment. Since the MdTRA protein interacts with its own pre-mRNA through TRA-2, our findings suggest that the conserved amino acid residues in the TRA-CAM domain may be crucial for the interaction between MdTRA and TRA-2, enhancing MdTRA recruitment on its pre-mRNA to induce female-specific splicing of tra in the housefly. © 2018 S. Karger AG, Basel.

New insights into the interplay between the lysine transporter LysP and the pH sensor CadC in Escherichia coli.

PubMed

Rauschmeier, Martina; Schüppel, Valentina; Tetsch, Larissa; Jung, Kirsten

2014-01-09

The coordination of signal transduction and substrate transport represents a sophisticated way to integrate information on metabolite fluxes into transcriptional regulation. This widely distributed process involves protein-protein interactions between two integral membrane proteins. Here we report new insights into the molecular mechanism of the regulatory interplay between the lysine-specific permease LysP and the membrane-integrated pH sensor CadC, which together induce lysine-dependent adaptation of E. coli under acidic stress. In vivo analyses revealed that, in the absence of either stimulus, the two proteins form a stable association, which is modulated by lysine and low pH. In addition to its transmembrane helix, the periplasmic domain of CadC also participated in the interaction. Site-directed mutagenesis pinpointed Arg265 and Arg268 in CadC as well as Asp275 and Asp278 in LysP as potential periplasmic interaction sites. Moreover, a systematic analysis of 100 LysP variants with single-site replacements indicated that the lysine signal is transduced from co-sensor to sensor via lysine-dependent conformational changes (upon substrate binding and/or transport) of LysP. Our results suggest a scenario in which CadC is inhibited by LysP via intramembrane and periplasmic contacts under non-inducing conditions. Upon induction, lysine-dependent conformational changes in LysP transduce the lysine signal via a direct conformational coupling to CadC without resolving the interaction completely. Moreover, concomitant pH-dependent protonation of periplasmic amino acids in both proteins dissolves their electrostatic connections resulting in further destabilization of the CadC/LysP interaction. © 2013.

The Dual Estrogen Receptor α Inhibitory Effects of the Tissue-Selective Estrogen Complex for Endometrial and Breast Safety

PubMed Central

Han, Sang Jun; Begum, Khurshida; Foulds, Charles E.; Hamilton, Ross A.; Bailey, Suzanna; Malovannaya, Anna; Chan, Doug; Qin, Jun

2016-01-01

The conjugated estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) is designed to minimize the undesirable effects of estrogen in the uterus and breast tissues and to allow the beneficial effects of estrogen in other estrogen-target tissues, such as the bone and brain. However, the molecular mechanism underlying endometrial and breast safety during TSEC use is not fully understood. Estrogen receptor α (ERα)–estrogen response element (ERE)–DNA pull-down assays using HeLa nuclear extracts followed by mass spectrometry–immunoblotting analyses revealed that, upon TSEC treatment, ERα interacted with transcriptional repressors rather than coactivators. Therefore, the TSEC-mediated recruitment of transcriptional repressors suppresses ERα-mediated transcription in the breast and uterus. In addition, TSEC treatment also degraded ERα protein in uterine tissue and breast cancer cells, but not in bone cells. Interestingly, ERα-ERE-DNA pull-down assays also revealed that, upon TSEC treatment, ERα interacted with the F-box protein 45 (FBXO45) E3 ubiquitin ligase. The loss-of- and gain-of-FBXO45 function analyses indicated that FBXO45 is involved in TSEC-mediated degradation of the ERα protein in endometrial and breast cells. In preclinical studies, these synergistic effects of TSEC on ERα inhibition also suppressed the estrogen-dependent progression of endometriosis. Therefore, the endometrial and breast safety effects of TSEC are associated with synergy between the selective recruitment of transcriptional repressors to ERα and FBXO45-mediated degradation of the ERα protein. PMID:26487511

Three members of Medicago truncatula ST family (MtST4, MtST5 and MtST6) are specifically induced by hormones involved in biotic interactions.

PubMed

Albornos, Lucía; Martín, Ignacio; Hernández-Nistal, Josefina; Labrador, Emilia; Dopico, Berta

2018-06-01

In this work, we study the function of the Medicago truncatula ST4, ST5 and ST6 proteins that belong to a protein family of unknown function characterized by the DUF2775 domain. Thus, we analyse their promoter sequence and activity, their transcript accumulation, and their subcellular location. The analysis of the three promoters showed different combination of cis-acting regulatory elements and they presented different activity pattern. Throughout development only ST6 mRNAs have been detected in most of the stages analysed, while ST4 was faintly detected in the roots and in the flowers and ST5 was always absent. The addition of MeJA, ET and SA revealed specific responses of the STs, the ST4 transcript accumulation increased by MeJA; the ST5 by MeJA and ET when applied together; and the ST6 by ET and by SA. Finally, the ST4 and ST5 proteins were in the cell wall whereas the ST6 had a dual location. From these results, we can conclude that the ST4, ST5 and ST6 RNAs are specifically and differentially up-regulated by MeJA, ET and SA, plant regulators also involved in the plant defence, pointing that ST4, ST5 and ST6 proteins might be involved in specific biotic interactions through different signalling pathways. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Soybean TCP transcription factors: Evolution, classification, protein interaction and stress and hormone responsiveness.

PubMed

Feng, Zhi-Juan; Xu, Sheng-Chun; Liu, Na; Zhang, Gu-Wen; Hu, Qi-Zan; Gong, Ya-Ming

2018-06-01

TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, a family of plant-specific proteins, play crucial roles in plant growth and development and stress response. However, systematical information is unknown regarding the TCP gene family in soybean. In the present study, a total of 54 GmTCPs were identified in soybean, which were grouped into 11 groups with the typical TCP conserved domains. Phylogenetic relationship, protein motif and gene structure analyses distinguished the GmTCPs into two homology classes: Class I and Class II. Class II was then differentiated into two subclasses: CIN and CYC/TB1. Unique cis-element number and composition existed in the promoter regions which might be involved in the gene transcriptional regulation of different GmTCPs. Tissue expression analysis demonstrated the diverse spatiotemporal expression profiles of GmTCPs. Furthermore, the interaction protein of one previously functionally unknown TCP protein-GmTCP8 was investigated. Yeast two-hybrid assay showed the interaction between GmTCP8 and an abscisic acid receptor (GmPYL10). QRT-PCR assays indicated the distinct expression profiles of GmTCPs in response to abiotic stresses (heat, drought and salt) and stress-related signals (abscisic acid, brassinolide, salicylicacid and methyl jasmonate). These results will facilitate to uncover the possible roles of GmTCPs under abiotic stress and hormone signal responses in soybean. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions.

PubMed

Wang, Ludi; Clarke, Lisa A; Eason, Russell J; Parker, Christopher C; Qi, Baoxiu; Scott, Rod J; Doughty, James

2017-01-01

The establishment of pollen-pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen-stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen-stigma interaction. Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP-B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth. Our results revealed that pollen hydration is severely impaired when multiple PCP-Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied. These results demonstrate that AtPCP-Bs are key regulators of the hydration 'checkpoint' in establishment of pollen-stigma compatibility. In addition, we propose that interspecies diversity of PCP-Bs may contribute to reproductive barriers in the Brassicaceae. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature ▿ †

PubMed Central

Wei, Dahai; Zhang, Xiaobo

2010-01-01

The virus-host interaction is essential to understanding the role that viruses play in ecological and geochemical processes in deep-sea vent ecosystems. Virus-induced changes in cellular gene expression and host physiology have been studied extensively. However, the molecular mechanism of interaction between a bacteriophage and its host at high temperature remains poorly understood. In the present study, the virus-induced gene expression profile of Geobacillus sp. E263, a thermophile isolated from a deep-sea hydrothermal ecosystem, was characterized. Based on proteomic analysis and random arbitrarily primed PCR (RAP-PCR) of Geobacillus sp. E263 cultured under non-bacteriophage GVE2 infection and GVE2 infection conditions, there were two types of protein/gene profiles in response to GVE2 infection. Twenty differentially expressed genes and proteins were revealed that could be grouped into 3 different categories based on cellular function, suggesting a coordinated response to infection. These differentially expressed genes and proteins were further confirmed by Northern blot analysis. To characterize the host proteins in response to virus infection, aspartate aminotransferase (AST) was inactivated to construct the AST mutant of Geobacillus sp. E263. The results showed that the AST protein was essential in virus infection. Thus, transcriptional and proteomic analyses and functional analysis revealed previously unknown host responses to deep-sea thermophilic virus infection. PMID:20015994

Do Viruses Exchange Genes across Superkingdoms of Life?

PubMed

Malik, Shahana S; Azem-E-Zahra, Syeda; Kim, Kyung Mo; Caetano-Anollés, Gustavo; Nasir, Arshan

2017-01-01

Viruses can be classified into archaeoviruses, bacterioviruses, and eukaryoviruses according to the taxonomy of the infected host. The host-constrained perception of viruses implies preference of genetic exchange between viruses and cellular organisms of their host superkingdoms and viral origins from host cells either via escape or reduction. However, viruses frequently establish non-lytic interactions with organisms and endogenize into the genomes of bacterial endosymbionts that reside in eukaryotic cells. Such interactions create opportunities for genetic exchange between viruses and organisms of non-host superkingdoms. Here, we take an atypical approach to revisit virus-cell interactions by first identifying protein fold structures in the proteomes of archaeoviruses, bacterioviruses, and eukaryoviruses and second by tracing their spread in the proteomes of superkingdoms Archaea, Bacteria, and Eukarya. The exercise quantified protein structural homologies between viruses and organisms of their host and non-host superkingdoms and revealed likely candidates for virus-to-cell and cell-to-virus gene transfers. Unexpected lifestyle-driven genetic affiliations between bacterioviruses and Eukarya and eukaryoviruses and Bacteria were also predicted in addition to a large cohort of protein folds that were universally shared by viral and cellular proteomes and virus-specific protein folds not detected in cellular proteomes. These protein folds provide unique insights into viral origins and evolution that are generally difficult to recover with traditional sequence alignment-dependent evolutionary analyses owing to the fast mutation rates of viral gene sequences.

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

PubMed

Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

Molecular modelling studies on the ORL1-receptor and ORL1-agonists

NASA Astrophysics Data System (ADS)

Bröer, Britta M.; Gurrath, Marion; Höltje, Hans-Dieter

2003-11-01

The ORL1 ( opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.

High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p

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

Mohr, Georg; Del Campo, Mark; Turner, Kathryn G.

The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionallymore » important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.« less

Usefulness of ELISA Methods for Assessing LPS Interactions with Proteins and Peptides

PubMed Central

Martínez-Sernández, Victoria; Orbegozo-Medina, Ricardo A.; Romarís, Fernanda; Paniagua, Esperanza; Ubeira, Florencio M.

2016-01-01

Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, can trigger severe inflammatory responses during bacterial infections, possibly leading to septic shock. One approach to combatting endotoxic shock is to neutralize the most conserved part and major mediator of LPS activity (lipid A) with LPS-binding proteins or peptides. Although several available assays evaluate the biological activity of these molecules on LPS (e.g. inhibition of LPS-induced TNF-α production in macrophages), the development of simple and cost-effective methods that would enable preliminary screening of large numbers of potential candidate molecules is of great interest. Moreover, it would be also desirable that such methods could provide information about the possible biological relevance of the interactions between proteins and LPS, which may enhance or neutralize LPS-induced inflammatory responses. In this study, we designed and evaluated different types of ELISA that could be used to study possible interactions between LPS and any protein or peptide. We also analysed the usefulness and limitations of the different ELISAs. Specifically, we tested the capacity of several proteins and peptides to bind FITC-labeled LPSs from Escherichia coli serotypes O111:B4 and O55:B5 in an indirect ELISA and in two competitive ELISAs including casein hydrolysate (hCAS) and biotinylated polymyxin B (captured by deglycosylated avidin; PMX) as LPS-binding agents in the solid phase. We also examined the influence of pH, detergents and different blocking agents on LPS binding. Our results showed that the competitive hCAS-ELISA performed under mildly acidic conditions can be used as a general method for studying LPS interactions, while the more restrictive PMX-ELISA may help to identify proteins/peptides that are likely to have neutralizing properties in vitro or in vivo. PMID:27249227

Microfluidic Devices for Studying Biomolecular Interactions

NASA Technical Reports Server (NTRS)

Wilson, Wilbur W.; Garcia, Carlos d.; Henry, Charles S.

2006-01-01

Microfluidic devices for monitoring biomolecular interactions have been invented. These devices are basically highly miniaturized liquid-chromatography columns. They are intended to be prototypes of miniature analytical devices of the laboratory on a chip type that could be fabricated rapidly and inexpensively and that, because of their small sizes, would yield analytical results from very small amounts of expensive analytes (typically, proteins). Other advantages to be gained by this scaling down of liquid-chromatography columns may include increases in resolution and speed, decreases in the consumption of reagents, and the possibility of performing multiple simultaneous and highly integrated analyses by use of multiple devices of this type, each possibly containing multiple parallel analytical microchannels. The principle of operation is the same as that of a macroscopic liquid-chromatography column: The column is a channel packed with particles, upon which are immobilized molecules of the protein of interest (or one of the proteins of interest if there are more than one). Starting at a known time, a solution or suspension containing molecules of the protein or other substance of interest is pumped into the channel at its inlet. The liquid emerging from the outlet of the channel is monitored to detect the molecules of the dissolved or suspended substance(s). The time that it takes these molecules to flow from the inlet to the outlet is a measure of the degree of interaction between the immobilized and the dissolved or suspended molecules. Depending on the precise natures of the molecules, this measure can be used for diverse purposes: examples include screening for solution conditions that favor crystallization of proteins, screening for interactions between drugs and proteins, and determining the functions of biomolecules.

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

PubMed

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

PubMed

Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

2016-08-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

PubMed Central

Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

2016-01-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

Genetic and physical interaction of the B-cell systemic lupus erythematosus-associated genes BANK1 and BLK.

PubMed

Castillejo-López, Casimiro; Delgado-Vega, Angélica M; Wojcik, Jerome; Kozyrev, Sergey V; Thavathiru, Elangovan; Wu, Ying-Yu; Sánchez, Elena; Pöllmann, David; López-Egido, Juan R; Fineschi, Serena; Domínguez, Nicolás; Lu, Rufei; James, Judith A; Merrill, Joan T; Kelly, Jennifer A; Kaufman, Kenneth M; Moser, Kathy L; Gilkeson, Gary; Frostegård, Johan; Pons-Estel, Bernardo A; D'Alfonso, Sandra; Witte, Torsten; Callejas, José Luis; Harley, John B; Gaffney, Patrick M; Martin, Javier; Guthridge, Joel M; Alarcón-Riquelme, Marta E

2012-01-01

Altered signalling in B cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signalling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterise the role of BANK1 and BLK in SLE, a genetic interaction analysis was performed hypothesising that genetic interactions could reveal functional pathways relevant to disease pathogenesis. The GPAT16 method was used to analyse the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localisation, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK. Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, the possibility that BANK1 and BLK could also show a protein-protein interaction was tested. The co-immunoprecipitation and co-localisation of BLK and BANK1 were demonstrated. In a Daudi cell line and primary naive B cells endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies. This study shows a genetic interaction between BANK1 and BLK, and demonstrates that these molecules interact physically. The results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signalling pathway.

Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

PubMed

Roberts, Thomas C; Johansson, Henrik J; McClorey, Graham; Godfrey, Caroline; Blomberg, K Emelie M; Coursindel, Thibault; Gait, Michael J; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A

2015-12-01

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle. © The Author 2015. Published by Oxford University Press.

The organization of RNA contacts by PTB for regulation of FAS splicing

PubMed Central

Mickleburgh, Ian; Kafasla, Panagiota; Cherny, Dmitry; Llorian, Miriam; Curry, Stephen; Jackson, Richard J.; Smith, Christopher W.J.

2014-01-01

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained. PMID:24957602

Identification of novel candidate drivers connecting different dysfunctional levels for lung adenocarcinoma using protein-protein interactions and a shortest path approach

NASA Astrophysics Data System (ADS)

Chen, Lei; Huang, Tao; Zhang, Yu-Hang; Jiang, Yang; Zheng, Mingyue; Cai, Yu-Dong

2016-07-01

Tumors are formed by the abnormal proliferation of somatic cells with disordered growth regulation under the influence of tumorigenic factors. Recently, the theory of “cancer drivers” connects tumor initiation with several specific mutations in the so-called cancer driver genes. According to the differentiation of four basic levels between tumor and adjacent normal tissues, the cancer drivers can be divided into the following: (1) Methylation level, (2) microRNA level, (3) mutation level, and (4) mRNA level. In this study, a computational method is proposed to identify novel lung adenocarcinoma drivers based on dysfunctional genes on the methylation, microRNA, mutation and mRNA levels. First, a large network was constructed using protein-protein interactions. Next, we searched all of the shortest paths connecting dysfunctional genes on different levels and extracted new candidate genes lying on these paths. Finally, the obtained candidate genes were filtered by a permutation test and an additional strict selection procedure involving a betweenness ratio and an interaction score. Several candidate genes remained, which are deemed to be related to two different levels of cancer. The analyses confirmed our assertions that some have the potential to contribute to the tumorigenesis process on multiple levels.

Identification of mud crab reovirus VP12 and its interaction with the voltage-dependent anion-selective channel protein of mud crab Scylla paramamosain.

PubMed

Xu, Hai-Dong; Su, Hong-Jun; Zou, Wei-Bin; Liu, Shan-Shan; Yan, Wen-Rui; Wang, Qian-Qian; Yuan, Li-Li; Chan, Siuming Francis; Yu, Xiao-Qiang; He, Jian-Guo; Weng, Shao-Ping

2015-05-01

Mud crab reovirus (MCRV) is the causative agent of a severe disease in cultured mud crab (Scylla paramamosain), which has caused huge economic losses in China. MCRV is a double-stranded RNA virus with 12 genomic segments. In this paper, SDS-PAGE, mass spectrometry and Western blot analyses revealed that the VP12 protein encoded by S12 gene is a structural protein of MCRV. Immune electron microscopy assay indicated that MCRV VP12 is a component of MCRV outer shell capsid. Yeast two hybrid cDNA library of mud crab was constructed and mud crab voltage-dependent anion-selective channel (mcVDAC) was obtained by MCRV VP12 screening. The full length of mcVDAC was 1180 bp with an open reading frame (ORF) of 849 bp encoding a 282 amino acid protein. The mcVDAC had a constitutive expression pattern in different tissues of mud crab. The interaction between MCRV VP12 and mcVDAC was determined by co-immunoprecipitation assay. The results of this study have provided an insight on the mechanisms of MCRV infection and the interactions between the virus and mud crab. Copyright © 2015 Elsevier Ltd. All rights reserved.

Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

PubMed Central

Tanny, Jason C.; Kirkpatrick, Donald S.; Gerber, Scott A.; Gygi, Steven P.; Moazed, Danesh

2004-01-01

Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo. PMID:15282295

PRIC320, a transcription coactivator, isolated from peroxisome proliferator-binding protein complex.

PubMed

Surapureddi, Sailesh; Viswakarma, Navin; Yu, Songtao; Guo, Dongsheng; Rao, M Sambasiva; Reddy, Janardan K

2006-05-05

Ciprofibrate, a potent peroxisome proliferator, induces pleiotropic responses in liver by activating peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor. Transcriptional regulation by liganded nuclear receptors involves the participation of coregulators that form multiprotein complexes possibly to achieve cell and gene specific transcription. SDS-PAGE and matrix-assisted laser desorption/ionization reflection time-of-flight mass spectrometric analyses of ciprofibrate-binding proteins from liver nuclear extracts obtained using ciprofibrate-Sepharose affinity matrix resulted in the identification of a new high molecular weight nuclear receptor coactivator, which we designated PRIC320. The full-length human cDNA encoding this protein has an open-reading frame that codes for a 320kDa protein containing 2882 amino acids. PRIC320 contains five LXXLL signature motifs that mediate interaction with nuclear receptors. PRIC320 binds avidly to nuclear receptors PPARalpha, CAR, ERalpha, and RXR, but only minimally with PPARgamma. PRIC320 also interacts with transcription cofactors CBP, PRIP, and PBP. Immunoprecipitation-immunoblotting as well as cellular localization studies confirmed the interaction between PPARalpha and PRIC320. PRIC320 acts as a transcription coactivator by stimulating PPARalpha-mediated transcription. We conclude that ciprofibrate, a PPARalpha ligand, binds a multiprotein complex and PRIC320 cloned from this complex functions as a nuclear receptor coactivator.

RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins

PubMed Central

2014-01-01

Background Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism. PMID:24393626

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

New insight into protein-nanomaterial interactions with UV-visible spectroscopy and chemometrics: human serum albumin and silver nanoparticles.

PubMed

Wang, Yong; Ni, Yongnian

2014-01-21

In recent years, great efforts have focused on the exploration and fabrication of protein nanoconjugates due to potential applications in many fields including bioanalytical science, biosensors, biocatalysis, biofuel cells and bio-based nanodevices. An important aspect of our understanding of protein nanoconjugates is to quantitatively understand how proteins interact with nanomaterials. In this report, human serum albumin (HSA) and citrate-coated silver nanoparticles (AgNPs) are selected as a case study of protein-nanomaterial interactions. UV-visible spectroscopy together with multivariate curve resolution by alternating least squares (MCR-ALS) algorithm is first exploited for the detailed study of AgNPs-HSA interactions. Introduction of the chemometrics tool allows extracting the kinetic profiles, spectra and distribution diagrams of two major absorbing pure species (AgNPs and AgNPs-HSA conjugate). These resolved profiles are then analysed to give the thermodynamic, kinetic and structural information of HSA binding to AgNPs. Transmission electron microscopy, circular dichroism spectroscopy and Fourier transform infrared spectroscopy are used to further characterize the complex system. Moreover, a sensitive spectroscopic biosensor for HSA is fabricated with the MCR-ALS resolved concentration of absorbing pure species. It is found that the linear range for the HSA nanosensor was from 1.9 nM to 45.0 nM with a detection limit of 0.9 nM. It is believed that the proposed method will play an important role in the fabrication and optimization of a robust nanobiosensor or cross-reactive sensors array for the detection and identification of biocomponents.

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets

PubMed Central

De Kumar, Bony; Parker, Hugo J.; Paulson, Ariel; Parrish, Mark E.; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D.; Unruh, Jay R.; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

2017-01-01

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. PMID:28784834

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4.

PubMed

Sehar, Ujala; Mehmood, Muhammad Aamer; Hussain, Khadim; Nawaz, Salman; Nadeem, Shahid; Siddique, Muhammad Hussnain; Nadeem, Habibullah; Gull, Munazza; Ahmad, Niaz; Sohail, Iqra; Gill, Saba Shahid; Majeed, Summera

2013-01-01

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.

Proteins analysed as virtual knots

NASA Astrophysics Data System (ADS)

Alexander, Keith; Taylor, Alexander J.; Dennis, Mark R.

2017-02-01

Long, flexible physical filaments are naturally tangled and knotted, from macroscopic string down to long-chain molecules. The existence of knotting in a filament naturally affects its configuration and properties, and may be very stable or disappear rapidly under manipulation and interaction. Knotting has been previously identified in protein backbone chains, for which these mechanical constraints are of fundamental importance to their molecular functionality, despite their being open curves in which the knots are not mathematically well defined; knotting can only be identified by closing the termini of the chain somehow. We introduce a new method for resolving knotting in open curves using virtual knots, which are a wider class of topological objects that do not require a classical closure and so naturally capture the topological ambiguity inherent in open curves. We describe the results of analysing proteins in the Protein Data Bank by this new scheme, recovering and extending previous knotting results, and identifying topological interest in some new cases. The statistics of virtual knots in protein chains are compared with those of open random walks and Hamiltonian subchains on cubic lattices, identifying a regime of open curves in which the virtual knotting description is likely to be important.

Proteins analysed as virtual knots

PubMed Central

Alexander, Keith; Taylor, Alexander J.; Dennis, Mark R.

2017-01-01

Long, flexible physical filaments are naturally tangled and knotted, from macroscopic string down to long-chain molecules. The existence of knotting in a filament naturally affects its configuration and properties, and may be very stable or disappear rapidly under manipulation and interaction. Knotting has been previously identified in protein backbone chains, for which these mechanical constraints are of fundamental importance to their molecular functionality, despite their being open curves in which the knots are not mathematically well defined; knotting can only be identified by closing the termini of the chain somehow. We introduce a new method for resolving knotting in open curves using virtual knots, which are a wider class of topological objects that do not require a classical closure and so naturally capture the topological ambiguity inherent in open curves. We describe the results of analysing proteins in the Protein Data Bank by this new scheme, recovering and extending previous knotting results, and identifying topological interest in some new cases. The statistics of virtual knots in protein chains are compared with those of open random walks and Hamiltonian subchains on cubic lattices, identifying a regime of open curves in which the virtual knotting description is likely to be important. PMID:28205562

Evolution of the Max and Mlx networks in animals.

PubMed

McFerrin, Lisa G; Atchley, William R

2011-01-01

Transcription factors (TFs) are essential for the regulation of gene expression and often form emergent complexes to perform vital roles in cellular processes. In this paper, we focus on the parallel Max and Mlx networks of TFs because of their critical involvement in cell cycle regulation, proliferation, growth, metabolism, and apoptosis. A basic-helix-loop-helix-zipper (bHLHZ) domain mediates the competitive protein dimerization and DNA binding among Max and Mlx network members to form a complex system of cell regulation. To understand the importance of these network interactions, we identified the bHLHZ domain of Max and Mlx network proteins across the animal kingdom and carried out several multivariate statistical analyses. The presence and conservation of Max and Mlx network proteins in animal lineages stemming from the divergence of Metazoa indicate that these networks have ancient and essential functions. Phylogenetic analysis of the bHLHZ domain identified clear relationships among protein families with distinct points of radiation and divergence. Multivariate discriminant analysis further isolated specific amino acid changes within the bHLHZ domain that classify proteins, families, and network configurations. These analyses on Max and Mlx network members provide a model for characterizing the evolution of TFs involved in essential networks.

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

DOE PAGES

Vild, Cody J.; Li, Yan; Guo, Emily Z.; ...

2015-01-30

Disassembly of the endosomal sorting complex required for transport (ESCRT) machinery from biological membranes is a critical final step in cellular processes that require the ESCRT function. This reaction is catalyzed by VPS4, an AAA-ATPase whose activity is tightly regulated by a host of proteins, including LIP5 and the ESCRT-III proteins. In this paper, we present structural and functional analyses of molecular interactions between human VPS4, LIP5, and the ESCRT-III proteins. The N-terminal domain of LIP5 (LIP5NTD) is required for LIP5-mediated stimulation of VPS4, and the ESCRT-III protein CHMP5 strongly inhibits the stimulation. Both of these observations are distinct frommore » what was previously described for homologous yeast proteins. The crystal structure of LIP5NTD in complex with the MIT (microtubule-interacting and transport)-interacting motifs of CHMP5 and a second ESCRT-III protein, CHMP1B, was determined at 1 Å resolution. It reveals an ESCRT-III binding induced moderate conformational change in LIP5NTD, which results from insertion of a conserved CHMP5 tyrosine residue (Tyr 182) at the core of LIP5NTD structure. Finally, mutation of Tyr 182 partially relieves the inhibition displayed by CHMP5. Together, these results suggest a novel mechanism of VPS4 regulation in metazoans, where CHMP5 functions as a negative allosteric switch to control LIP5-mediated stimulation of VPS4.« less

C-terminal, endoplasmic reticulum-lumenal domain of prosurfactant protein C - structural features and membrane interactions.

PubMed

Casals, Cristina; Johansson, Hanna; Saenz, Alejandra; Gustafsson, Magnus; Alfonso, Carlos; Nordling, Kerstin; Johansson, Jan

2008-02-01

Surfactant protein C (SP-C) constitutes the transmembrane part of prosurfactant protein C (proSP-C) and is alpha-helical in its native state. The C-terminal part of proSP-C (CTC) is localized in the endoplasmic reticulum lumen and binds to misfolded (beta-strand) SP-C, thereby preventing its aggregation and amyloid fibril formation. In this study, we investigated the structure of recombinant human CTC and the effects of CTC-membrane interaction on protein structure. CTC forms noncovalent trimers and supratrimeric oligomers. It contains two intrachain disulfide bridges, and its secondary structure is significantly affected by urea or heat only after disulfide reduction. The postulated Brichos domain of CTC, with homologs found in proteins associated with amyloid and proliferative disease, is up to 1000-fold more protected from limited proteolysis than the rest of CTC. The protein exposes hydrophobic surfaces, as determined by CTC binding to the environment-sensitive fluorescent probe 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate). Fluorescence energy transfer experiments further reveal close proximity between bound 1,1'-bis(4-anilino-5,5'-naphthalenesulfonate) and tyrosine residues in CTC, some of which are conserved in all Brichos domains. CTC binds to unilamellar phospholipid vesicles with low micromolar dissociation constants, and differential scanning calorimetry and CD analyses indicate that membrane-bound CTC is less structurally ordered than the unbound protein. The exposed hydrophobic surfaces and the structural disordering that result from interactions with phospholipid membranes suggest a mechanism whereby CTC binds to misfolded SP-C in the endoplasmic reticulum membrane.

Echinostoma caproni: identification of enolase in excretory/secretory products, molecular cloning, and functional expression.

PubMed

Marcilla, Antonio; Pérez-García, Ana; Espert, Ana; Bernal, Dolores; Muñoz-Antolí, Carla; Esteban, José Guillermo; Toledo, Rafael

2007-09-01

In order to investigate molecules that could be involved in host-trematode relationships, we have analysed the excretory/secretory products (ESP) of Echinostoma caproni following a proteomic approach. Actin, Gluthathione S-transferase (GST) and enolase have been identified in the ESP. Enolase, observed to be one of the most abundant proteins, was further characterized. The molecular cloning and in vitro expression in Escherichia coli of E. caproni enolase allowed us to determine that the protein contains 431 amino acids and a theoretical MW of 46272 Da. E. caproni enolase shows high homology to other trematode enolases. The recombinant protein binds specifically to human plasminogen in vitro, as observed for the native protein, confirming its properties as a host-interacting molecule.

Automation of NMR structure determination of proteins.

PubMed

Altieri, Amanda S; Byrd, R Andrew

2004-10-01

The automation of protein structure determination using NMR is coming of age. The tedious processes of resonance assignment, followed by assignment of NOE (nuclear Overhauser enhancement) interactions (now intertwined with structure calculation), assembly of input files for structure calculation, intermediate analyses of incorrect assignments and bad input data, and finally structure validation are all being automated with sophisticated software tools. The robustness of the different approaches continues to deal with problems of completeness and uniqueness; nevertheless, the future is very bright for automation of NMR structure generation to approach the levels found in X-ray crystallography. Currently, near completely automated structure determination is possible for small proteins, and the prospect for medium-sized and large proteins is good. Copyright 2004 Elsevier Ltd.

The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

PubMed

Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

2017-12-15

Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants.

PubMed

Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

2014-01-01

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

PubMed Central

Nietzsche, Madlen; Schießl, Ingrid; Börnke, Frederik

2014-01-01

In plants, SNF1-related kinase (SnRK1) responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic α subunit associates with a regulatory β subunit and an activating γ subunit. Several different metabolites as well as the hormone abscisic acid (ABA) have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF) 581 could interact with both isoforms of the SnRK1α subunit (AKIN10/11) of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation. PMID:24600465

Toxicoproteomic analysis of human lung epithelial cells exposed to steel industry ambient particulate matter (PM) reveals possible mechanism of PM related carcinogenesis.

PubMed

Senthil Kumar, S; Muthuselvam, P; Pugalenthi, V; Subramanian, N; Ramkumar, K M; Suresh, T; Suzuki, T; Rajaguru, P

2018-08-01

Toxicoproteomic analysis of steel industry ambient particulate matter (PM) that contain high concentrations of PAHs and metals was done by treating human lung cancer cell-line, A549 and the cell lysates were analysed using quantitative label-free nano LC-MS/MS. A total of 18,562 peptides representing 1576 proteins were identified and quantified, with 196 proteins had significantly altered expression in the treated cells. Enrichment analyses revealed that proteins associated to redox homeostsis, metabolism, and cellular energy generation were inhibited while, proteins related to DNA damage and repair and other stresses were over expressed. Altered activities of several tumor associated proteins were observed. Protein-protein interaction network and biological pathway analysis of these differentially expressed proteins were carried out to obtain a systems level view of proteome changes. Together it could be inferred that PM exposure induced oxidative stress which could have lead into DNA damage and tumor related changes. However, lowering of cellular metabolism, and energy production could reduce its ability to overcome these stress. This kind of disequilibrium between the DNA damage and ability of the cells to repair the DNA damage may lead into genomic instability that is capable of acting as the driving force during PM induced carcinogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Mishra, Arjun K.; Agnihotri, Pragati; Srivastava, Vijay Kumar

Highlights: • L. donovani spermidine synthase and S-adenosylmethionine decarboxylase have been cloned and purified. • S-adenosylmethionine decarboxylase has autocatalytic property. • GST pull down assay shows the two proteins to form a metabolon. • Isothermal titration calorimetry shows that binding was exothermic having K{sub d} value of 0.4 μM. • Interaction confirmed by fluorescence spectroscopy and size exclusion chromatography. - Abstract: Polyamine biosynthesis pathway has long been considered an essential drug target for trypanosomatids including Leishmania. S-adenosylmethionine decarboxylase (AdoMetDc) and spermidine synthase (SpdSyn) are enzymes of this pathway that catalyze successive steps, with the product of the former, decarboxylated S-adenosylmethioninemore » (dcSAM), acting as an aminopropyl donor for the latter enzyme. Here we have explored the possibility of and identified the protein–protein interaction between SpdSyn and AdoMetDc. The protein–protein interaction has been identified using GST pull down assay. Isothermal titration calorimetry reveals that the interaction is thermodynamically favorable. Fluorescence spectroscopy studies also confirms the interaction, with SpdSyn exhibiting a change in tertiary structure with increasing concentrations of AdoMetDc. Size exclusion chromatography suggests the presence of the complex as a hetero-oligomer. Taken together, these results suggest that the enzymes indeed form a heteromer. Computational analyses suggest that this complex differs significantly from the corresponding human complex, implying that this complex could be a better therapeutic target than the individual enzymes.« less

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

PubMed Central

Law, Michael J.; Linde, Michael E.; Chambers, Eric J.; Oubridge, Chris; Katsamba, Phinikoula S.; Nilsson, Lennart; Haworth, Ian S.; Laird-Offringa, Ite A.

2006-01-01

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes. PMID:16407334

Computational analysis of protein-protein interfaces involving an alpha helix: insights for terphenyl-like molecules binding.

PubMed

Isvoran, Adriana; Craciun, Dana; Martiny, Virginie; Sperandio, Olivier; Miteva, Maria A

2013-06-14

Protein-Protein Interactions (PPIs) are key for many cellular processes. The characterization of PPI interfaces and the prediction of putative ligand binding sites and hot spot residues are essential to design efficient small-molecule modulators of PPI. Terphenyl and its derivatives are small organic molecules known to mimic one face of protein-binding alpha-helical peptides. In this work we focus on several PPIs mediated by alpha-helical peptides. We performed computational sequence- and structure-based analyses in order to evaluate several key physicochemical and surface properties of proteins known to interact with alpha-helical peptides and/or terphenyl and its derivatives. Sequence-based analysis revealed low sequence identity between some of the analyzed proteins binding alpha-helical peptides. Structure-based analysis was performed to calculate the volume, the fractal dimension roughness and the hydrophobicity of the binding regions. Besides the overall hydrophobic character of the binding pockets, some specificities were detected. We showed that the hydrophobicity is not uniformly distributed in different alpha-helix binding pockets that can help to identify key hydrophobic hot spots. The presence of hydrophobic cavities at the protein surface with a more complex shape than the entire protein surface seems to be an important property related to the ability of proteins to bind alpha-helical peptides and low molecular weight mimetics. Characterization of similarities and specificities of PPI binding sites can be helpful for further development of small molecules targeting alpha-helix binding proteins.

JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

PubMed Central

Drerup, Catherine M.; Nechiporuk, Alex V.

2013-01-01

Retrograde axonal transport requires an intricate interaction between the dynein motor and its cargo. What mediates this interaction is largely unknown. Using forward genetics and a novel in vivo imaging approach, we identified JNK-interacting protein 3 (Jip3) as a direct mediator of dynein-based retrograde transport of activated (phosphorylated) c-Jun N-terminal Kinase (JNK) and lysosomes. Zebrafish jip3 mutants (jip3nl7) displayed large axon terminal swellings that contained high levels of activated JNK and lysosomes, but not other retrograde cargos such as late endosomes and autophagosomes. Using in vivo analysis of axonal transport, we demonstrated that the terminal accumulations of activated JNK and lysosomes were due to a decreased frequency of retrograde movement of these cargos in jip3nl7, whereas anterograde transport was largely unaffected. Through rescue experiments with Jip3 engineered to lack the JNK binding domain and exogenous expression of constitutively active JNK, we further showed that loss of Jip3–JNK interaction underlies deficits in pJNK retrograde transport, which subsequently caused axon terminal swellings but not lysosome accumulation. Lysosome accumulation, rather, resulted from loss of lysosome association with dynein light intermediate chain (dynein accessory protein) in jip3nl7, as demonstrated by our co-transport analyses. Thus, our results demonstrate that Jip3 is necessary for the retrograde transport of two distinct cargos, active JNK and lysosomes. Furthermore, our data provide strong evidence that Jip3 in fact serves as an adapter protein linking these cargos to dynein. PMID:23468645

Structure and function of homodomain-leucine zipper (HD-Zip) proteins.

PubMed

Elhiti, Mohamed; Stasolla, Claudio

2009-02-01

Homeodomain-leucine zipper (HD-Zip) proteins are transcription factors unique to plants and are encoded by more than 25 genes in Arabidopsis thaliana. Based on sequence analyses these proteins have been classified into four distinct groups: HD-Zip I-IV. HD-Zip proteins are characterized by the presence of two functional domains; a homeodomain (HD) responsible for DNA binding and a leucine zipper domain (Zip) located immediately C-terminal to the homeodomain and involved in protein-protein interaction. Despite sequence similarities HD-ZIP proteins participate in a variety of processes during plant growth and development. HD-Zip I proteins are generally involved in responses related to abiotic stress, abscisic acid (ABA), blue light, de-etiolation and embryogenesis. HD-Zip II proteins participate in light response, shade avoidance and auxin signalling. Members of the third group (HD-Zip III) control embryogenesis, leaf polarity, lateral organ initiation and meristem function. HD-Zip IV proteins play significant roles during anthocyanin accumulation, differentiation of epidermal cells, trichome formation and root development.

FOX-2 Dependent Splicing of Ataxin-2 Transcript Is Affected by Ataxin-1 Overexpression

PubMed Central

Welzel, Franziska; Kaehler, Christian; Isau, Melanie; Hallen, Linda; Lehrach, Hans; Krobitsch, Sylvia

2012-01-01

Alternative splicing is a fundamental posttranscriptional mechanism for controlling gene expression, and splicing defects have been linked to various human disorders. The splicing factor FOX-2 is part of a main protein interaction hub in a network related to human inherited ataxias, however, its impact remains to be elucidated. Here, we focused on the reported interaction between FOX-2 and ataxin-1, the disease-causing protein in spinocerebellar ataxia type 1. In this line, we further evaluated this interaction by yeast-2-hybrid analyses and co-immunoprecipitation experiments in mammalian cells. Interestingly, we discovered that FOX-2 localization and splicing activity is affected in the presence of nuclear ataxin-1 inclusions. Moreover, we observed that FOX-2 directly interacts with ataxin-2, a protein modulating spinocerebellar ataxia type 1 pathogenesis. Finally, we provide evidence that splicing of pre-mRNA of ataxin-2 depends on FOX-2 activity, since reduction of FOX-2 levels led to increased skipping of exon 18 in ataxin-2 transcripts. Most striking, we observed that ataxin-1 overexpression has an effect on this splicing event as well. Thus, our results demonstrate that FOX-2 is involved in splicing of ataxin-2 transcripts and that this splicing event is altered by overexpression of ataxin-1. PMID:22666429

Stringent DDI-based prediction of H. sapiens-M. tuberculosis H37Rv protein-protein interactions.

PubMed

Zhou, Hufeng; Rezaei, Javad; Hugo, Willy; Gao, Shangzhi; Jin, Jingjing; Fan, Mengyuan; Yong, Chern-Han; Wozniak, Michal; Wong, Limsoon

2013-01-01

H. sapiens-M. tuberculosis H37Rv protein-protein interaction (PPI) data are very important information to illuminate the infection mechanism of M. tuberculosis H37Rv. But current H. sapiens-M. tuberculosis H37Rv PPI data are very scarce. This seriously limits the study of the interaction between this important pathogen and its host H. sapiens. Computational prediction of H. sapiens-M. tuberculosis H37Rv PPIs is an important strategy to fill in the gap. Domain-domain interaction (DDI) based prediction is one of the frequently used computational approaches in predicting both intra-species and inter-species PPIs. However, the performance of DDI-based host-pathogen PPI prediction has been rather limited. We develop a stringent DDI-based prediction approach with emphasis on (i) differences between the specific domain sequences on annotated regions of proteins under the same domain ID and (ii) calculation of the interaction strength of predicted PPIs based on the interacting residues in their interaction interfaces. We compare our stringent DDI-based approach to a conventional DDI-based approach for predicting PPIs based on gold standard intra-species PPIs and coherent informative Gene Ontology terms assessment. The assessment results show that our stringent DDI-based approach achieves much better performance in predicting PPIs than the conventional approach. Using our stringent DDI-based approach, we have predicted a small set of reliable H. sapiens-M. tuberculosis H37Rv PPIs which could be very useful for a variety of related studies. We also analyze the H. sapiens-M. tuberculosis H37Rv PPIs predicted by our stringent DDI-based approach using cellular compartment distribution analysis, functional category enrichment analysis and pathway enrichment analysis. The analyses support the validity of our prediction result. Also, based on an analysis of the H. sapiens-M. tuberculosis H37Rv PPI network predicted by our stringent DDI-based approach, we have discovered some important properties of domains involved in host-pathogen PPIs. We find that both host and pathogen proteins involved in host-pathogen PPIs tend to have more domains than proteins involved in intra-species PPIs, and these domains have more interaction partners than domains on proteins involved in intra-species PPI. The stringent DDI-based prediction approach reported in this work provides a stringent strategy for predicting host-pathogen PPIs. It also performs better than a conventional DDI-based approach in predicting PPIs. We have predicted a small set of accurate H. sapiens-M. tuberculosis H37Rv PPIs which could be very useful for a variety of related studies.

Functional display of family 11 endoxylanases on the surface of phage M13.

PubMed

Beliën, T; Hertveldt, K; Van den Brande, K; Robben, J; Van Campenhout, S; Volckaert, G

2005-02-09

Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors.

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

PubMed

Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

2016-08-31

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

PubMed Central

Klockenbusch, Cordula; Kast, Juergen

2010-01-01

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879

Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling

PubMed Central

Warncke, Jan D.; Vakonakis, Ioannis

2016-01-01

SUMMARY During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family. PMID:27582258

MsViz: A Graphical Software Tool for In-Depth Manual Validation and Quantitation of Post-translational Modifications.

PubMed

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

2017-08-04

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

Double quick, double click reversible peptide "stapling".

PubMed

Grison, Claire M; Burslem, George M; Miles, Jennifer A; Pilsl, Ludwig K A; Yeo, David J; Imani, Zeynab; Warriner, Stuart L; Webb, Michael E; Wilson, Andrew J

2017-07-01

The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( h Cys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

PubMed Central

Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

2011-01-01

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

Quantitative analysis of the protein corona on FePt nanoparticles formed by transferrin binding

PubMed Central

Jiang, Xiue; Weise, Stefan; Hafner, Margit; Röcker, Carlheinz; Zhang, Feng; Parak, Wolfgang J.; Nienhaus, G. Ulrich

2010-01-01

Nanoparticles are finding a rapidly expanding range of applications in research and technology, finally entering our daily life in medical, cosmetic or food products. Their ability to invade all regions of an organism including cells and cellular organelles offers new strategies for medical diagnosis and therapy (nanomedicine), but their safe use requires a deep knowledge about their interactions with biological systems at the molecular level. Upon incorporation, nanoparticles are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a ‘protein corona’. These nanoparticle–protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here we have quantitatively analysed the adsorption of human transferrin onto small (radius approx. 5 nm) polymer-coated FePt nanoparticles by using fluorescence correlation spectroscopy. Transferrin binds to the negatively charged nanoparticles with an affinity of approximately 26 µM in a cooperative fashion and forms a monolayer with a thickness of 7 nm. By using confocal fluorescence microscopy, we have observed that the uptake of FePt nanoparticles by HeLa cells is suppressed by the protein corona compared with the bare nanoparticles. PMID:19776149

Chronic hypoxia stress-induced differential modulation of heat-shock protein 70 and presynaptic proteins.

PubMed

Fei, Guanghe; Guo, Conghui; Sun, Hong-Shuo; Feng, Zhong-Ping

2007-01-01

Chronic hypoxia exposure can cause neurobehavioral dysfunction, but the underlying cellular and molecular mechanisms remain unclear. Here, we found that adult Lymnaea stagnalis snails maintained in low O(2) (approximately 5%) for 4 days developed slowed reactions to light stimuli, and reduced righting movement. Semiquantitative immunoblotting analyses showed that hypoxia exposure induced increased expression of heat-shock protein (HSP)70 in ganglion preparations, and suppressed expression of the presynaptic proteins syntaxin I, synaptic vesicle protein 2 (SV2) and synaptotagmin I. Detailed time course analyses showed that an early moderate increase developed within 6 h, preceding a substantial up-regulation of HSP70 after 4 days; an early reduction of syntaxin I in the first 24 h; a delayed reduction of synaptotagmin I after 4 days; and a biphasic change in SV2. Using a double-stranded RNA interference approach, we demonstrated that preventing the hypoxia inducible HSP70 enhanced down-regulation of syntaxin and synaptotagmin, and aggravated motor and sensory suppression. Co-immunoprecipitation analysis revealed an interaction between HSP70 and syntaxin. We have thus provided the first evidence that early induction of HSP70 by chronic hypoxia is critical for maintaining expression levels of presynaptic proteins. These findings implicate a new molecular mechanism underlying chronic hypoxia-induced neurobehavioral adaptation and impairment.

Characterization of Hepatitis C Virus Core Protein Multimerization and Membrane Envelopment: Revelation of a Cascade of Core-Membrane Interactions ▿

PubMed Central

Ai, Li-Shuang; Lee, Yu-Wen; Chen, Steve S.-L.

2009-01-01

The molecular basis underlying hepatitis C virus (HCV) core protein maturation and morphogenesis remains elusive. We characterized the concerted events associated with core protein multimerization and interaction with membranes. Analyses of core proteins expressed from a subgenomic system showed that the signal sequence located between the core and envelope glycoprotein E1 is critical for core association with endoplasmic reticula (ER)/late endosomes and the core's envelopment by membranes, which was judged by the core's acquisition of resistance to proteinase K digestion. Despite exerting an inhibitory effect on the core's association with membranes, (Z-LL)2-ketone, a specific inhibitor of signal peptide peptidase (SPP), did not affect core multimeric complex formation, suggesting that oligomeric core complex formation proceeds prior to or upon core attachment to membranes. Protease-resistant core complexes that contained both innate and processed proteins were detected in the presence of (Z-LL)2-ketone, implying that core envelopment occurs after intramembrane cleavage. Mutations of the core that prevent signal peptide cleavage or coexpression with an SPP loss-of-function D219A mutant decreased the core's envelopment, demonstrating that SPP-mediated cleavage is required for core envelopment. Analyses of core mutants with a deletion in domain I revealed that this domain contains sequences crucial for core envelopment. The core proteins expressed by infectious JFH1 and Jc1 RNAs in Huh7 cells also assembled into a multimeric complex, associated with ER/late-endosomal membranes, and were enveloped by membranes. Treatment with (Z-LL)2-ketone or coexpression with D219A mutant SPP interfered with both core envelopment and infectious HCV production, indicating a critical role of core envelopment in HCV morphogenesis. The results provide mechanistic insights into the sequential and coordinated processes during the association of the HCV core protein with membranes in the early phase of virus maturation and morphogenesis. PMID:19605478

Septin dynamics are essential for exocytosis.

PubMed

Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A; Whitelegge, Julian P; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

2015-02-27

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

What interactions drive the salivary mucosal pellicle formation?

PubMed Central

Gibbins, Hannah L.; Yakubov, Gleb E.; Proctor, Gordon B.; Wilson, Stephen; Carpenter, Guy H.

2014-01-01

The bound salivary pellicle is essential for protection of both the enamel and mucosa in the oral cavity. The enamel pellicle formation is well characterised, however the mucosal pellicle proteins have only recently been clarified and what drives their formation is still unclear. The aim of this study was to examine the salivary pellicle on particles with different surface properties (hydrophobic or hydrophilic with a positive or negative charge), to determine a suitable model to mimic the mucosal pellicle. A secondary aim was to use the model to test how transglutaminase may alter pellicle formation. Particles were incubated with resting whole mouth saliva, parotid saliva and submandibular/sublingual saliva. Following incubation and two PBS and water washes bound salivary proteins were eluted with two concentrations of SDS, which were later analysed using SDS-PAGE and Western blotting. Experiments were repeated with purified transglutaminase to determine how this epithelial-derived enzyme may alter the bound pellicle. Protein pellicles varied according to the starting salivary composition and the particle chemistry. Amylase, the single most abundant protein in saliva, did not bind to any particle indicating specific protein binding. Most proteins bound through hydrophobic interactions and a few according to their charges. The hydrophobic surface most closely matched the known salivary mucosal pellicle by containing mucins, cystatin and statherin but an absence of amylase and proline-rich proteins. This surface was further used to examine the effect of added transglutaminase. At the concentrations used only statherin showed any evidence of crosslinking with itself or another saliva protein. In conclusion, the formation of the salivary mucosal pellicle is probably mediated, at least in part, by hydrophobic interactions to the epithelial cell surface. PMID:24921197

Pathoproteomics of testicular tissue deficient in the GARP component VPS54: the wobbler mouse model of globozoospermia.

PubMed

Jockusch, Harald; Holland, Ashling; Staunton, Lisa; Schmitt-John, Thomas; Heimann, Peter; Dowling, Paul; Ohlendieck, Kay

2014-04-01

In human globozoospermia, round-headed spermatozoa lack an acrosome and therefore cannot properly interact with oocytes. In the wobbler (WR) mouse, an L967Q missense mutation in the vesicular protein-sorting factor VPS54 causes motor neuron degeneration and globozoospermia. Although electron microscopy of WR testis shows all major components of spermatogenesis, they appear in a deranged morphology that prevents the formation of the acrosome. In order to determine proteome-wide changes, affected testes were analysed by 2D-DIGE and MS. The concentration of 8 proteins was increased and that of 35 proteins decreased as compared to wild type. Mass spectrometric analysis identified proteins with an altered concentration to be associated with metabolite transport, fatty acid metabolism, cellular interactions, microtubule assembly and stress response (chaperones Hsp70-2 and Hsp90α). Minor changes were observed for proteins involved in cell redox homeostasis, cytoskeleton formation, PTMs, detoxification and metabolism. The most dramatically decreased protein in WR testis was identified as fatty acid binding protein FABP3, as confirmed by immunoblot analysis. We conclude that a missense mutation in VPS54, an essential component of the Golgi-associated retrograde protein complex, not only prevents the formation of an acrosome but also initiates a cascade of metabolic abnormalities and a stress reaction. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards Establishment of a Rice Stress Response Interactome

PubMed Central

Seo, Young-Su; Chern, Mawsheng; Bartley, Laura E.; Han, Muho; Jung, Ki-Hong; Lee, Insuk; Walia, Harkamal; Richter, Todd; Xu, Xia; Cao, Peijian; Bai, Wei; Ramanan, Rajeshwari; Amonpant, Fawn; Arul, Loganathan; Canlas, Patrick E.; Ruan, Randy; Park, Chang-Jin; Chen, Xuewei; Hwang, Sohyun; Jeon, Jong-Seong; Ronald, Pamela C.

2011-01-01

Rice (Oryza sativa) is a staple food for more than half the world and a model for studies of monocotyledonous species, which include cereal crops and candidate bioenergy grasses. A major limitation of crop production is imposed by a suite of abiotic and biotic stresses resulting in 30%–60% yield losses globally each year. To elucidate stress response signaling networks, we constructed an interactome of 100 proteins by yeast two-hybrid (Y2H) assays around key regulators of the rice biotic and abiotic stress responses. We validated the interactome using protein–protein interaction (PPI) assays, co-expression of transcripts, and phenotypic analyses. Using this interactome-guided prediction and phenotype validation, we identified ten novel regulators of stress tolerance, including two from protein classes not previously known to function in stress responses. Several lines of evidence support cross-talk between biotic and abiotic stress responses. The combination of focused interactome and systems analyses described here represents significant progress toward elucidating the molecular basis of traits of agronomic importance. PMID:21533176

Construction of reliable protein-protein interaction networks with a new interaction generality measure.

PubMed

Saito, Rintaro; Suzuki, Harukazu; Hayashizaki, Yoshihide

2003-04-12

Recent screening techniques have made large amounts of protein-protein interaction data available, from which biologically important information such as the function of uncharacterized proteins, the existence of novel protein complexes, and novel signal-transduction pathways can be discovered. However, experimental data on protein interactions contain many false positives, making these discoveries difficult. Therefore computational methods of assessing the reliability of each candidate protein-protein interaction are urgently needed. We developed a new 'interaction generality' measure (IG2) to assess the reliability of protein-protein interactions using only the topological properties of their interaction-network structure. Using yeast protein-protein interaction data, we showed that reliable protein-protein interactions had significantly lower IG2 values than less-reliable interactions, suggesting that IG2 values can be used to evaluate and filter interaction data to enable the construction of reliable protein-protein interaction networks.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

In Silico Molecular Modeling and Docking Studies on Novel Mutants (E229V, H225P and D230C) of the Nucleotide-Binding Domain of Homo sapiens Hsp70.

PubMed

Elengoe, Asita; Hamdan, Salehhuddin

2017-12-01

In this study, we explored the possibility of determining the synergistic interactions between nucleotide-binding domain (NBD) of Homo sapiens heat-shock 70 kDa protein (Hsp70) and E1A 32 kDa of adenovirus serotype 5 motif (PNLVP) in the efficiency of killing of tumor cells in cancer treatment. At present, the protein interaction between NBD and PNLVP motif is still unknown, but believed to enhance the rate of virus replication in tumor cells. Three mutant models (E229V, H225P and D230C) were built and simulated, and their interactions with PNLVP motif were studied. The PNLVP motif showed the binding energy and intermolecular energy values with the novel E229V mutant at -7.32 and -11.2 kcal/mol. The E229V mutant had the highest number of hydrogen bonds (7). Based on the root mean square deviation, root mean square fluctuation, hydrogen bonds, salt bridge, secondary structure, surface-accessible solvent area, potential energy and distance matrices analyses, it was proved that the E229V had the strongest and most stable interaction with the PNLVP motif among all the four protein-ligand complex structures. The knowledge of this protein-ligand complex model would help in designing Hsp70 structure-based drug for cancer therapy.

Study of interactions between metal ions and protein model compounds by energy decomposition analyses and the AMOEBA force field

NASA Astrophysics Data System (ADS)

Jing, Zhifeng; Qi, Rui; Liu, Chengwen; Ren, Pengyu

2017-10-01

The interactions between metal ions and proteins are ubiquitous in biology. The selective binding of metal ions has a variety of regulatory functions. Therefore, there is a need to understand the mechanism of protein-ion binding. The interactions involving metal ions are complicated in nature, where short-range charge-penetration, charge transfer, polarization, and many-body effects all contribute significantly, and a quantitative description of all these interactions is lacking. In addition, it is unclear how well current polarizable force fields can capture these energy terms and whether these polarization models are good enough to describe the many-body effects. In this work, two energy decomposition methods, absolutely localized molecular orbitals and symmetry-adapted perturbation theory, were utilized to study the interactions between Mg2+/Ca2+ and model compounds for amino acids. Comparison of individual interaction components revealed that while there are significant charge-penetration and charge-transfer effects in Ca complexes, these effects can be captured by the van der Waals (vdW) term in the AMOEBA force field. The electrostatic interaction in Mg complexes is well described by AMOEBA since the charge penetration is small, but the distance-dependent polarization energy is problematic. Many-body effects were shown to be important for protein-ion binding. In the absence of many-body effects, highly charged binding pockets will be over-stabilized, and the pockets will always favor Mg and thus lose selectivity. Therefore, many-body effects must be incorporated in the force field in order to predict the structure and energetics of metalloproteins. Also, the many-body effects of charge transfer in Ca complexes were found to be non-negligible. The absorption of charge-transfer energy into the additive vdW term was a main source of error for the AMOEBA many-body interaction energies.