Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets*

PubMed Central

Yu, Xueping; Ivanic, Joseph; Memišević, Vesna; Wallqvist, Anders; Reifman, Jaques

2011-01-01

We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not necessarily imply that one detection methodology was better or worse, but rather that, to a large extent, the insights reflected the methodological biases themselves. Consequently, interpreting the protein interaction data within their experimental or cellular context provided the best avenue for overcoming biases and inferring biological knowledge. PMID:21876202

A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex.

PubMed

Arsenovic, Paul T; Bathula, Kranthidhar; Conway, Daniel E

2017-04-11

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy

PubMed Central

Allen, David G.; Whitehead, Nicholas P.; Froehner, Stanley C.

2015-01-01

Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca2+-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca2+ entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease. PMID:26676145

The binary protein-protein interaction landscape of Escherichia coli

PubMed Central

Rajagopala, Seesandra V.; Vlasblom, James; Arnold, Roland; Franca-Koh, Jonathan; Pakala, Suman B.; Phanse, Sadhna; Ceol, Arnaud; Häuser, Roman; Siszler, Gabriella; Wuchty, Stefan; Emili, Andrew; Babu, Mohan; Aloy, Patrick; Pieper, Rembert; Uetz, Peter

2014-01-01

Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (~70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, approximately doubling the number of known binary PPIs in E. coli. Integration of binary PPIs and genetic interactions revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that could be mapped within multi-protein complexes were informative regarding internal topology and indicated that interactions within complexes are significantly more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily significant model microbe. PMID:24561554

Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

PubMed

Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Yang, Jincai; Xie, Wei; Hu, Po; Hu, Xiaohua

2017-01-01

How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI) data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment). It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

Protein-protein interactions within photosystem II under photoprotection: the synergy between CP29 minor antenna, subunit S (PsbS) and zeaxanthin at all-atom resolution.

PubMed

Daskalakis, Vangelis

2018-05-07

The assembly and disassembly of protein complexes within cells are crucial life-sustaining processes. In photosystem II (PSII) of higher plants, there is a delicate yet obscure balance between light harvesting and photo-protection under fluctuating light conditions, that involves protein-protein complexes. Recent breakthroughs in molecular dynamics (MD) simulations are combined with new approaches herein to provide structural and energetic insight into such a complex between the CP29 minor antenna and the PSII subunit S (PsbS). The microscopic model involves extensive sampling of bound and dissociated states at atomic resolution in the presence of photo-protective zeaxanthin (Zea), and reveals well defined protein-protein cross-sections. The complex is placed within PSII, and macroscopic connections are emerging (PsbS-CP29-CP24-CP47) along the energy transfer pathways from the antenna to the PSII core. These connections explain macroscopic observations in the literature, while the previously obscured atomic scale details are now revealed. The implications of these findings are discussed in the context of the Non-Photochemical Quenching (NPQ) of chlorophyll fluorescence, the down-regulatory mechanism of photosynthesis, that enables the protection of PSII against excess excitation load. Zea is found at the PsbS-CP29 cross-section and a pH-dependent equilibrium between PsbS dimer/monomers and the PsbS-CP29 dissociation/association is identified as the target for engineering tolerant plants with increased crop and biomass yields. Finally, the new MD based approaches can be used to probe protein-protein interactions in general, and the PSII structure provided can initiate large scale molecular simulations of the photosynthetic apparatus, under NPQ conditions.

A constraint logic programming approach to associate 1D and 3D structural components for large protein complexes.

PubMed

Dal Palù, Alessandro; Pontelli, Enrico; He, Jing; Lu, Yonggang

2007-01-01

The paper describes a novel framework, constructed using Constraint Logic Programming (CLP) and parallelism, to determine the association between parts of the primary sequence of a protein and alpha-helices extracted from 3D low-resolution descriptions of large protein complexes. The association is determined by extracting constraints from the 3D information, regarding length, relative position and connectivity of helices, and solving these constraints with the guidance of a secondary structure prediction algorithm. Parallelism is employed to enhance performance on large proteins. The framework provides a fast, inexpensive alternative to determine the exact tertiary structure of unknown proteins.

Genome-wide predicting disease-related protein complexes by walking on the heterogeneous network based on data integration and laplacian normalization.

PubMed

Liu, Zhiming; Luo, Jiawei

2017-08-01

Associating protein complexes to human inherited diseases is critical for better understanding of biological processes and functional mechanisms of the disease. Many protein complexes have been identified and functionally annotated by computational and purification methods so far, however, the particular roles they were playing in causing disease have not yet been well determined. In this study, we present a novel method to identify associations between protein complexes and diseases. First, we construct a disease-protein heterogeneous network based on data integration and laplacian normalization. Second, we apply a random walk with restart on heterogeneous network (RWRH) algorithm on this network to quantify the strength of the association between proteins and the query disease. Third, we sum over the scores of member proteins to obtain a summary score for each candidate protein complex, and then rank all candidate protein complexes according to their scores. With a series of leave-one-out cross-validation experiments, we found that our method not only possesses high performance but also demonstrates robustness regarding the parameters and the network structure. We test our approach with breast cancer and select top 20 highly ranked protein complexes, 17 of the selected protein complexes are evidenced to be connected with breast cancer. Our proposed method is effective in identifying disease-related protein complexes based on data integration and laplacian normalization. Copyright © 2017. Published by Elsevier Ltd.

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing.

PubMed

Kim, Yong-Eun; Park, Chungoo; Kim, Kyoon Eon; Kim, Kee K

2018-04-30

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing. Copyright © 2018 Elsevier Inc. All rights reserved.

Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

PubMed Central

Hyung, Suk-Joon; Ruotolo, Brandon T.

2013-01-01

Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

C1orf163/RESA1 is a novel mitochondrial intermembrane space protein connected to respiratory chain assembly.

PubMed

Kozjak-Pavlovic, Vera; Prell, Florian; Thiede, Bernd; Götz, Monika; Wosiek, Dominik; Ott, Christine; Rudel, Thomas

2014-02-20

Oxidative phosphorylation (OXPHOS) in mitochondria takes place at the inner membrane, which folds into numerous cristae. The stability of cristae depends, among other things, on the mitochondrial intermembrane space bridging complex. Its components include inner mitochondrial membrane protein mitofilin and outer membrane protein Sam50. We identified a conserved, uncharacterized protein, C1orf163 [SEL1 repeat containing 1 protein (SELRC1)], as one of the proteins significantly reduced after the knockdown of Sam50 and mitofilin. We show that C1orf163 is a mitochondrial soluble intermembrane space protein. Sam50 depletion affects moderately the import and assembly of C1orf163 into two protein complexes of approximately 60kDa and 150kDa. We observe that the knockdown of C1orf163 leads to reduction of levels of proteins belonging to the OXPHOS complexes. The activity of complexes I and IV is reduced in C1orf163-depleted cells, and we observe the strongest defects in the assembly of complex IV. Therefore, we propose C1orf163 to be a novel factor important for the assembly of respiratory chain complexes in human mitochondria and suggest to name it RESA1 (for RESpiratory chain Assembly 1). Copyright © 2013 Elsevier Ltd. All rights reserved.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

PubMed

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-09-25

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Bio-AIMS Collection of Chemoinformatics Web Tools based on Molecular Graph Information and Artificial Intelligence Models.

PubMed

Munteanu, Cristian R; Gonzalez-Diaz, Humberto; Garcia, Rafael; Loza, Mabel; Pazos, Alejandro

2015-01-01

The molecular information encoding into molecular descriptors is the first step into in silico Chemoinformatics methods in Drug Design. The Machine Learning methods are a complex solution to find prediction models for specific biological properties of molecules. These models connect the molecular structure information such as atom connectivity (molecular graphs) or physical-chemical properties of an atom/group of atoms to the molecular activity (Quantitative Structure - Activity Relationship, QSAR). Due to the complexity of the proteins, the prediction of their activity is a complicated task and the interpretation of the models is more difficult. The current review presents a series of 11 prediction models for proteins, implemented as free Web tools on an Artificial Intelligence Model Server in Biosciences, Bio-AIMS (http://bio-aims.udc.es/TargetPred.php). Six tools predict protein activity, two models evaluate drug - protein target interactions and the other three calculate protein - protein interactions. The input information is based on the protein 3D structure for nine models, 1D peptide amino acid sequence for three tools and drug SMILES formulas for two servers. The molecular graph descriptor-based Machine Learning models could be useful tools for in silico screening of new peptides/proteins as future drug targets for specific treatments.

An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

NASA Astrophysics Data System (ADS)

Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R.; Campuzano, Iain D. G.; Loo, Joseph A.

2018-02-01

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

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

Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay

2011-01-01

The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, wemore » detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.« less

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

PubMed

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

PubMed Central

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-01-01

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135

Community of protein complexes impacts disease association

PubMed Central

Wang, Qianghu; Liu, Weisha; Ning, Shangwei; Ye, Jingrun; Huang, Teng; Li, Yan; Wang, Peng; Shi, Hongbo; Li, Xia

2012-01-01

One important challenge in the post-genomic era is uncovering the relationships among distinct pathophenotypes by using molecular signatures. Given the complex functional interdependencies between cellular components, a disease is seldom the consequence of a defect in a single gene product, instead reflecting the perturbations of a group of closely related gene products that carry out specific functions together. Therefore, it is meaningful to explore how the community of protein complexes impacts disease associations. Here, by integrating a large amount of information from protein complexes and the cellular basis of diseases, we built a human disease network in which two diseases are linked if they share common disease-related protein complex. A systemic analysis revealed that linked disease pairs exhibit higher comorbidity than those that have no links, and that the stronger association two diseases have based on protein complexes, the higher comorbidity they are prone to display. Moreover, more connected diseases tend to be malignant, which have high prevalence. We provide novel disease associations that cannot be identified through previous analysis. These findings will potentially provide biologists and clinicians new insights into the etiology, classification and treatment of diseases. PMID:22549411

Synaptogenesis Is Modulated by Heparan Sulfate in Caenorhabditis elegans

PubMed Central

Lázaro-Peña, María I.; Díaz-Balzac, Carlos A.; Bülow, Hannes E.; Emmons, Scott W.

2018-01-01

The nervous system regulates complex behaviors through a network of neurons interconnected by synapses. How specific synaptic connections are genetically determined is still unclear. Male mating is the most complex behavior in Caenorhabditis elegans. It is composed of sequential steps that are governed by > 3000 chemical connections. Here, we show that heparan sulfates (HS) play a role in the formation and function of the male neural network. HS, sulfated in position 3 by the HS modification enzyme HST-3.1/HS 3-O-sulfotransferase and attached to the HS proteoglycan glypicans LON-2/glypican and GPN-1/glypican, functions cell-autonomously and nonautonomously for response to hermaphrodite contact during mating. Loss of 3-O sulfation resulted in the presynaptic accumulation of RAB-3, a molecule that localizes to synaptic vesicles, and disrupted the formation of synapses in a component of the mating circuits. We also show that the neural cell adhesion protein NRX-1/neurexin promotes and the neural cell adhesion protein NLG-1/neuroligin inhibits the formation of the same set of synapses in a parallel pathway. Thus, neural cell adhesion proteins and extracellular matrix components act together in the formation of synaptic connections. PMID:29559501

A novel method for identifying disease associated protein complexes based on functional similarity protein complex networks.

PubMed

Le, Duc-Hau

2015-01-01

Protein complexes formed by non-covalent interaction among proteins play important roles in cellular functions. Computational and purification methods have been used to identify many protein complexes and their cellular functions. However, their roles in terms of causing disease have not been well discovered yet. There exist only a few studies for the identification of disease-associated protein complexes. However, they mostly utilize complicated heterogeneous networks which are constructed based on an out-of-date database of phenotype similarity network collected from literature. In addition, they only apply for diseases for which tissue-specific data exist. In this study, we propose a method to identify novel disease-protein complex associations. First, we introduce a framework to construct functional similarity protein complex networks where two protein complexes are functionally connected by either shared protein elements, shared annotating GO terms or based on protein interactions between elements in each protein complex. Second, we propose a simple but effective neighborhood-based algorithm, which yields a local similarity measure, to rank disease candidate protein complexes. Comparing the predictive performance of our proposed algorithm with that of two state-of-the-art network propagation algorithms including one we used in our previous study, we found that it performed statistically significantly better than that of these two algorithms for all the constructed functional similarity protein complex networks. In addition, it ran about 32 times faster than these two algorithms. Moreover, our proposed method always achieved high performance in terms of AUC values irrespective of the ways to construct the functional similarity protein complex networks and the used algorithms. The performance of our method was also higher than that reported in some existing methods which were based on complicated heterogeneous networks. Finally, we also tested our method with prostate cancer and selected the top 100 highly ranked candidate protein complexes. Interestingly, 69 of them were evidenced since at least one of their protein elements are known to be associated with prostate cancer. Our proposed method, including the framework to construct functional similarity protein complex networks and the neighborhood-based algorithm on these networks, could be used for identification of novel disease-protein complex associations.

PDBToSDF: Create ligand structure files from PDB file.

PubMed

Muppalaneni, Naresh Babu; Rao, Allam Appa

2011-01-01

Protein Data Bank (PDB) file contains atomic data for protein and ligand in protein-ligand complexes. Structure data file (SDF) contains data for atoms, bonds, connectivity and coordinates of molecule for ligands. We describe PDBToSDF as a tool to separate the ligand data from pdb file for the calculation of ligand properties like molecular weight, number of hydrogen bond acceptors, hydrogen bond receptors easily.

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches

PubMed Central

Muñoz, Victor; Cerminara, Michele

2016-01-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico. All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. PMID:27574021

Usher syndrome: molecular links of pathogenesis, proteins and pathways.

PubMed

Kremer, Hannie; van Wijk, Erwin; Märker, Tina; Wolfrum, Uwe; Roepman, Ronald

2006-10-15

Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in the synaptic processes of both cell types. The association of other proteins with the complex indicates functional links to a number of basic cell-biological processes. Prominently present is the connection to the dynamics of the actin cytoskeleton, involved in cellular morphology, cell polarity and cell-cell interactions. The Usher protein complex can also be linked to the cadherins/catenins in the adherens junction-associated protein complexes, suggesting a role in cell polarity and tissue organization. A third link can be established to the integrin transmembrane signaling network. The Usher interactome, as outlined in this review, participates in pathways common in inner ear and retina that are disrupted in the Usher syndrome.

The length but not the sequence of peptide linker modules exerts the primary influence on the conformations of protein domains in cellulosome multi-enzyme complexes.

PubMed

Różycki, Bartosz; Cazade, Pierre-André; O'Mahony, Shane; Thompson, Damien; Cieplak, Marek

2017-08-16

Cellulosomes are large multi-protein catalysts produced by various anaerobic microorganisms to efficiently degrade plant cell-wall polysaccharides down into simple sugars. X-ray and physicochemical structural characterisations show that cellulosomes are composed of numerous protein domains that are connected by unstructured polypeptide segments, yet the properties and possible roles of these 'linker' peptides are largely unknown. We have performed coarse-grained and all-atom molecular dynamics computer simulations of a number of cellulosomal linkers of different lengths and compositions. Our data demonstrates that the effective stiffness of the linker peptides, as quantified by the equilibrium fluctuations in the end-to-end distances, depends primarily on the length of the linker and less so on the specific amino acid sequence. The presence of excluded volume - provided by the domains that are connected - dampens the motion of the linker residues and reduces the effective stiffness of the linkers. Simultaneously, the presence of the linkers alters the conformations of the protein domains that are connected. We demonstrate that short, stiff linkers induce significant rearrangements in the folded domains of the mini-cellulosome composed of endoglucanase Cel8A in complex with scaffoldin ScafT (Cel8A-ScafT) of Clostridium thermocellum as well as in a two-cohesin system derived from the scaffoldin ScaB of Acetivibrio cellulolyticus. We give experimentally testable predictions on structural changes in protein domains that depend on the length of linkers.

Protein-protein interaction networks (PPI) and complex diseases

PubMed Central

Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

2014-01-01

The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

Interactions among tobacco sieve element occlusion (SEO) proteins.

PubMed

Jekat, Stephan B; Ernst, Antonia M; Zielonka, Sascia; Noll, Gundula A; Prüfer, Dirk

2012-12-01

Angiosperms transport their photoassimilates through sieve tubes, which comprise longitudinally-connected sieve elements. In dicots and also some monocots, the sieve elements contain parietal structural proteins known as phloem proteins or P-proteins. Following injury, P proteins disperse and accumulate as viscous plugs at the sieve plates to prevent the loss of valuable transport sugars. Tobacco (Nicotiana tabacum) P-proteins are multimeric complexes comprising subunits encoded by members of the SEO (sieve element occlusion) gene family. The existence of multiple subunits suggests that P-protein assembly involves interactions between SEO proteins, but this process is largely uncharacterized and it is unclear whether the different subunits perform unique roles or are redundant. We therefore extended our analysis of the tobacco P-proteins NtSEO1 and NtSEO2 to investigate potential interactions between them, and found that both proteins can form homomeric and heteromeric complexes in planta.

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis.

PubMed

Mahmoud, Shima; Planes, María Dolores; Cabedo, Marc; Trujillo, Cristina; Rienzo, Alessandro; Caballero-Molada, Marcos; Sharma, Sukesh C; Montesinos, Consuelo; Mulet, José Miguel; Serrano, Ramón

2017-07-01

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H + -ATPase Pma1 (which drives nutrient and K + uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K + uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K + transport. © 2017 Federation of European Biochemical Societies.

Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments.

PubMed

Rocha, M S

2015-09-01

In this review we focus on the idea of establishing connections between the mechanical properties of DNA-ligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in particular when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA-ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch DNA-ligand complexes and to obtain "force × extension" data, from which the mechanical properties of the complexes can be determined. We also discuss the characteristics of the main types of interactions that can occur between DNA and ligands, from covalent binding to simple electrostatic driven interactions. Finally, we present a historical survey of the attempts to connect mechanics to physical chemistry for DNA-ligand systems, emphasizing a recently developed fitting approach useful to connect the persistence length of DNA-ligand complexes to the physicochemical properties of the interaction. Such an approach in principle can be used for any type of ligand, from drugs to proteins, even if multiple binding modes are present.

LINCing complex functions at the nuclear envelope

PubMed Central

Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

2013-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

Faster Mass Spectrometry-based Protein Inference: Junction Trees are More Efficient than Sampling and Marginalization by Enumeration

PubMed Central

Serang, Oliver; Noble, William Stafford

2012-01-01

The problem of identifying the proteins in a complex mixture using tandem mass spectrometry can be framed as an inference problem on a graph that connects peptides to proteins. Several existing protein identification methods make use of statistical inference methods for graphical models, including expectation maximization, Markov chain Monte Carlo, and full marginalization coupled with approximation heuristics. We show that, for this problem, the majority of the cost of inference usually comes from a few highly connected subgraphs. Furthermore, we evaluate three different statistical inference methods using a common graphical model, and we demonstrate that junction tree inference substantially improves rates of convergence compared to existing methods. The python code used for this paper is available at http://noble.gs.washington.edu/proj/fido. PMID:22331862

Keeping the LINC: the importance of nucleocytoskeletal coupling in intracellular force transmission and cellular function.

PubMed

Lombardi, Maria L; Lammerding, Jan

2011-12-01

Providing a stable physical connection between the nucleus and the cytoskeleton is essential for a wide range of cellular functions and it could also participate in mechanosensing by transmitting intra- and extra-cellular mechanical stimuli via the cytoskeleton to the nucleus. Nesprins and SUN proteins, located at the nuclear envelope, form the LINC (linker of nucleoskeleton and cytoskeleton) complex that connects the nucleus to the cytoskeleton; underlying nuclear lamins contribute to anchoring LINC complex components at the nuclear envelope. Disruption of the LINC complex or loss of lamins can result in disturbed perinuclear actin and intermediate filament networks and causes severe functional defects, including impaired nuclear positioning, cell polarization and cell motility. Recent studies have identified the LINC complex as the major force-transmitting element at the nuclear envelope and suggest that many of the aforementioned defects can be attributed to disturbed force transmission between the nucleus and the cytoskeleton. Thus mutations in nesprins, SUN proteins or lamins, which have been linked to muscular dystrophies and cardiomyopathies, may weaken or completely eliminate LINC complex function at the nuclear envelope and result in impaired intracellular force transmission, thereby disrupting critical cellular functions.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity

PubMed Central

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-01-01

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity. PMID:22955885

Modular synchronization in complex networks.

PubMed

Oh, E; Rho, K; Hong, H; Kahng, B

2005-10-01

We study the synchronization transition (ST) of a modified Kuramoto model on two different types of modular complex networks. It is found that the ST depends on the type of intermodular connections. For the network with decentralized (centralized) intermodular connections, the ST occurs at finite coupling constant (behaves abnormally). Such distinct features are found in the yeast protein interaction network and the Internet, respectively. Moreover, by applying the finite-size scaling analysis to an artificial network with decentralized intermodular connections, we obtain the exponent associated with the order parameter of the ST to be beta approximately 1 different from beta(MF) approximately 1/2 obtained from the scale-free network with the same degree distribution but the absence of modular structure, corresponding to the mean field value.

The diminishing role of hubs in dynamical processes on complex networks.

PubMed

Quax, Rick; Apolloni, Andrea; Sloot, Peter M A

2013-11-06

It is notoriously difficult to predict the behaviour of a complex self-organizing system, where the interactions among dynamical units form a heterogeneous topology. Even if the dynamics of each microscopic unit is known, a real understanding of their contributions to the macroscopic system behaviour is still lacking. Here, we develop information-theoretical methods to distinguish the contribution of each individual unit to the collective out-of-equilibrium dynamics. We show that for a system of units connected by a network of interaction potentials with an arbitrary degree distribution, highly connected units have less impact on the system dynamics when compared with intermediately connected units. In an equilibrium setting, the hubs are often found to dictate the long-term behaviour. However, we find both analytically and experimentally that the instantaneous states of these units have a short-lasting effect on the state trajectory of the entire system. We present qualitative evidence of this phenomenon from empirical findings about a social network of product recommendations, a protein-protein interaction network and a neural network, suggesting that it might indeed be a widespread property in nature.

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

PubMed

Muñoz, Victor; Cerminara, Michele

2016-09-01

Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).

Genes2Networks: connecting lists of gene symbols using mammalian protein interactions databases.

PubMed

Berger, Seth I; Posner, Jeremy M; Ma'ayan, Avi

2007-10-04

In recent years, mammalian protein-protein interaction network databases have been developed. The interactions in these databases are either extracted manually from low-throughput experimental biomedical research literature, extracted automatically from literature using techniques such as natural language processing (NLP), generated experimentally using high-throughput methods such as yeast-2-hybrid screens, or interactions are predicted using an assortment of computational approaches. Genes or proteins identified as significantly changing in proteomic experiments, or identified as susceptibility disease genes in genomic studies, can be placed in the context of protein interaction networks in order to assign these genes and proteins to pathways and protein complexes. Genes2Networks is a software system that integrates the content of ten mammalian interaction network datasets. Filtering techniques to prune low-confidence interactions were implemented. Genes2Networks is delivered as a web-based service using AJAX. The system can be used to extract relevant subnetworks created from "seed" lists of human Entrez gene symbols. The output includes a dynamic linkable three color web-based network map, with a statistical analysis report that identifies significant intermediate nodes used to connect the seed list. Genes2Networks is powerful web-based software that can help experimental biologists to interpret lists of genes and proteins such as those commonly produced through genomic and proteomic experiments, as well as lists of genes and proteins associated with disease processes. This system can be used to find relationships between genes and proteins from seed lists, and predict additional genes or proteins that may play key roles in common pathways or protein complexes.

A Global Protein Kinase and Phosphatase Interaction Network in Yeast

PubMed Central

Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

2011-01-01

The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture

PubMed Central

Ding, Zhao-Ying; Huang, Yu-Cheng; Lee, Myong-Chol; Tseng, Min-Jen; Chi, Ya-Hui

2017-01-01

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture. PMID:28716842

Proteins Encoded in Genomic Regions Associated with Immune-Mediated Disease Physically Interact and Suggest Underlying Biology

PubMed Central

Rossin, Elizabeth J.; Lage, Kasper; Raychaudhuri, Soumya; Xavier, Ramnik J.; Tatar, Diana; Benita, Yair

2011-01-01

Genome-wide association studies (GWAS) have defined over 150 genomic regions unequivocally containing variation predisposing to immune-mediated disease. Inferring disease biology from these observations, however, hinges on our ability to discover the molecular processes being perturbed by these risk variants. It has previously been observed that different genes harboring causal mutations for the same Mendelian disease often physically interact. We sought to evaluate the degree to which this is true of genes within strongly associated loci in complex disease. Using sets of loci defined in rheumatoid arthritis (RA) and Crohn's disease (CD) GWAS, we build protein–protein interaction (PPI) networks for genes within associated loci and find abundant physical interactions between protein products of associated genes. We apply multiple permutation approaches to show that these networks are more densely connected than chance expectation. To confirm biological relevance, we show that the components of the networks tend to be expressed in similar tissues relevant to the phenotypes in question, suggesting the network indicates common underlying processes perturbed by risk loci. Furthermore, we show that the RA and CD networks have predictive power by demonstrating that proteins in these networks, not encoded in the confirmed list of disease associated loci, are significantly enriched for association to the phenotypes in question in extended GWAS analysis. Finally, we test our method in 3 non-immune traits to assess its applicability to complex traits in general. We find that genes in loci associated to height and lipid levels assemble into significantly connected networks but did not detect excess connectivity among Type 2 Diabetes (T2D) loci beyond chance. Taken together, our results constitute evidence that, for many of the complex diseases studied here, common genetic associations implicate regions encoding proteins that physically interact in a preferential manner, in line with observations in Mendelian disease. PMID:21249183

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions.

PubMed

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel; Søgaard-Andersen, Lotte; Mignot, Tâm

2015-07-20

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate-bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA-MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein-cytoskeleton interactions are a universally conserved feature. © 2015 Treuner-Lange et al.

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

PubMed

Youn, Ji-Young; Dunham, Wade H; Hong, Seo Jung; Knight, James D R; Bashkurov, Mikhail; Chen, Ginny I; Bagci, Halil; Rathod, Bhavisha; MacLeod, Graham; Eng, Simon W M; Angers, Stéphane; Morris, Quaid; Fabian, Marc; Côté, Jean-François; Gingras, Anne-Claude

2018-02-01

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

Structural requirements for the assembly of LINC complexes and their function in cellular mechanical stiffness

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

Stewart-Hutchinson, P.J.; Hale, Christopher M.; Wirtz, Denis

The evolutionary-conserved interactions between KASH and SUN domain-containing proteins within the perinuclear space establish physical connections, called LINC complexes, between the nucleus and the cytoskeleton. Here, we show that the KASH domains of Nesprins 1, 2 and 3 interact promiscuously with luminal domains of Sun1 and Sun2. These constructs disrupt endogenous LINC complexes as indicated by the displacement of endogenous Nesprins from the nuclear envelope. We also provide evidence that KASH domains most probably fit a pocket provided by SUN domains and that post-translational modifications are dispensable for that interaction. We demonstrate that the disruption of endogenous LINC complexes affectmore » cellular mechanical stiffness to an extent that compares to the loss of mechanical stiffness previously reported in embryonic fibroblasts derived from mouse lacking A-type lamins, a mouse model of muscular dystrophies and cardiomyopathies. These findings support a model whereby physical connections between the nucleus and the cytoskeleton are mediated by interactions between diverse combinations of Sun proteins and Nesprins through their respective evolutionary-conserved domains. Furthermore, they emphasize, for the first time, the relevance of LINC complexes in cellular mechanical stiffness suggesting a possible involvement of their disruption in various laminopathies, a group of human diseases linked to mutations of A-type lamins.« less

Triangle network motifs predict complexes by complementing high-error interactomes with structural information.

PubMed

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-06-27

A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN.

Triangle network motifs predict complexes by complementing high-error interactomes with structural information

PubMed Central

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-01-01

Background A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. Results We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Conclusion Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN. PMID:19558694

Corolla Is a Novel Protein That Contributes to the Architecture of the Synaptonemal Complex of Drosophila

PubMed Central

Collins, Kimberly A.; Unruh, Jay R.; Slaughter, Brian D.; Yu, Zulin; Lake, Cathleen M.; Nielsen, Rachel J.; Box, Kimberly S.; Miller, Danny E.; Blumenstiel, Justin P.; Perera, Anoja G.; Malanowski, Kathryn E.; Hawley, R. Scott

2014-01-01

In most organisms the synaptonemal complex (SC) connects paired homologs along their entire length during much of meiotic prophase. To better understand the structure of the SC, we aim to identify its components and to determine how each of these components contributes to SC function. Here, we report the identification of a novel SC component in Drosophila melanogaster female oocytes, which we have named Corolla. Using structured illumination microscopy, we demonstrate that Corolla is a component of the central region of the SC. Consistent with its localization, we show by yeast two-hybrid analysis that Corolla strongly interacts with Cona, a central element protein, demonstrating the first direct interaction between two inner-synaptonemal complex proteins in Drosophila. These observations help provide a more complete model of SC structure and function in Drosophila females. PMID:24913682

Taking structure searches to the next dimension.

PubMed

Schafferhans, Andrea; Rost, Burkhard

2014-07-08

Structure comparisons are now the first step when a new experimental high-resolution protein structure has been determined. In this issue of Structure, Wiederstein and colleagues describe their latest tool for comparing structures, which gives us the unprecedented power to discover crucial structural connections between whole complexes of proteins in the full structural database in real time. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evidence for the Existence of One Antenna-Associated, Lipid-Dissolved and Two Protein-Bound Pools of Diadinoxanthin Cycle Pigments in Diatoms[C][W

PubMed Central

Lepetit, Bernard; Volke, Daniela; Gilbert, Matthias; Wilhelm, Christian; Goss, Reimund

2010-01-01

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls. PMID:20935178

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.

Split green fluorescent protein as a modular binding partner for protein crystallization.

PubMed

Nguyen, Hau B; Hung, Li-Wei; Yeates, Todd O; Terwilliger, Thomas C; Waldo, Geoffrey S

2013-12-01

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

Split green fluorescent protein as a modular binding partner for protein crystallization

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

Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.

2013-12-01

A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was testedmore » by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization.« less

Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

PubMed Central

Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

2016-01-01

Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

Network based approaches reveal clustering in protein point patterns

NASA Astrophysics Data System (ADS)

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

2014-03-01

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

LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex

PubMed Central

Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni

2014-01-01

Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275

[Fanconi anemia: genes and function(s) revisited].

PubMed

Papadopoulo, Dora; Moustacchi, Ethel

2005-01-01

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Toroidal surface complexes of bacteriophage {phi}12 are responsible for host-cell attachment

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

Leo-Macias, Alejandra; Katz, Garrett; Wei Hui

2011-06-05

Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage {phi}12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of {phi}12 demonstrates that the gene for the hexameric spike is located in {phi}12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimatesmore » derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity. - Research Highlights: > Subtomogram averaging reveals enhanced detail of a {phi}12 cystovirus surface protein complex. > The surface protein complex has a toroidal shape and six-fold symmetry. > It is encoded by the medium-size genome segment. > The proteins of the surface complex most likely are one copy of P3a and three copies of P3c.« less

Nonlinear network model analysis of vibrational energy transfer and localisation in the Fenna-Matthews-Olson complex

NASA Astrophysics Data System (ADS)

Morgan, Sarah E.; Cole, Daniel J.; Chin, Alex W.

2016-11-01

Collective protein modes are expected to be important for facilitating energy transfer in the Fenna-Matthews-Olson (FMO) complex of photosynthetic green sulphur bacteria, however to date little work has focussed on the microscopic details of these vibrations. The nonlinear network model (NNM) provides a computationally inexpensive approach to studying vibrational modes at the microscopic level in large protein structures, whilst incorporating anharmonicity in the inter-residue interactions which can influence protein dynamics. We apply the NNM to the entire trimeric FMO complex and find evidence for the existence of nonlinear discrete breather modes. These modes tend to transfer energy to the highly connected core pigments, potentially opening up alternative excitation energy transfer routes through their influence on pigment properties. Incorporating localised modes based on these discrete breathers in the optical spectra calculations for FMO using ab initio site energies and excitonic couplings can substantially improve their agreement with experimental results.

A General, Adaptive, Roadmap-Based Algorithm for Protein Motion Computation.

PubMed

Molloy, Kevin; Shehu, Amarda

2016-03-01

Precious information on protein function can be extracted from a detailed characterization of protein equilibrium dynamics. This remains elusive in wet and dry laboratories, as function-modulating transitions of a protein between functionally-relevant, thermodynamically-stable and meta-stable structural states often span disparate time scales. In this paper we propose a novel, robotics-inspired algorithm that circumvents time-scale challenges by drawing analogies between protein motion and robot motion. The algorithm adapts the popular roadmap-based framework in robot motion computation to handle the more complex protein conformation space and its underlying rugged energy surface. Given known structures representing stable and meta-stable states of a protein, the algorithm yields a time- and energy-prioritized list of transition paths between the structures, with each path represented as a series of conformations. The algorithm balances computational resources between a global search aimed at obtaining a global view of the network of protein conformations and their connectivity and a detailed local search focused on realizing such connections with physically-realistic models. Promising results are presented on a variety of proteins that demonstrate the general utility of the algorithm and its capability to improve the state of the art without employing system-specific insight.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

PubMed

Bai, Fang; Morcos, Faruck; Cheng, Ryan R; Jiang, Hualiang; Onuchic, José N

2016-12-13

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

Emerging connections between RNA and autophagy.

PubMed

Frankel, Lisa B; Lubas, Michal; Lund, Anders H

2017-01-02

Macroautophagy/autophagy is a key catabolic process, essential for maintaining cellular homeostasis and survival through the removal and recycling of unwanted cellular material. Emerging evidence has revealed intricate connections between the RNA and autophagy research fields. While a majority of studies have focused on protein, lipid and carbohydrate catabolism via autophagy, accumulating data supports the view that several types of RNA and associated ribonucleoprotein complexes are specifically recruited to phagophores (precursors to autophagosomes) and subsequently degraded in the lysosome/vacuole. Moreover, recent studies have revealed a substantial number of novel autophagy regulators with RNA-related functions, indicating roles for RNA and associated proteins not only as cargo, but also as regulators of this process. In this review, we discuss widespread evidence of RNA catabolism via autophagy in yeast, plants and animals, reviewing the molecular mechanisms and biological importance in normal physiology, stress and disease. In addition, we explore emerging evidence of core autophagy regulation mediated by RNA-binding proteins and noncoding RNAs, and point to gaps in our current knowledge of the connection between RNA and autophagy. Finally, we discuss the pathological implications of RNA-protein aggregation, primarily in the context of neurodegenerative disease.

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

A Schiff base connectivity switch in sensory rhodopsin signaling

PubMed Central

Sineshchekov, Oleg A.; Sasaki, Jun; Phillips, Brian J.; Spudich, John L.

2008-01-01

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI–HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI–HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pKa. These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition. PMID:18852467

A Schiff base connectivity switch in sensory rhodopsin signaling.

PubMed

Sineshchekov, Oleg A; Sasaki, Jun; Phillips, Brian J; Spudich, John L

2008-10-21

Sensory rhodopsin I (SRI) in Halobacterium salinarum acts as a receptor for single-quantum attractant and two-quantum repellent phototaxis, transmitting light stimuli via its bound transducer HtrI. Signal-inverting mutations in the SRI-HtrI complex reverse the single-quantum response from attractant to repellent. Fast intramolecular charge movements reported here reveal that the unphotolyzed SRI-HtrI complex exists in two conformational states, which differ by their connection of the retinylidene Schiff base in the SRI photoactive site to inner or outer half-channels. In single-quantum photochemical reactions, the conformer with the Schiff base connected to the cytoplasmic (CP) half-channel generates an attractant signal, whereas the conformer with the Schiff base connected to the extracellular (EC) half-channel generates a repellent signal. In the wild-type complex the conformer equilibrium is poised strongly in favor of that with CP-accessible Schiff base. Signal-inverting mutations shift the equilibrium in favor of the EC-accessible Schiff base form, and suppressor mutations shift the equilibrium back toward the CP-accessible Schiff base form, restoring the wild-type phenotype. Our data show that the sign of the behavioral response directly correlates with the state of the connectivity switch, not with the direction of proton movements or changes in acceptor pK(a). These findings identify a shared fundamental process in the mechanisms of transport and signaling by the rhodopsin family. Furthermore, the effects of mutations in the HtrI subunit of the complex on SRI Schiff base connectivity indicate that the two proteins are tightly coupled to form a single unit that undergoes a concerted conformational transition.

Molecular Dynamics Study of HIV-1 RT-DNA-Nevirapine Complexes Explains NNRTI Inhibition, and Resistance by Connection Mutations

PubMed Central

Vijayan, R.S.K.; Arnold, Eddy; Das, Kalyan

2015-01-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. PMID:24174331

Single-molecule pull-down (SiMPull) for new-age biochemistry: methodology and biochemical applications of single-molecule pull-down (SiMPull) for probing biomolecular interactions in crude cell extracts.

PubMed

Aggarwal, Vasudha; Ha, Taekjip

2014-11-01

Macromolecular interactions play a central role in many biological processes. Protein-protein interactions have mostly been studied by co-immunoprecipitation, which cannot provide quantitative information on all possible molecular connections present in the complex. We will review a new approach that allows cellular proteins and biomolecular complexes to be studied in real-time at the single-molecule level. This technique is called single-molecule pull-down (SiMPull), because it integrates principles of conventional immunoprecipitation with the powerful single-molecule fluorescence microscopy. SiMPull is used to count how many of each protein is present in the physiological complexes found in cytosol and membranes. Concurrently, it serves as a single-molecule biochemical tool to perform functional studies on the pulled-down proteins. In this review, we will focus on the detailed methodology of SiMPull, its salient features and a wide range of biological applications in comparison with other biosensing tools. © 2014 WILEY Periodicals, Inc.

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

PubMed

Bausewein, Thomas; Mills, Deryck J; Langer, Julian D; Nitschke, Beate; Nussberger, Stephan; Kühlbrandt, Werner

2017-08-10

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

STRIPAK complexes: structure, biological function, and involvement in human diseases.

PubMed

Hwang, Juyeon; Pallas, David C

2014-02-01

The mammalian striatin family consists of three proteins, striatin, S/G2 nuclear autoantigen, and zinedin. Striatin family members have no intrinsic catalytic activity, but rather function as scaffolding proteins. Remarkably, they organize multiple diverse, large signaling complexes that participate in a variety of cellular processes. Moreover, they appear to be regulatory/targeting subunits for the major eukaryotic serine/threonine protein phosphatase 2A. In addition, striatin family members associate with germinal center kinase III kinases as well as other novel components, earning these assemblies the name striatin-interacting phosphatase and kinase (STRIPAK) complexes. Recently, there has been a great increase in functional and mechanistic studies aimed at identifying and understanding the roles of STRIPAK and STRIPAK-like complexes in cellular processes of multiple organisms. These studies have identified novel STRIPAK and STRIPAK-like complexes and have explored their roles in specific signaling pathways. Together, the results of these studies have sparked increased interest in striatin family complexes because they have revealed roles in signaling, cell cycle control, apoptosis, vesicular trafficking, Golgi assembly, cell polarity, cell migration, neural and vascular development, and cardiac function. Moreover, STRIPAK complexes have been connected to clinical conditions, including cardiac disease, diabetes, autism, and cerebral cavernous malformation. In this review, we discuss the expression, localization, and protein domain structure of striatin family members. Then we consider the diverse complexes these proteins and their homologs form in various organisms, emphasizing what is known regarding function and regulation. Finally, we explore possible roles of striatin family complexes in disease, especially cerebral cavernous malformation. Copyright © 2013 Elsevier Ltd. All rights reserved.

The small G-protein MglA connects to the MreB actin cytoskeleton at bacterial focal adhesions

PubMed Central

Treuner-Lange, Anke; Macia, Eric; Guzzo, Mathilde; Hot, Edina; Faure, Laura M.; Jakobczak, Beata; Espinosa, Leon; Alcor, Damien; Ducret, Adrien; Keilberg, Daniela; Castaing, Jean Philippe; Lacas Gervais, Sandra; Franco, Michel

2015-01-01

In Myxococcus xanthus the gliding motility machinery is assembled at the leading cell pole to form focal adhesions, translocated rearward to propel the cell, and disassembled at the lagging pole. We show that MglA, a Ras-like small G-protein, is an integral part of this machinery. In this function, MglA stimulates the assembly of the motility complex by directly connecting it to the MreB actin cytoskeleton. Because the nucleotide state of MglA is regulated spatially and MglA only binds MreB in the guanosine triphosphate–bound form, the motility complexes are assembled at the leading pole and dispersed at the lagging pole where the guanosine triphosphatase activating protein MglB disrupts the MglA–MreB interaction. Thus, MglA acts as a nucleotide-dependent molecular switch to regulate the motility machinery spatially. The function of MreB in motility is independent of its function in peptidoglycan synthesis, representing a coopted function. Our findings highlight a new function for the MreB cytoskeleton and suggest that G-protein–cytoskeleton interactions are a universally conserved feature. PMID:26169353

14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

PubMed Central

Xu, Jennifer; Steele-Ogus, Melissa; Alas, Germain C. M.

2017-01-01

ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes. PMID:28932813

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

Protein complexes and functional modules in molecular networks

NASA Astrophysics Data System (ADS)

Spirin, Victor; Mirny, Leonid A.

2003-10-01

Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

Mitochondrial Protein Interaction Mapping Identifies Regulators of Respiratory Chain Function.

PubMed

Floyd, Brendan J; Wilkerson, Emily M; Veling, Mike T; Minogue, Catie E; Xia, Chuanwu; Beebe, Emily T; Wrobel, Russell L; Cho, Holly; Kremer, Laura S; Alston, Charlotte L; Gromek, Katarzyna A; Dolan, Brendan K; Ulbrich, Arne; Stefely, Jonathan A; Bohl, Sarah L; Werner, Kelly M; Jochem, Adam; Westphall, Michael S; Rensvold, Jarred W; Taylor, Robert W; Prokisch, Holger; Kim, Jung-Ja P; Coon, Joshua J; Pagliarini, David J

2016-08-18

Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal functions for these proteins (termed MXPs), we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron-transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function. Copyright © 2016 Elsevier Inc. All rights reserved.

Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators.

PubMed

Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter

2011-12-09

Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.

Plant cytoskeleton: DELLA connects gibberellins to microtubules.

PubMed

Dixit, Ram

2013-06-03

A new study reveals that DELLA proteins directly interact with the prefoldin complex, thus regulating tubulin subunit availability in a gibberellin-dependent manner. This finding provides a mechanistic link between the growth-promoting plant hormone gibberellin and cortical microtubule organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Locating overlapping dense subgraphs in gene (protein) association networks and predicting novel protein functional groups among these subgraphs

NASA Astrophysics Data System (ADS)

Palla, Gergely; Derenyi, Imre; Farkas, Illes J.; Vicsek, Tamas

2006-03-01

Most tasks in a cell are performed not by individual proteins, but by functional groups of proteins (either physically interacting with each other or associated in other ways). In gene (protein) association networks these groups show up as sets of densely connected nodes. In the yeast, Saccharomyces cerevisiae, known physically interacting groups of proteins (called protein complexes) strongly overlap: the total number of proteins contained by these complexes by far underestimates the sum of their sizes (2750 vs. 8932). Thus, most functional groups of proteins, both physically interacting and other, are likely to share many of their members with other groups. However, current algorithms searching for dense groups of nodes in networks usually exclude overlaps. With the aim to discover both novel functions of individual proteins and novel protein functional groups we combine in protein association networks (i) a search for overlapping dense subgraphs based on the Clique Percolation Method (CPM) (Palla, G., et.al. Nature 435, 814-818 (2005), http://angel.elte.hu/clustering), which explicitly allows for overlaps among the groups, and (ii) a verification and characterization of the identified groups of nodes (proteins) with the help of standard annotation databases listing known functions.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

PubMed

Patel, Jennifer T; Bottrill, Andrew; Prosser, Suzanna L; Jayaraman, Sangeetha; Straatman, Kees; Fry, Andrew M; Shackleton, Sue

2014-01-01

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

Programmable polyproteams built using twin peptide superglues

PubMed Central

Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D.; Yan, Jun; Robinson, Carol V.; Howarth, Mark

2016-01-01

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable “polyproteams” should enable exploration of a new area of biological space. PMID:26787909

Programmable polyproteams built using twin peptide superglues.

PubMed

Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D; Gayet, Raphaël V; Yan, Jun; Robinson, Carol V; Howarth, Mark

2016-02-02

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.

Joining the dots - protein-RNA interactions mediating local mRNA translation in neurons.

PubMed

Gallagher, Christopher; Ramos, Andres

2018-06-01

Establishing and maintaining the complex network of connections required for neuronal communication requires the transport and in situ translation of large groups of mRNAs to create local proteomes. In this Review, we discuss the regulation of local mRNA translation in neurons and the RNA-binding proteins that recognise RNA zipcode elements and connect the mRNAs to the cellular transport networks, as well as regulate their translation control. However, mRNA recognition by the regulatory proteins is mediated by the combinatorial action of multiple RNA-binding domains. This increases the specificity and affinity of the interaction, while allowing the protein to recognise a diverse set of targets and mediate a range of mechanisms for translational regulation. The structural and molecular understanding of the interactions can be used together with novel microscopy and transcriptome-wide data to build a mechanistic framework for the regulation of local mRNA translation. © 2018 Federation of European Biochemical Societies.

Exploring the evolution of protein function in Archaea.

PubMed

Goncearenco, Alexander; Berezovsky, Igor N

2012-05-30

Despite recent progress in studies of the evolution of protein function, the questions what were the first functional protein domains and what were their basic building blocks remain unresolved. Previously, we introduced the concept of elementary functional loops (EFLs), which are the functional units of enzymes that provide elementary reactions in biochemical transformations. They are presumably descendants of primordial catalytic peptides. We analyzed distant evolutionary connections between protein functions in Archaea based on the EFLs comprising them. We show examples of the involvement of EFLs in new functional domains, as well as reutilization of EFLs and functional domains in building multidomain structures and protein complexes. Our analysis of the archaeal superkingdom yields the dominating mechanisms in different periods of protein evolution, which resulted in several levels of the organization of biochemical function. First, functional domains emerged as combinations of prebiotic peptides with the very basic functions, such as nucleotide/phosphate and metal cofactor binding. Second, domain recombination brought to the evolutionary scene the multidomain proteins and complexes. Later, reutilization and de novo design of functional domains and elementary functional loops complemented evolution of protein function.

Evolution of SH2 domains and phosphotyrosine signalling networks

PubMed Central

Liu, Bernard A.; Nash, Piers D.

2012-01-01

Src homology 2 (SH2) domains mediate selective protein–protein interactions with tyrosine phosphorylated proteins, and in doing so define specificity of phosphotyrosine (pTyr) signalling networks. SH2 domains and protein-tyrosine phosphatases expand alongside protein-tyrosine kinases (PTKs) to coordinate cellular and organismal complexity in the evolution of the unikont branch of the eukaryotes. Examination of conserved families of PTKs and SH2 domain proteins provides fiduciary marks that trace the evolutionary landscape for the development of complex cellular systems in the proto-metazoan and metazoan lineages. The evolutionary provenance of conserved SH2 and PTK families reveals the mechanisms by which diversity is achieved through adaptations in tissue-specific gene transcription, altered ligand binding, insertions of linear motifs and the gain or loss of domains following gene duplication. We discuss mechanisms by which pTyr-mediated signalling networks evolve through the development of novel and expanded families of SH2 domain proteins and the elaboration of connections between pTyr-signalling proteins. These changes underlie the variety of general and specific signalling networks that give rise to tissue-specific functions and increasingly complex developmental programmes. Examination of SH2 domains from an evolutionary perspective provides insight into the process by which evolutionary expansion and modification of molecular protein interaction domain proteins permits the development of novel protein-interaction networks and accommodates adaptation of signalling networks. PMID:22889907

Endocytosis and Endosomal Trafficking in Plants.

PubMed

Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

2016-04-29

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

Role of protein fluctuation correlations in electron transfer in photosynthetic complexes.

PubMed

Nesterov, Alexander I; Berman, Gennady P

2015-04-01

We consider the dependence of the electron transfer in photosynthetic complexes on correlation properties of random fluctuations of the protein environment. The electron subsystem is modeled by a finite network of connected electron (exciton) sites. The fluctuations of the protein environment are modeled by random telegraph processes, which act either collectively (correlated) or independently (uncorrelated) on the electron sites. We derived an exact closed system of first-order linear differential equations with constant coefficients, for the average density matrix elements and for their first moments. Under some conditions, we obtained analytic expressions for the electron transfer rates and found the range of parameters for their applicability by comparing with the exact numerical simulations. We also compared the correlated and uncorrelated regimes and demonstrated numerically that the uncorrelated fluctuations of the protein environment can, under some conditions, either increase or decrease the electron transfer rates.

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

PubMed Central

Ungar, Daniel; Oka, Toshihiko; Brittle, Elizabeth E.; Vasile, Eliza; Lupashin, Vladimir V.; Chatterton, Jon E.; Heuser, John E.; Krieger, Monty; Waters, M. Gerard

2002-01-01

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. “Deep etch” EM of purified COG revealed an ∼37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function. PMID:11980916

Distributions of vesicular glutamate transporters 1 and 2 in the visual system of tree shrews (Tupaia belangeri)1

PubMed Central

Balaram, P; Isaamullah, M; Petry, HM; Bickford, ME; Kaas, JH

2014-01-01

Vesicular glutamate transporter (VGLUT) proteins regulate the storage and release of glutamate from synapses of excitatory neurons. Two isoforms, VGLUT1 and VGLUT2, are found in most glutamatergic projections across the mammalian visual system, and appear to differentially identify subsets of excitatory projections between visual structures. To expand current knowledge on the distribution of VGLUT isoforms in highly visual mammals, we examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the lateral geniculate nucleus (LGN), superior colliculus, pulvinar complex, and primary visual cortex (V1) in tree shrews (Tupaia belangeri), which are closely related to primates but classified as a separate order (Scandentia). We found that VGLUT1 was distributed in intrinsic and corticothalamic connections, whereas VGLUT2 was predominantly distributed in subcortical and thalamocortical connections. VGLUT1 and VGLUT2 were coexpressed in the LGN and in the pulvinar complex, as well as in restricted layers of V1, suggesting a greater heterogeneity in the range of efferent glutamatergic projections from these structures. These findings provide further evidence that VGLUT1 and VGLUT2 identify distinct populations of excitatory neurons in visual brain structures across mammals. Observed variations in individual projections may highlight the evolution of these connections through the mammalian lineage. PMID:25521420

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

PubMed

McGarry, Ken; McDonald, Sharon

2018-06-01

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

Molecular dynamics study of HIV-1 RT-DNA-nevirapine complexes explains NNRTI inhibition and resistance by connection mutations.

PubMed

Vijayan, R S K; Arnold, Eddy; Das, Kalyan

2014-05-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of several highly active antiretroviral therapy regimens. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns molecular dynamics (MD) simulations, followed by essential dynamics, free-energy landscape analyses, and network analyses of RT-DNA, RT-DNA-nevirapine (NVP), and N348I/T369I mutant RT-DNA-NVP complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon NVP binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-NVP complex suggesting enhanced rigidity of RT upon NVP binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. Copyright © 2013 Wiley Periodicals, Inc.

The SH2 Domain–Containing Proteins in 21 Species Establish the Provenance and Scope of Phosphotyrosine Signaling in Eukaryotes

PubMed Central

Liu, Bernard A.; Shah, Eshana; Jablonowski, Karl; Stergachis, Andrew; Engelmann, Brett; Nash, Piers D.

2014-01-01

The Src homology 2 (SH2) domains are participants in metazoan signal transduction, acting as primary mediators for regulated protein-protein interactions with tyrosine-phosphorylated substrates. Here, we describe the origin and evolution of SH2 domain proteins by means of sequence analysis from 21 eukaryotic organisms from the basal unicellular eukaryotes, where SH2 domains first appeared, through the multicellular animals and increasingly complex metazoans. On the basis of our results, SH2 domains and phosphotyrosine signaling emerged in the early Unikonta, and the numbers of SH2 domains expanded in the choanoflagellate and metazoan lineages with the development of tyrosine kinases, leading to rapid elaboration of phosphotyrosine signaling in early multicellular animals. Our results also indicated that SH2 domains coevolved and the number of the domains expanded alongside protein tyrosine kinases and tyrosine phosphatases, thereby coupling phosphotyrosine signaling to downstream signaling networks. Gene duplication combined with domain gain or loss produced novel SH2-containing proteins that function within phosphotyrosine signaling, which likely have contributed to diversity and complexity in metazoans. We found that intra- and intermolecular interactions within and between SH2 domain proteins increased in prevalence along with organismal complexity and may function to generate more highly connected and robust phosphotyrosine signaling networks. PMID:22155787

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

@TOME-2: a new pipeline for comparative modeling of protein-ligand complexes.

PubMed

Pons, Jean-Luc; Labesse, Gilles

2009-07-01

@TOME 2.0 is new web pipeline dedicated to protein structure modeling and small ligand docking based on comparative analyses. @TOME 2.0 allows fold recognition, template selection, structural alignment editing, structure comparisons, 3D-model building and evaluation. These tasks are routinely used in sequence analyses for structure prediction. In our pipeline the necessary software is efficiently interconnected in an original manner to accelerate all the processes. Furthermore, we have also connected comparative docking of small ligands that is performed using protein-protein superposition. The input is a simple protein sequence in one-letter code with no comment. The resulting 3D model, protein-ligand complexes and structural alignments can be visualized through dedicated Web interfaces or can be downloaded for further studies. These original features will aid in the functional annotation of proteins and the selection of templates for molecular modeling and virtual screening. Several examples are described to highlight some of the new functionalities provided by this pipeline. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/AT2/

Efficient and accurate Greedy Search Methods for mining functional modules in protein interaction networks.

PubMed

He, Jieyue; Li, Chaojun; Ye, Baoliu; Zhong, Wei

2012-06-25

Most computational algorithms mainly focus on detecting highly connected subgraphs in PPI networks as protein complexes but ignore their inherent organization. Furthermore, many of these algorithms are computationally expensive. However, recent analysis indicates that experimentally detected protein complexes generally contain Core/attachment structures. In this paper, a Greedy Search Method based on Core-Attachment structure (GSM-CA) is proposed. The GSM-CA method detects densely connected regions in large protein-protein interaction networks based on the edge weight and two criteria for determining core nodes and attachment nodes. The GSM-CA method improves the prediction accuracy compared to other similar module detection approaches, however it is computationally expensive. Many module detection approaches are based on the traditional hierarchical methods, which is also computationally inefficient because the hierarchical tree structure produced by these approaches cannot provide adequate information to identify whether a network belongs to a module structure or not. In order to speed up the computational process, the Greedy Search Method based on Fast Clustering (GSM-FC) is proposed in this work. The edge weight based GSM-FC method uses a greedy procedure to traverse all edges just once to separate the network into the suitable set of modules. The proposed methods are applied to the protein interaction network of S. cerevisiae. Experimental results indicate that many significant functional modules are detected, most of which match the known complexes. Results also demonstrate that the GSM-FC algorithm is faster and more accurate as compared to other competing algorithms. Based on the new edge weight definition, the proposed algorithm takes advantages of the greedy search procedure to separate the network into the suitable set of modules. Experimental analysis shows that the identified modules are statistically significant. The algorithm can reduce the computational time significantly while keeping high prediction accuracy.

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.

The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis

PubMed Central

Kellner, Nikola; Heimel, Kai; Obhof, Theresa; Finkernagel, Florian; Kämper, Jörg

2014-01-01

The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking. PMID:24391515

Repulsive Guidance Molecule is a structural bridge between Neogenin and Bone Morphogenetic Protein

PubMed Central

Healey, Eleanor G.; Bishop, Benjamin; Elegheert, Jonathan; Bell, Christian H.; Padilla-Parra, Sergi; Siebold, Christian

2015-01-01

Repulsive guidance molecules (RGMs) control crucial processes spanning cell motility, adhesion, immune cell regulation and systemic iron metabolism. RGMs signal via two fundamental signaling cascades: the Neogenin (NEO1) and the Bone Morphogenetic Protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a novel protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the ternary BMP2–RGM–NEO1 complex crystal structure, which combined with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub linking BMP and NEO1 and physically connecting these fundamental signaling pathways. PMID:25938661

Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

PubMed Central

Steiner, G; Hartmuth, K; Skriner, K; Maurer-Fogy, I; Sinski, A; Thalmann, E; Hassfeld, W; Barta, A; Smolen, J S

1992-01-01

RA33 is a nuclear autoantigen with an apparent molecular mass of 33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. Images PMID:1522214

Emerging functions of multi-protein complex Mediator with special emphasis on plants.

PubMed

Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

2017-10-01

Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

Staining Method for Protein Analysis by Capillary Gel Electrophoresis

PubMed Central

Wu, Shuqing; Lu, Joann J; Wang, Shili; Peck, Kristy L.; Li, Guigen; Liu, Shaorong

2009-01-01

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS–protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS–protein–dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS–protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli. PMID:17874848

Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

PubMed Central

Garvin, Michael R.; Bielawski, Joseph P.; Gharrett, Anthony J.

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm. PMID:21969854

Positive Darwinian selection in the piston that powers proton pumps in complex I of the mitochondria of Pacific salmon.

PubMed

Garvin, Michael R; Bielawski, Joseph P; Gharrett, Anthony J

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm.

GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

PubMed

Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

2016-12-01

In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

Convergent roles of de novo mutations and common variants in schizophrenia in tissue-specific and spatiotemporal co-expression network.

PubMed

Jia, Peilin; Chen, Xiangning; Fanous, Ayman H; Zhao, Zhongming

2018-05-24

Genetic components susceptible to complex disease such as schizophrenia include a wide spectrum of variants, including common variants (CVs) and de novo mutations (DNMs). Although CVs and DNMs differ by origin, it remains elusive whether and how they interact at the gene, pathway, and network levels that leads to the disease. In this work, we characterized the genes harboring schizophrenia-associated CVs (CVgenes) and the genes harboring DNMs (DNMgenes) using measures from network, tissue-specific expression profile, and spatiotemporal brain expression profile. We developed an algorithm to link the DNMgenes and CVgenes in spatiotemporal brain co-expression networks. DNMgenes tended to have central roles in the human protein-protein interaction (PPI) network, evidenced in their high degree and high betweenness values. DNMgenes and CVgenes connected with each other significantly more often than with other genes in the networks. However, only CVgenes remained significantly connected after adjusting for their degree. In our gene co-expression PPI network, we found DNMgenes and CVgenes connected in a tissue-specific fashion, and such a pattern was similar to that in GTEx brain but not in other GTEx tissues. Importantly, DNMgene-CVgene subnetworks were enriched with pathways of chromatin remodeling, MHC protein complex binding, and neurotransmitter activities. In summary, our results unveiled that both DNMgenes and CVgenes contributed to a core set of biologically important pathways and networks, and their interactions may attribute to the risk for schizophrenia. Our results also suggested a stronger biological effect of DNMgenes than CVgenes in schizophrenia.

Revealing networks from dynamics: an introduction

NASA Astrophysics Data System (ADS)

Timme, Marc; Casadiego, Jose

2014-08-01

What can we learn from the collective dynamics of a complex network about its interaction topology? Taking the perspective from nonlinear dynamics, we briefly review recent progress on how to infer structural connectivity (direct interactions) from accessing the dynamics of the units. Potential applications range from interaction networks in physics, to chemical and metabolic reactions, protein and gene regulatory networks as well as neural circuits in biology and electric power grids or wireless sensor networks in engineering. Moreover, we briefly mention some standard ways of inferring effective or functional connectivity.

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

[Technical peculiarities of the argon-plasma welding of gastrointestinal walls wounds in experimental environment].

PubMed

Terekhov, G V; Furmanov, Iu A; Gvozdetskiĭ, V S; Savitskaia, I M

2008-06-01

A new method of the live biological tissues connection, using thermal energy of a high-temperature argon plasma, constituting perspective trend of application of a new nonsuture methods of the tissues connection, original for the world practice, was elaborated in the Department of Experimental Surgery together with the Institute of welding named after Academician E. O. Paton NAS of Ukraine. The argon-plasma welding application secure safe adhesion of the connecting surfaces formation due to the protein complexes temperature denaturation occurrence. The absence of foreign bodies in the connection zone as well as the presence of the plasma flow bacterocidal properties secure, while application of this new method, a significant lowering of a bacterial soiling of the formatted anastomoses, not interfering with the tissue natural regeneration process course.

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

PubMed

Villa, Fabrizio; Simon, Aline C; Ortiz Bazan, Maria Angeles; Kilkenny, Mairi L; Wirthensohn, David; Wightman, Mel; Matak-Vinkovíc, Dijana; Pellegrini, Luca; Labib, Karim

2016-08-04

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Nanoscale Dewetting Transition in Protein Complex Folding

PubMed Central

Hua, Lan; Huang, Xuhui; Liu, Pu; Zhou, Ruhong; Berne, Bruce J.

2011-01-01

In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding. PMID:17608515

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance.

PubMed

Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Huddler, Donald; Muda, Marco; Dixon, Jack E

2002-03-15

Dock, the Drosophila orthologue of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, we purified proteins in a complex with the SH2 domain of Dock from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. We demonstrate that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. We conclude that DSH3PX1 is a substrate for DAck in vivo and in vitro and define one of the major in vitro sites of DSH3PX1 phosphorylation to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. We demonstrate that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance.

Identification of Crosslinked Peptides after Click-based Enrichment Using Sequential CID and ETD Tandem Mass Spectrometry

PubMed Central

Chowdhury, Saiful M.; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

2010-01-01

Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides. PMID:19496583

Distinct functions for IFT140 and IFT20 in opsin transport.

PubMed Central

Crouse, Jacquelin A.; Lopes, Vanda S.; SanAgustin, Jovenal T.; Keady, Brian T.; Williams, David S.; Pazour, Gregory J.

2014-01-01

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. PMID:24619649

The Evolving Contribution of Mass Spectrometry to Integrative Structural Biology

NASA Astrophysics Data System (ADS)

Faini, Marco; Stengel, Florian; Aebersold, Ruedi

2016-06-01

Protein complexes are key catalysts and regulators for the majority of cellular processes. Unveiling their assembly and structure is essential to understanding their function and mechanism of action. Although conventional structural techniques such as X-ray crystallography and NMR have solved the structure of important protein complexes, they cannot consistently deal with dynamic and heterogeneous assemblies, limiting their applications to small scale experiments. A novel methodological paradigm, integrative structural biology, aims at overcoming such limitations by combining complementary data sources into a comprehensive structural model. Recent applications have shown that a range of mass spectrometry (MS) techniques are able to generate interaction and spatial restraints (cross-linking MS) information on native complexes or to study the stoichiometry and connectivity of entire assemblies (native MS) rapidly, reliably, and from small amounts of substrate. Although these techniques by themselves do not solve structures, they do provide invaluable structural information and are thus ideally suited to contribute to integrative modeling efforts. The group of Brian Chait has made seminal contributions in the use of mass spectrometric techniques to study protein complexes. In this perspective, we honor the contributions of the Chait group and discuss concepts and milestones of integrative structural biology. We also review recent examples of integration of structural MS techniques with an emphasis on cross-linking MS. We then speculate on future MS applications that would unravel the dynamic nature of protein complexes upon diverse cellular states.

ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

PubMed

Baudier, Jacques

2018-05-01

In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

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

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

Distributions of vesicular glutamate transporters 1 and 2 in the visual system of tree shrews (Tupaia belangeri).

PubMed

Balaram, P; Isaamullah, M; Petry, H M; Bickford, M E; Kaas, J H

2015-08-15

Vesicular glutamate transporter (VGLUT) proteins regulate the storage and release of glutamate from synapses of excitatory neurons. Two isoforms, VGLUT1 and VGLUT2, are found in most glutamatergic projections across the mammalian visual system, and appear to differentially identify subsets of excitatory projections between visual structures. To expand current knowledge on the distribution of VGLUT isoforms in highly visual mammals, we examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the lateral geniculate nucleus (LGN), superior colliculus, pulvinar complex, and primary visual cortex (V1) in tree shrews (Tupaia belangeri), which are closely related to primates but classified as a separate order (Scandentia). We found that VGLUT1 was distributed in intrinsic and corticothalamic connections, whereas VGLUT2 was predominantly distributed in subcortical and thalamocortical connections. VGLUT1 and VGLUT2 were coexpressed in the LGN and in the pulvinar complex, as well as in restricted layers of V1, suggesting a greater heterogeneity in the range of efferent glutamatergic projections from these structures. These findings provide further evidence that VGLUT1 and VGLUT2 identify distinct populations of excitatory neurons in visual brain structures across mammals. Observed variations in individual projections may highlight the evolution of these connections through the mammalian lineage. © 2015 Wiley Periodicals, Inc.

CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres.

PubMed

Hoischen, Christian; Yavas, Sibel; Wohland, Thorsten; Diekmann, Stephan

2018-01-01

Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (Kd) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.

Discovering disease-associated genes in weighted protein-protein interaction networks

NASA Astrophysics Data System (ADS)

Cui, Ying; Cai, Meng; Stanley, H. Eugene

2018-04-01

Although there have been many network-based attempts to discover disease-associated genes, most of them have not taken edge weight - which quantifies their relative strength - into consideration. We use connection weights in a protein-protein interaction (PPI) network to locate disease-related genes. We analyze the topological properties of both weighted and unweighted PPI networks and design an improved random forest classifier to distinguish disease genes from non-disease genes. We use a cross-validation test to confirm that weighted networks are better able to discover disease-associated genes than unweighted networks, which indicates that including link weight in the analysis of network properties provides a better model of complex genotype-phenotype associations.

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

PubMed

Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid; Rapoport, Tom A

2017-05-12

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

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

Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, wemore » report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.« less

Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

PubMed Central

Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

2016-01-01

Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

New Markov-Shannon Entropy models to assess connectivity quality in complex networks: from molecular to cellular pathway, Parasite-Host, Neural, Industry, and Legal-Social networks.

PubMed

Riera-Fernández, Pablo; Munteanu, Cristian R; Escobar, Manuel; Prado-Prado, Francisco; Martín-Romalde, Raquel; Pereira, David; Villalba, Karen; Duardo-Sánchez, Aliuska; González-Díaz, Humberto

2012-01-21

Graph and Complex Network theory is expanding its application to different levels of matter organization such as molecular, biological, technological, and social networks. A network is a set of items, usually called nodes, with connections between them, which are called links or edges. There are many different experimental and/or theoretical methods to assign node-node links depending on the type of network we want to construct. Unfortunately, the use of a method for experimental reevaluation of the entire network is very expensive in terms of time and resources; thus the development of cheaper theoretical methods is of major importance. In addition, different methods to link nodes in the same type of network are not totally accurate in such a way that they do not always coincide. In this sense, the development of computational methods useful to evaluate connectivity quality in complex networks (a posteriori of network assemble) is a goal of major interest. In this work, we report for the first time a new method to calculate numerical quality scores S(L(ij)) for network links L(ij) (connectivity) based on the Markov-Shannon Entropy indices of order k-th (θ(k)) for network nodes. The algorithm may be summarized as follows: (i) first, the θ(k)(j) values are calculated for all j-th nodes in a complex network already constructed; (ii) A Linear Discriminant Analysis (LDA) is used to seek a linear equation that discriminates connected or linked (L(ij)=1) pairs of nodes experimentally confirmed from non-linked ones (L(ij)=0); (iii) the new model is validated with external series of pairs of nodes; (iv) the equation obtained is used to re-evaluate the connectivity quality of the network, connecting/disconnecting nodes based on the quality scores calculated with the new connectivity function. This method was used to study different types of large networks. The linear models obtained produced the following results in terms of overall accuracy for network reconstruction: Metabolic networks (72.3%), Parasite-Host networks (93.3%), CoCoMac brain cortex co-activation network (89.6%), NW Spain fasciolosis spreading network (97.2%), Spanish financial law network (89.9%) and World trade network for Intelligent & Active Food Packaging (92.8%). In order to seek these models, we studied an average of 55,388 pairs of nodes in each model and a total of 332,326 pairs of nodes in all models. Finally, this method was used to solve a more complicated problem. A model was developed to score the connectivity quality in the Drug-Target network of US FDA approved drugs. In this last model the θ(k) values were calculated for three types of molecular networks representing different levels of organization: drug molecular graphs (atom-atom bonds), protein residue networks (amino acid interactions), and drug-target network (compound-protein binding). The overall accuracy of this model was 76.3%. This work opens a new door to the computational reevaluation of network connectivity quality (collation) for complex systems in molecular, biomedical, technological, and legal-social sciences as well as in world trade and industry. Copyright © 2011 Elsevier Ltd. All rights reserved.

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis.

PubMed

Feichtinger, René G; Neureiter, Daniel; Skaria, Tom; Wessler, Silja; Cover, Timothy L; Mayr, Johannes A; Zimmermann, Franz A; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas ("intestinal" and "diffuse"), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.

Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis

PubMed Central

Skaria, Tom; Wessler, Silja; Cover, Timothy L.; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

2017-01-01

Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection. PMID:28744336

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

PubMed Central

Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

2016-01-01

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

[Lysozyme in the treatment of juvenile laryngeal papillomatosis. A new concept in its etiopathogenesis].

PubMed

Altamar-Ríos, J

1990-01-01

The A. inform about the results achieved with lysozyme chlorhydrate in the treatment of 15 patients with juvenile laryngeal papillomatosis. The lysozyme is an electropositive enzyme which synthesis is related to the degree of proteins and vitamin B complex ingestion. Lysozyme is a component of the immunitary inespecific system, serving to prevent against HPV-DNA at the level of the secretory film of the mucociliary apparatus of the respiratory mucous membrane. Furthermore, lysozyme hydrolyzes the mucopolysaccharide of the connective tissue and inhibits the virus-DNA replication. 100-300 mgr daily during 30-60 days simultaneously with hyperproteic diet and vitamin B complex (after correction of the nutrimental deficiencies) brought about the evanishment of papillomatosis. The A. suggest that the predisposition to infection by virus DNA is primarily of immunitary origin, because of lysozyme deficiency, and secondary due to a low intake of proteins and vitamin B complex.

Active-State Model of a Dopamine D2 Receptor - Gαi Complex Stabilized by Aripiprazole-Type Partial Agonists

PubMed Central

Kling, Ralf C.; Tschammer, Nuska; Lanig, Harald; Clark, Timothy; Gmeiner, Peter

2014-01-01

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy. PMID:24932547

[Mechanisms of genoprotective action of a phytoecdysteroid drug(BTK-8L) in chromatin damage by tetrachloromethane].

PubMed

Gubskiĭ, Iu I; Levitskiĭ, E L; Kholodova, Iu D; Goriushko, A G; Primak, R G; Vistunova, I E; Sachenko, L G

1993-01-01

Hepatoprotective action of prophylactic injection of aqueous solution of preparation BTK-8L from plant ecdysteroids to experimental animals with the liver damage by tetrachloromethane was revealed. This effect at least partially was connected with the genoprotective action of the given preparation. As a result, normalization of free radical chromatin lipid peroxidation reaction, modified at the intoxication, as well as partial correction of physical and chemical properties of chromatin protein-lipid complex were those molecular mechanisms of genoprotective action of BTK-8L, which were manifested by the influence of the preparation on such indices which characterized the depth structure of the complex as microviscosity and energy transfer from the protein to the lipid probe. Investigation of the interaction of the preparation with chromatin fractions in vitro and comparison of this interaction with the analogous process in model systems allowed revealing determinative participation of chromatin proteins and lipids in the given process. The preparation interacted more intensively with the active chromatin fraction, which contained a more marked protein-lipid complex, as comparing to the repressed one. Injection of the preparation also normalized such indices as relation between the chromatine fractions and protein/DNA ratio in them. On the contrary, injection of the alcoholic solution of the preparation to experimental animals, aggravated genotoxic tetrachloromethane action.

Getting to the core of cadherin complex function in Caenorhabditis elegans.

PubMed

Hardin, Jeff

2015-01-01

The classic cadherin-catenin complex (CCC) mediates cell-cell adhesion in metazoans. Although substantial insights have been gained by studying the CCC in vertebrate tissue culture, analyzing requirements for and regulation of the CCC in vertebrates remains challenging. Caenorhabditis elegans is a powerful system for connecting the molecular details of CCC function with functional requirements in a living organism. Recent data, using an "angstroms to embryos" approach, have elucidated functions for key residues, conserved across all metazoans, that mediate cadherin/β-catenin binding. Other recent work reveals a novel, potentially ancestral, role for the C. elegans p120ctn homologue in regulating polarization of blastomeres in the early embryo via Cdc42 and the partitioning-defective (PAR)/atypical protein kinase C (aPKC) complex. Finally, recent work suggests that the CCC is trafficked to the cell surface via the clathrin adaptor protein complex 1 (AP-1) in surprising ways. These studies continue to underscore the value of C. elegans as a model system for identifying conserved molecular mechanisms involving the CCC.

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

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

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

Rational assembly of nanoparticle superlattices with designed lattice symmetries

DOEpatents

Gang, Oleg; Lu, Fang; Tagawa, Miho

2017-09-05

A method for lattice design via multivalent linkers (LDML) is disclosed that introduces a rationally designed symmetry of connections between particles in order to achieve control over the morphology of their assembly. The method affords the inclusion of different programmable interactions within one linker that allow an assembly of different types of particles. The designed symmetry of connections is preferably provided utilizing DNA encoding. The linkers may include fabricated "patchy" particles, DNA scaffold constructs and Y-shaped DNA linkers, anisotropic particles, which are preferably functionalized with DNA, multimeric protein-DNA complexes, and particles with finite numbers of DNA linkers.

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed Central

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-01-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure. Images PMID:207893

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-05-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient- and Exercise-Induced Stress

PubMed Central

Vassilopoulos, Athanassios; Pennington, J. Daniel; Andresson, Thorkell; Rees, David M.; Bosley, Allen D.; Fearnley, Ian M.; Ham, Amy; Flynn, Charles Robb; Hill, Salisha; Rose, Kristie Lindsey; Kim, Hyun-Seok; Walker, John E.

2014-01-01

Abstract Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCPK139 directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins. Antioxid. Redox Signal. 21, 551–564. PMID:24252090

Elastic Network Model of a Nuclear Transport Complex

NASA Astrophysics Data System (ADS)

Ryan, Patrick; Liu, Wing K.; Lee, Dockjin; Seo, Sangjae; Kim, Young-Jin; Kim, Moon K.

2010-05-01

The structure of Kap95p was obtained from the Protein Data Bank (www.pdb.org) and analyzed RanGTP plays an important role in both nuclear protein import and export cycles. In the nucleus, RanGTP releases macromolecular cargoes from importins and conversely facilitates cargo binding to exportins. Although the crystal structure of the nuclear import complex formed by importin Kap95p and RanGTP was recently identified, its molecular mechanism still remains unclear. To understand the relationship between structure and function of a nuclear transport complex, a structure-based mechanical model of Kap95p:RanGTP complex is introduced. In this model, a protein structure is simply modeled as an elastic network in which a set of coarse-grained point masses are connected by linear springs representing biochemical interactions at atomic level. Harmonic normal mode analysis (NMA) and anharmonic elastic network interpolation (ENI) are performed to predict the modes of vibrations and a feasible pathway between locked and unlocked conformations of Kap95p, respectively. Simulation results imply that the binding of RanGTP to Kap95p induces the release of the cargo in the nucleus as well as prevents any new cargo from attaching to the Kap95p:RanGTP complex.

Network based transcription factor analysis of regenerating axolotl limbs

PubMed Central

2011-01-01

Background Studies on amphibian limb regeneration began in the early 1700's but we still do not completely understand the cellular and molecular events of this unique process. Understanding a complex biological process such as limb regeneration is more complicated than the knowledge of the individual genes or proteins involved. Here we followed a systems biology approach in an effort to construct the networks and pathways of protein interactions involved in formation of the accumulation blastema in regenerating axolotl limbs. Results We used the human orthologs of proteins previously identified by our research team as bait to identify the transcription factor (TF) pathways and networks that regulate blastema formation in amputated axolotl limbs. The five most connected factors, c-Myc, SP1, HNF4A, ESR1 and p53 regulate ~50% of the proteins in our data. Among these, c-Myc and SP1 regulate 36.2% of the proteins. c-Myc was the most highly connected TF (71 targets). Network analysis showed that TGF-β1 and fibronectin (FN) lead to the activation of these TFs. We found that other TFs known to be involved in epigenetic reprogramming, such as Klf4, Oct4, and Lin28 are also connected to c-Myc and SP1. Conclusions Our study provides a systems biology approach to how different molecular entities inter-connect with each other during the formation of an accumulation blastema in regenerating axolotl limbs. This approach provides an in silico methodology to identify proteins that are not detected by experimental methods such as proteomics but are potentially important to blastema formation. We found that the TFs, c-Myc and SP1 and their target genes could potentially play a central role in limb regeneration. Systems biology has the potential to map out numerous other pathways that are crucial to blastema formation in regeneration-competent limbs, to compare these to the pathways that characterize regeneration-deficient limbs and finally, to identify stem cell markers in regeneration. PMID:21418574

The Major Histocompatibility Complex and Autism Spectrum Disorder

PubMed Central

Needleman, Leigh A.; McAllister, A. Kimberley

2015-01-01

Autism spectrum disorder (ASD) is a complex disorder that appears to be caused by interactions between genetic changes and environmental insults during early development. A wide range of factors have been linked to the onset of ASD, but recently both genetic associations and environmental factors point to a central role for immune- related genes and immune responses to environmental stimuli. Specifically, many of the proteins encoded by the major histocompatibility complex (MHC) play a vital role in the formation, refinement, maintenance, and plasticity of the brain. Manipulations of levels of MHC molecules have illustrated how disrupted MHC signaling can significantly alter brain connectivity and function. Thus, an emerging hypothesis in our field is that disruptions in MHC expression in the developing brain caused by mutations and/or immune dysregulation may contribute to the altered brain connectivity and function characteristic of ASD. This review provides an overview of the structure and function of the three classes of MHC molecules in the immune system, healthy brain, and their possible involvement in ASD. PMID:22760919

In silico modeling of the yeast protein and protein family interaction network

NASA Astrophysics Data System (ADS)

Goh, K.-I.; Kahng, B.; Kim, D.

2004-03-01

Understanding of how protein interaction networks of living organisms have evolved or are organized can be the first stepping stone in unveiling how life works on a fundamental ground. Here we introduce an in silico ``coevolutionary'' model for the protein interaction network and the protein family network. The essential ingredient of the model includes the protein family identity and its robustness under evolution, as well as the three previously proposed: gene duplication, divergence, and mutation. This model produces a prototypical feature of complex networks in a wide range of parameter space, following the generalized Pareto distribution in connectivity. Moreover, we investigate other structural properties of our model in detail with some specific values of parameters relevant to the yeast Saccharomyces cerevisiae, showing excellent agreement with the empirical data. Our model indicates that the physical constraints encoded via the domain structure of proteins play a crucial role in protein interactions.

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

PubMed Central

Pereira, Bruno; Videira, Arnaldo

2013-01-01

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. PMID:23648483

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

PubMed Central

Roux, Kyle J.; Crisp, Melissa L.; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L.; Burke, Brian

2009-01-01

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin–Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1–3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning. PMID:19164528

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.

PubMed

Roux, Kyle J; Crisp, Melissa L; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L; Burke, Brian

2009-02-17

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.

Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

PubMed

Kuhl, D; Kennedy, T E; Barzilai, A; Kandel, E R

1992-12-01

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis.

PubMed

Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M Leticia; Toye, Ashley M

2015-01-01

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. Copyright© Ferrata Storti Foundation.

The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

PubMed Central

Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

2015-01-01

Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

The intriguing plant nuclear lamina.

PubMed

Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

2014-01-01

The nuclear lamina is a complex protein mesh attached to the inner nuclear membrane (INM), which is also associated with nuclear pore complexes. It provides mechanical support to the nucleus and nuclear envelope, and as well as facilitating the connection of the nucleoskeleton to the cytoskeleton, it is also involved in chromatin organization, gene regulation, and signaling. In metazoans, the nuclear lamina consists of a polymeric layer of lamins and other interacting proteins responsible for its association with the INM and chromatin. In plants, field emission scanning electron microscopy of nuclei, and thin section transmission electron microscopy of isolated nucleoskeletons, reveals the lamina to have a similar structure to that of metazoans. Moreover, although plants lack lamin genes and the genes encoding most lamin-binding proteins, the main functions of the lamina are fulfilled in plants. Hence, it would appear that the plant lamina is not based on lamins and that other proteins substitute for lamins in plant cells. The nuclear matrix constituent proteins are the best characterized structural proteins in the plant lamina. Although these proteins do not display strong sequence similarity to lamins, their predicted secondary structure and sub-nuclear distribution, as well as their influence on nuclear size and shape, and on heterochromatin organization, suggest they could be functional lamin analogs. In this review we shall summarize what is currently known about the organization and composition of the plant nuclear lamina and its interacting complexes, and we will discuss the activity of this structure in the plant cell and its nucleus.

The balance of protein expression and degradation: an ESCRTs point of view.

PubMed

Babst, Markus; Odorizzi, Greg

2013-08-01

Endosomal sorting complexes required for transport (ESCRTs) execute the biogenesis of late endosomal multivesicular bodies (MVBs). The ESCRT pathway has traditionally been viewed as a means by which transmembrane proteins are degraded in vacuoles/lysosomes. More recent studies aimed at understanding the broader functions of ESCRTs have uncovered unexpected links with pathways that control cellular metabolism. Central to this communication is TORC1, the kinase complex that controls many of the catabolic and anabolic systems. The connection between TORC1 activity and ESCRTs allows cells to quickly adapt to the stress of nutrient limitations until the longer-term autophagic pathway is activated. Increasing evidence also points to ESCRTs regulating RNA interference (RNAi) pathways that control translation. Copyright © 2013 Elsevier Ltd. All rights reserved.

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

PubMed

Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

2015-11-23

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

PubMed

Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

2018-01-01

The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

PubMed

Worby, C A; Simonson-Leff, N; Clemens, J C; Kruger, R P; Muda, M; Dixon, J E

2001-11-09

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Dietary Fiber-Induced Changes in the Structure and Thermal Properties of Gluten Proteins Studied by Fourier Transform-Raman Spectroscopy and Thermogravimetry.

PubMed

Nawrocka, Agnieszka; Szymańska-Chargot, Monika; Miś, Antoni; Wilczewska, Agnieszka Z; Markiewicz, Karolina H

2016-03-16

Interactions between gluten proteins and dietary fiber supplements at the stage of bread dough formation are crucial in the baking industry. The dietary fiber additives are regarded as a source of polysaccharides and antioxidants, which have positive effects on human health. The fiber enrichment of bread causes a significant reduction in its quality, which is connected with changes in the structure of gluten proteins. Changes in the structure of gluten proteins and their thermal properties induced by seven commercial dietary fibers (fruit, vegetable, and cereal) were studied by FT-Raman spectroscopy and thermogravimetry (TGA), respectively. For this aim the bread dough at 500 FU consistency was made of a blend of wheat starch and wheat gluten as well as the fiber, the content of which ranged from 3 to 18% w/w. The obtained results revealed that all dietary fibers apart from oat caused similar changes in the secondary structure of gluten proteins. The most noticeable changes were observed in the regions connected with hydrogen-bonded β-sheets (1614 and 1684 cm(-1)) and β-turns (1640 and 1657 cm(-1)). Other changes observed in the gluten structure, concerning other β-structures, conformation of disulfide bridges, and aromatic amino acid microenvironment, depend on the fibers' chemical composition. The results concerning structural changes suggested that the observed formation of hydrogen bonds in the β-structures can be connected with aggregation or abnormal folding. This hypothesis was confirmed by thermogravimetric results. Changes in weight loss indicated the formation of a more complex and strong gluten network.

Expression, Purification and Characterization of GMZ2'.10C, a Complex Disulphide-Bonded Fusion Protein Vaccine Candidate against the Asexual and Sexual Life-Stages of the Malaria-Causing Plasmodium falciparum Parasite.

PubMed

Mistarz, Ulrik H; Singh, Susheel K; Nguyen, Tam T T N; Roeffen, Will; Yang, Fen; Lissau, Casper; Madsen, Søren M; Vrang, Astrid; Tiendrebeogo, Régis W; Kana, Ikhlaq H; Sauerwein, Robert W; Theisen, Michael; Rand, Kasper D

2017-09-01

Production and characterization of a chimeric fusion protein (GMZ2'.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 (MSP3), and the highly disulphide bonded Pfs48/45 (10C). GMZ2'.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite. GMZ2'.10C was produced in Lactococcus lactis and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS. CP-GMZ2'.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2'.10C. CP-GMZ2'.10C and IP-GMZ2'.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus Pfs48/45 was analysed by tandem mass spectrometry and was established for GMZ2'.10C and two reference fusion proteins encompassing similar parts of Pfs48/45. GMZ2'.10C, combining GMZ2' and correctly-folded Pfs48/45 can be produced by the Lactoccus lactis P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of Pfs48/45 was revealed experimentally, providing an important guideline for employing the Pfs48/45 antigen in vaccine design.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

PubMed

Yavuz, Halenur; Kattan, Iman; Hernandez, Javier Matias; Hofnagel, Oliver; Witkowska, Agata; Raunser, Stefan; Walla, Peter Jomo; Jahn, Reinhard

2018-04-17

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N-termini to their membrane-proximal C-termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates, where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

PubMed Central

Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

2013-01-01

Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial. PMID:23658807

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration

PubMed Central

Musiek, Erik S.; Lim, Miranda M.; Yang, Guangrui; Bauer, Adam Q.; Qi, Laura; Lee, Yool; Roh, Jee Hoon; Ortiz-Gonzalez, Xilma; Dearborn, Joshua T.; Culver, Joseph P.; Herzog, Erik D.; Hogenesch, John B.; Wozniak, David F.; Dikranian, Krikor; Giasson, Benoit I.; Weaver, David R.; Holtzman, David M.; FitzGerald, Garret A.

2013-01-01

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator–like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration. PMID:24270424

Interdependence of free zinc changes and protein complex assembly - insights into zinc signal regulation.

PubMed

Kocyła, Anna; Adamczyk, Justyna; Krężel, Artur

2018-01-24

Cellular zinc (Zn(ii)) is bound with proteins that are part of the proteomes of all domains of life. It is mostly utilized as a catalytic or structural protein cofactor, which results in a vast number of binding architectures. The Zn(ii) ion is also important for the formation of transient protein complexes with a Zn(ii)-dependent quaternary structure that is formed upon cellular zinc signals. The mechanisms by which proteins associate with and dissociate from Zn(ii) and the connection with cellular Zn(ii) changes remain incompletely understood. In this study, we aimed to examine how zinc protein domains with various Zn(ii)-binding architectures are formed under free Zn(ii) concentration changes and how formation of the Zn(ii)-dependent assemblies is related to the protein concentration and reactivity. To accomplish these goals we chose four zinc domains with different Zn(ii)-to-protein binding stoichiometries: classical zinc finger (ZnP), LIM domain (Zn 2 P), zinc hook (ZnP 2 ) and zinc clasp (ZnP 1 P 2 ) folds. Our research demonstrated a lack of changes in the saturation level of intraprotein zinc binding sites, despite various peptide concentrations, while homo- and heterodimers indicated a concentration-dependent tendency. In other words, at a certain free Zn(ii) concentration, the fraction of a formed dimeric complex increases or decreases with subunit concentration changes. Secondly, even small or local changes in free Zn(ii) may significantly affect protein saturation depending on its architecture, function and subcellular concentration. In our paper, we indicate the importance of interdependence of free Zn(ii) availability and protein subunit concentrations for cellular zinc signal regulation.

Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

PubMed Central

2012-01-01

Background The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. Results Our findings demonstrate that a miRNA’s functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. Conclusions We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment. PMID:22929553

Protein quality control in organelles - AAA/FtsH story.

PubMed

Janska, Hanna; Kwasniak, Malgorzata; Szczepanowska, Joanna

2013-02-01

This review focuses on organellar AAA/FtsH proteases, whose proteolytic and chaperone-like activity is a crucial component of the protein quality control systems of mitochondrial and chloroplast membranes. We compare the AAA/FtsH proteases from yeast, mammals and plants. The nature of the complexes formed by AAA/FtsH proteases and the current view on their involvement in degradation of non-native organellar proteins or assembly of membrane complexes are discussed. Additional functions of AAA proteases not directly connected with protein quality control found in yeast and mammals but not yet in plants are also described shortly. Following an overview of the molecular functions of the AAA/FtsH proteases we discuss physiological consequences of their inactivation in yeast, mammals and plants. The molecular basis of phenotypes associated with inactivation of the AAA/FtsH proteases is not fully understood yet, with the notable exception of those observed in m-AAA protease-deficient yeast cells, which are caused by impaired maturation of mitochondrial ribosomal protein. Finally, examples of cytosolic events affecting protein quality control in mitochondria and chloroplasts are given. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids. Copyright © 2012 Elsevier B.V. All rights reserved.

Mitochondrial Ion Channels in Cancer Transformation

PubMed Central

Madamba, Stephen M.; Damri, Kevin N.; Dejean, Laurent M.; Peixoto, Pablo M.

2015-01-01

Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process. PMID:26090338

@TOME-2: a new pipeline for comparative modeling of protein–ligand complexes

PubMed Central

Pons, Jean-Luc; Labesse, Gilles

2009-01-01

@TOME 2.0 is new web pipeline dedicated to protein structure modeling and small ligand docking based on comparative analyses. @TOME 2.0 allows fold recognition, template selection, structural alignment editing, structure comparisons, 3D-model building and evaluation. These tasks are routinely used in sequence analyses for structure prediction. In our pipeline the necessary software is efficiently interconnected in an original manner to accelerate all the processes. Furthermore, we have also connected comparative docking of small ligands that is performed using protein–protein superposition. The input is a simple protein sequence in one-letter code with no comment. The resulting 3D model, protein–ligand complexes and structural alignments can be visualized through dedicated Web interfaces or can be downloaded for further studies. These original features will aid in the functional annotation of proteins and the selection of templates for molecular modeling and virtual screening. Several examples are described to highlight some of the new functionalities provided by this pipeline. The server and its documentation are freely available at http://abcis.cbs.cnrs.fr/AT2/ PMID:19443448

Application of clustering methods: Regularized Markov clustering (R-MCL) for analyzing dengue virus similarity

NASA Astrophysics Data System (ADS)

Lestari, D.; Raharjo, D.; Bustamam, A.; Abdillah, B.; Widhianto, W.

2017-07-01

Dengue virus consists of 10 different constituent proteins and are classified into 4 major serotypes (DEN 1 - DEN 4). This study was designed to perform clustering against 30 protein sequences of dengue virus taken from Virus Pathogen Database and Analysis Resource (VIPR) using Regularized Markov Clustering (R-MCL) algorithm and then we analyze the result. By using Python program 3.4, R-MCL algorithm produces 8 clusters with more than one centroid in several clusters. The number of centroid shows the density level of interaction. Protein interactions that are connected in a tissue, form a complex protein that serves as a specific biological process unit. The analysis of result shows the R-MCL clustering produces clusters of dengue virus family based on the similarity role of their constituent protein, regardless of serotypes.

Data on the association of the nuclear envelope protein Sun1 with nucleoli.

PubMed

Moujaber, Ossama; Omran, Nawal; Kodiha, Mohamed; Pié, Brigitte; Cooper, Ellis; Presley, John F; Stochaj, Ursula

2017-08-01

SUN proteins participate in diverse cellular activities, many of which are connected to the nuclear envelope. Recently, the family member SUN1 has been linked to novel biological activities. These include the regulation of nucleoli, intranuclear compartments that assemble ribosomal subunits. We show that SUN1 associates with nucleoli in several mammalian epithelial cell lines. This nucleolar localization is not shared by all cell types, as SUN1 concentrates at the nuclear envelope in ganglionic neurons and non-neuronal satellite cells. Database analyses and Western blotting emphasize the complexity of SUN1 protein profiles in different mammalian cells. We constructed a STRING network which identifies SUN1-related proteins as part of a larger network that includes several nucleolar proteins. Taken together, the current data highlight the diversity of SUN1 proteins and emphasize the possible links between SUN1 and nucleoli.

Hidden Markov model-derived structural alphabet for proteins: the learning of protein local shapes captures sequence specificity.

PubMed

Camproux, A C; Tufféry, P

2005-08-05

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.

Functional properties of protein from frozen mantle and fin of jumbo squid Dosidicus gigas in function of pH and ionic strength.

PubMed

Rocha-Estrada, J G; Córdova-Murueta, J H; García-Carreño, F L

2010-10-01

Functional properties of protein from mantle and fin of the jumbo squid Dosidicus gigas were explained based on microscopic muscle fiber and protein fractions profiles as observed in SDS-PAGE. Fin has higher content of connective tissue and complex fiber arrangement, and we observed higher hardness of fin gels as expected. Myosin heavy chain (MHC) was found in sarcoplasmic, myofibril and soluble-in-alkali fractions of mantle and only in sarcoplasmic and soluble-in-alkali fractions of fin. An additive effect of salt concentration and pH affected the solubility and foaming properties. Fin and mantle proteins yielded similar results in solubility tests, but significant differences occurred for specific pH and concentrations of salt. Foaming capacity was proportional to solubility; foam stability was also affected by pH and salt concentration. Hardness and fracture strength of fin gels were significantly higher than mantle gels; gels from proteins of both tissues reached the highest level in the folding test. Structural and molecular properties, such as MHC and paramyosin solubility, arrangement of muscle fibers and the content of connective tissue were useful to explain the differences observed in these protein properties. High-strength gels can be formed from squid mantle or fin muscle. Fin displayed similar or better properties than mantle in all tests.

Effect of fish gelatin-gum arabic interactions on structural and functional properties of concentrated emulsions.

PubMed

Anvari, Mohammad; Joyner Melito, Helen S

2017-12-01

Concentrated emulsions containing both proteins and polysaccharides are the basis for many commercial products; however, the effects of protein-polysaccharide interactions on the functional properties of these complex systems are often poorly understood from a fundamental standpoint. Hence, the objective of this study was to determine the effects of fish gelatin (FG)-gum arabic (GA) complexation at different aqueous phase pH (3.6, 5.0, and 9.0) on concentrated emulsion structure-function relationships. Concentrated emulsions were prepared using FG-GA mixtures and characterized by rheometry and confocal scanning laser microscopy (CSLM). CSLM images showed that all samples were O/W emulsions; emulsions with lower pH showed smaller oil droplets, greater homogeneity in size distribution, and higher stability. This was attributed to an increased number of FG-GA complexes in the emulsification. Electrostatic attractive interactions and charge neutralization created biopolymer associations with increased emulsification capacity. Samples with FG-GA mixtures at lower pH showed higher elastic moduli under small deformation and exhibited greater deviation between apparent and complex viscosities under the Cox-Merz rule, indicating increased gel network extension and greater intermolecular connectivity between adsorbed layers of adjacent oil droplets. These results can be used to incorporate protein-polysaccharide complexes as a suitable emulsifier in materials comprising concentrated emulsions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

DOE PAGES

Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; ...

2013-07-15

In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Inmore » addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.« less

Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc03700g

PubMed Central

Sadaf, Aiman; Du, Yang; Santillan, Claudia; Mortensen, Jonas S.; Molist, Iago; Seven, Alpay B.; Hariharan, Parameswaran; Skiniotis, Georgios; Loland, Claus J.; Kobilka, Brian K.; Guan, Lan; Byrne, Bernadette

2017-01-01

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization. PMID:29619178

Normalized Cut Algorithm for Automated Assignment of Protein Domains

NASA Technical Reports Server (NTRS)

Samanta, M. P.; Liang, S.; Zha, H.; Biegel, Bryan A. (Technical Monitor)

2002-01-01

We present a novel computational method for automatic assignment of protein domains from structural data. At the core of our algorithm lies a recently proposed clustering technique that has been very successful for image-partitioning applications. This grap.,l-theory based clustering method uses the notion of a normalized cut to partition. an undirected graph into its strongly-connected components. Computer implementation of our method tested on the standard comparison set of proteins from the literature shows a high success rate (84%), better than most existing alternative In addition, several other features of our algorithm, such as reliance on few adjustable parameters, linear run-time with respect to the size of the protein and reduced complexity compared to other graph-theory based algorithms, would make it an attractive tool for structural biologists.

Features analysis for identification of date and party hubs in protein interaction network of Saccharomyces Cerevisiae.

PubMed

Mirzarezaee, Mitra; Araabi, Babak N; Sadeghi, Mehdi

2010-12-19

It has been understood that biological networks have modular organizations which are the sources of their observed complexity. Analysis of networks and motifs has shown that two types of hubs, party hubs and date hubs, are responsible for this complexity. Party hubs are local coordinators because of their high co-expressions with their partners, whereas date hubs display low co-expressions and are assumed as global connectors. However there is no mutual agreement on these concepts in related literature with different studies reporting their results on different data sets. We investigated whether there is a relation between the biological features of Saccharomyces Cerevisiae's proteins and their roles as non-hubs, intermediately connected, party hubs, and date hubs. We propose a classifier that separates these four classes. We extracted different biological characteristics including amino acid sequences, domain contents, repeated domains, functional categories, biological processes, cellular compartments, disordered regions, and position specific scoring matrix from various sources. Several classifiers are examined and the best feature-sets based on average correct classification rate and correlation coefficients of the results are selected. We show that fusion of five feature-sets including domains, Position Specific Scoring Matrix-400, cellular compartments level one, and composition pairs with two and one gaps provide the best discrimination with an average correct classification rate of 77%. We study a variety of known biological feature-sets of the proteins and show that there is a relation between domains, Position Specific Scoring Matrix-400, cellular compartments level one, composition pairs with two and one gaps of Saccharomyces Cerevisiae's proteins, and their roles in the protein interaction network as non-hubs, intermediately connected, party hubs and date hubs. This study also confirms the possibility of predicting non-hubs, party hubs and date hubs based on their biological features with acceptable accuracy. If such a hypothesis is correct for other species as well, similar methods can be applied to predict the roles of proteins in those species.

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains.

PubMed

Villa, Riccardo; Martorana, Alessandra M; Okuda, Suguru; Gourlay, Louise J; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel; Polissi, Alessandra

2013-03-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

PubMed Central

Villa, Riccardo; Martorana, Alessandra M.; Okuda, Suguru; Gourlay, Louise J.; Nardini, Marco; Sperandeo, Paola; Dehò, Gianni; Bolognesi, Martino; Kahne, Daniel

2013-01-01

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components. PMID:23292770

Glutathione, Glutaredoxins, and Iron.

PubMed

Berndt, Carsten; Lillig, Christopher Horst

2017-11-20

Glutathione (GSH) is the most abundant cellular low-molecular-weight thiol in the majority of organisms in all kingdoms of life. Therefore, functions of GSH and disturbed regulation of its concentration are associated with numerous physiological and pathological situations. Recent Advances: The function of GSH as redox buffer or antioxidant is increasingly being questioned. New functions, especially functions connected to the cellular iron homeostasis, were elucidated. Via the formation of iron complexes, GSH is an important player in all aspects of iron metabolism: sensing and regulation of iron levels, iron trafficking, and biosynthesis of iron cofactors. The variety of GSH coordinated iron complexes and their functions with a special focus on FeS-glutaredoxins are summarized in this review. Interestingly, GSH analogues that function as major low-molecular-weight thiols in organisms lacking GSH resemble the functions in iron homeostasis. Since these iron-related functions are most likely also connected to thiol redox chemistry, it is difficult to distinguish between mechanisms related to either redox or iron metabolisms. The ability of GSH to coordinate iron in different complexes with or without proteins needs further investigation. The discovery of new Fe-GSH complexes and their physiological functions will significantly advance our understanding of cellular iron homeostasis. Antioxid. Redox Signal. 27, 1235-1251.

Molecular Dynamics of the ZIKA Virus NS3 Helicase

NASA Astrophysics Data System (ADS)

Raubenolt, Bryan; Rick, Steven; The Rick Group Team

The recent outbreaks of the ZIKA virus (ZIKV) and its connection to microcephaly in newborns has raised its awareness as a global threat and many scientific research efforts are currently underway in attempt to create a vaccine. Molecular Dynamics is a powerful method of investigating the physical behavior of protein complexes. ZIKV is comprised of 3 structural and 7 nonstructural proteins. The NS3 helicase protein appears to play a significant role in the replication complex and its inhibition could be a crucial source of antiviral drug design. This research primarily focuses on studying the structural dynamics, over the course of few hundred nanoseconds, of NS3 helicase in the free state, as well as in complex form with human ssRNA, ATP, and an analogue of GTP. RMSD and RMSF plots of each simulation will provide details on the forces involved in the overall stability of the active and inactive states. Furthermore, free energy calculations on a per residue level will reveal the most interactive residues between states and ultimately the primary driving force behind these interactions. Together these analyses will provide highly relevant information on the binding surface chemistry and thus serve as the basis for potential drug design.

Coordinate Activation of Redox-Dependent ASK1/TGF-β Signaling by a Multiprotein Complex (MPK38, ASK1, SMADs, ZPR9, and TRX) Improves Glucose and Lipid Metabolism in Mice.

PubMed

Seong, Hyun-A; Manoharan, Ravi; Ha, Hyunjung

2016-03-10

To explore the molecular connections between redox-dependent apoptosis signal-regulating kinase 1 (ASK1) and transforming growth factor-β (TGF-β) signaling pathways and to examine the physiological processes in which coordinated regulation of these two signaling pathways plays a critical role. We provide evidence that the ASK1 and TGF-β signaling pathways are interconnected by a multiprotein complex harboring murine protein serine-threonine kinase 38 (MPK38), ASK1, Sma- and Mad-related proteins (SMADs), zinc-finger-like protein 9 (ZPR9), and thioredoxin (TRX) and demonstrate that the activation of either ASK1 or TGF-β activity is sufficient to activate both the redox-dependent ASK1 and TGF-β signaling pathways. Physiologically, the restoration of the downregulated activation levels of ASK1 and TGF-β signaling in genetically and diet-induced obese mice by adenoviral delivery of SMAD3 or ZPR9 results in the amelioration of adiposity, hyperglycemia, hyperlipidemia, and impaired ketogenesis. Our data suggest that the multiprotein complex linking ASK1 and TGF-β signaling pathways may be a potential target for redox-mediated metabolic complications.

Protein-protein interactions between SWCNT/chitosan/EGF and EGF receptor: a model of drug delivery system.

PubMed

Rungnim, Chompoonut; Rungrotmongkol, Thanyada; Kungwan, Nawee; Hannongbua, Supot

2016-09-01

Epidermal growth factor (EGF) was used as the targeting ligand to enhance the specificity of a cancer drug delivery system (DDS) via its specific interaction with the EGF receptor (EGFR) that is overexpressed on the surface of some cancer cells. To investigate the intermolecular interaction and binding affinity between the EGF-conjugated DDS and the EGFR, 50 ns molecular dynamics simulations were performed on the complex of tethered EGFR and EGF linked to single-wall carbon nanotube (SWCNT) through a biopolymer chitosan wrapping the tube outer surface (EGFR·EGF-CS-SWCNT-Drug complex), and compared to the EGFR·EGF complex and free EGFR. The binding pattern of the EGF-CS-SWCNT-Drug complex to the EGFR was broadly comparable to that for EGF, but the binding affinity of the EGF-CS-SWCNT-Drug complex was predicted to be somewhat better than that for EGF alone. Additionally, the chitosan chain could prevent undesired interactions of SWCNT at the binding pocket region. Therefore, EGF connected to SWCNT via a chitosan linker is a seemingly good formulation for developing a smart DDS served as part of an alternative cancer therapy.

Dynamics between actin and the VE-cadherin/catenin complex

PubMed Central

Abu Taha, Abdallah; Schnittler, Hans-J

2014-01-01

Endothelial adherens junctions are critical for physiological and pathological processes such as differentiation, maintenance of entire monolayer integrity, and the remodeling. The endothelial-specific VE-cadherin/catenin complex provides the backbone of adherens junctions and acts in close interaction with actin filaments and actin/myosin-mediated contractility to fulfill the junction demands. The functional connection between the cadherin/catenin complex and actin filaments might be either directly through α-catenins, or indirectly e.g., via linker proteins such as vinculin, p120ctn, α-actinin, or EPLIN. However, both junction integrity and dynamic remodeling have to be contemporarily coordinated. The actin-related protein complex ARP2/3 and its activating molecules, such as N-WASP and WAVE, have been shown to regulate the lammellipodia-mediated formation of cell junctions in both epithelium and endothelium. Recent reports now demonstrate a novel aspect of the ARP2/3 complex and the nucleating-promoting factors in the maintenance of endothelial barrier function and junction remodeling of established endothelial cell junctions. Those mechanisms open novel possibilities; not only in fulfilling physiological demands but obtained information may be of critical importance in pathologies such as wound healing, angiogenesis, inflammation, and cell diapedesis. PMID:24621569

Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

PubMed

Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

2018-07-04

The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

PubMed Central

Babayeva, Sima; Rocque, Brittany; Aoudjit, Lamine; Zilber, Yulia; Li, Jane; Baldwin, Cindy; Kawachi, Hiroshi; Takano, Tomoko; Torban, Elena

2013-01-01

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate. PMID:23824190

Determining Protein Complex Structures Based on a Bayesian Model of in Vivo Förster Resonance Energy Transfer (FRET) Data*

PubMed Central

Bonomi, Massimiliano; Pellarin, Riccardo; Kim, Seung Joong; Russel, Daniel; Sundin, Bryan A.; Riffle, Michael; Jaschob, Daniel; Ramsden, Richard; Davis, Trisha N.; Muller, Eric G. D.; Sali, Andrej

2014-01-01

The use of in vivo Förster resonance energy transfer (FRET) data to determine the molecular architecture of a protein complex in living cells is challenging due to data sparseness, sample heterogeneity, signal contributions from multiple donors and acceptors, unequal fluorophore brightness, photobleaching, flexibility of the linker connecting the fluorophore to the tagged protein, and spectral cross-talk. We addressed these challenges by using a Bayesian approach that produces the posterior probability of a model, given the input data. The posterior probability is defined as a function of the dependence of our FRET metric FRETR on a structure (forward model), a model of noise in the data, as well as prior information about the structure, relative populations of distinct states in the sample, forward model parameters, and data noise. The forward model was validated against kinetic Monte Carlo simulations and in vivo experimental data collected on nine systems of known structure. In addition, our Bayesian approach was validated by a benchmark of 16 protein complexes of known structure. Given the structures of each subunit of the complexes, models were computed from synthetic FRETR data with a distance root-mean-squared deviation error of 14 to 17 Å. The approach is implemented in the open-source Integrative Modeling Platform, allowing us to determine macromolecular structures through a combination of in vivo FRETR data and data from other sources, such as electron microscopy and chemical cross-linking. PMID:25139910

Studies of Biosilicification; The Role of Proteins, Carbohydrates and Model Compounds in Structure Control

DTIC Science & Technology

2005-12-31

No. carbons Pore volume data. Resolution of complex monosaccharide mixtures from plant cell wall isolates by high pH anion exchange chromatography. To...interwoven polysaccharide chains embedded in a gel matrix of galacturonic acid rich polysaccharides connected by calcium bridges. This network also...picomolar levels). Also, it allows the determination of intact monosaccharides without pre or post column derivatisation, decreasing the time of

KOSMOS: a universal morph server for nucleic acids, proteins and their complexes.

PubMed

Seo, Sangjae; Kim, Moon Ki

2012-07-01

KOSMOS is the first online morph server to be able to address the structural dynamics of DNA/RNA, proteins and even their complexes, such as ribosomes. The key functions of KOSMOS are the harmonic and anharmonic analyses of macromolecules. In the harmonic analysis, normal mode analysis (NMA) based on an elastic network model (ENM) is performed, yielding vibrational modes and B-factor calculations, which provide insight into the potential biological functions of macromolecules based on their structural features. Anharmonic analysis involving elastic network interpolation (ENI) is used to generate plausible transition pathways between two given conformations by optimizing a topology-oriented cost function that guarantees a smooth transition without steric clashes. The quality of the computed pathways is evaluated based on their various facets, including topology, energy cost and compatibility with the NMA results. There are also two unique features of KOSMOS that distinguish it from other morph servers: (i) the versatility in the coarse-graining methods and (ii) the various connection rules in the ENM. The models enable us to analyze macromolecular dynamics with the maximum degrees of freedom by combining a variety of ENMs from full-atom to coarse-grained, backbone and hybrid models with one connection rule, such as distance-cutoff, number-cutoff or chemical-cutoff. KOSMOS is available at http://bioengineering.skku.ac.kr/kosmos.

Network representation of protein interactions: Theory of graph description and analysis.

PubMed

Kurzbach, Dennis

2016-09-01

A methodological framework is presented for the graph theoretical interpretation of NMR data of protein interactions. The proposed analysis generalizes the idea of network representations of protein structures by expanding it to protein interactions. This approach is based on regularization of residue-resolved NMR relaxation times and chemical shift data and subsequent construction of an adjacency matrix that represents the underlying protein interaction as a graph or network. The network nodes represent protein residues. Two nodes are connected if two residues are functionally correlated during the protein interaction event. The analysis of the resulting network enables the quantification of the importance of each amino acid of a protein for its interactions. Furthermore, the determination of the pattern of correlations between residues yields insights into the functional architecture of an interaction. This is of special interest for intrinsically disordered proteins, since the structural (three-dimensional) architecture of these proteins and their complexes is difficult to determine. The power of the proposed methodology is demonstrated at the example of the interaction between the intrinsically disordered protein osteopontin and its natural ligand heparin. © 2016 The Protein Society.

Role of membrane contact sites in protein import into mitochondria

PubMed Central

Horvath, Susanne E; Rampelt, Heike; Oeljeklaus, Silke; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus

2015-01-01

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long-standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence-carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture. PMID:25514890

DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

PubMed Central

Putnam, Andrea A.

2013-01-01

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

Malleable machines in transcription regulation: the mediator complex.

PubMed

Tóth-Petróczy, Agnes; Oldfield, Christopher J; Simon, István; Takagi, Yuichiro; Dunker, A Keith; Uversky, Vladimir N; Fuxreiter, Monika

2008-12-01

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein-protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function.

Biogeoscience from a Metallomic and Proteomic Perspective

NASA Astrophysics Data System (ADS)

Anbar, A. D.; Shock, E.

2004-12-01

In the wake of the genomics revolution, life scientists are expanding their focus from the genome to the "proteome" - the assemblage of all proteins in a cell - and the "metallome" - the distribution of inorganic species in a cell. The proteome and metallome are tightly connected because proteins and protein products are intimately involved in the transport and homeostasis of inorganic elements, and because many enzymes depend on inorganic elements for catalytic activity. Together, they are at the heart of metabolic function. Unlike the relatively static genome, the proteome and metallome are extremely dynamic, changing rapidly in response to environmental cues. They are substantially more complex than the genome; for example, in humans, some 30,000 genes code for approximately 500,000 proteins. Metaphorically, the proteome and metallome constitute the complex, dynamic "language" by which the genome and the environment communicate. Therefore biogeochemists, like life scientists, are moving beyond a strictly genomic perspective. Research guided by proteomic and metallomic perspectives and methodologies should provide new insights into the connections between life and the inorganic Earth in modern environments, and the evolution of these connections through time. For example, biogeochemical research in modern environments, such as Yellowstone hot springs, is hindered by the gap between genomic determinations of metabolic potential in ecosystems and geochemical characterizations of the energetic boundary conditions faced by these ecosystems; genomics tells us "who is there" and geochemistry tells us "what they might be doing", but neither genomics nor geochemistry easily provide quantitative information about which metabolisms are actually active or a framework for understanding why ecosystems do not fully exploit the energy available in their surroundings. Such questions are fundamentally kinetic rather than thermodynamic and therefore demand that we characterize and understand the proteins and inorganic elements used by organisms to catalyze reactions and capture energy from their surroundings. Similar challenges are faced when attempting to map the evolutionary relationships inferred from phylogenetic analyses of genomes to ecological histories determined by geochemists and paleobiologists - for example, ongoing efforts to understand the evolutionary history of eukaryotes and metazoa - because the driving forces for the evolution and ecological radiation of organisms lie at the intersection of metabolism and environment, and hence in the gap between genomes and geochemistry. Future progress in understanding the biogeochemistry of modern and ancient environments will be spurred by integrating proteomic and metallomic methods and perspectives.

Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein.

PubMed

Daniel, Katrin; Tränkner, Daniel; Wojtasz, Lukasz; Shibuya, Hiroki; Watanabe, Yoshinori; Alsheimer, Manfred; Tóth, Attila

2014-05-22

Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions.

Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein

PubMed Central

2014-01-01

Background Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. Results We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. Conclusion CCDC79 is a meiosis-specific telomere associated protein. Based on our findings we propose that CCDC79 plays a role in meiosis-specific telomere functions. In particular, we favour the possibility that CCDC79 is involved in telomere-nuclear envelope attachment and/or the stabilization of meiotic telomeres. These conclusions are consistent with the findings of an independently initiated study that analysed CCDC79/TERB1 functions. PMID:24885367

TOPICAL REVIEW: The physics of chromatin

NASA Astrophysics Data System (ADS)

Schiessel, Helmut

2003-05-01

Recent progress has been made in the understanding of the physical properties of chromatin - the dense complex of DNA and histone proteins that occupies the nuclei of plant and animal cells. Here I will focus on the two lowest levels of the hierarchy of DNA folding into the chromatin complex. (i) The nucleosome, the chromatin repeating unit consisting of a globular aggregate of eight histone proteins with the DNA wrapped around it: its overcharging, the DNA unwrapping transition, the 'sliding' of the octamer along the DNA. (ii) The 30 nm chromatin fibre, the necklace-like structure of nucleosomes connected via linker DNA: its geometry, its mechanical properties under stretching and its response to changing ionic conditions. I will stress that chromatin combines two seemingly contradictory features: (1) high compaction of DNA within the nuclear envelope and, at the same time, (2) accessibility to genes, promoter regions and gene regulatory sequences.

Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

PubMed

Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M; Smith, Eric M; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

2017-12-01

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

PubMed

Iida, Shinji; Nakamura, Haruki; Higo, Junichi

2016-06-15

We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. © 2016 The Author(s).

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

deepNF: Deep network fusion for protein function prediction.

PubMed

Gligorijevic, Vladimir; Barot, Meet; Bonneau, Richard

2018-06-01

The prevalence of high-throughput experimental methods has resulted in an abundance of large-scale molecular and functional interaction networks. The connectivity of these networks provides a rich source of information for inferring functional annotations for genes and proteins. An important challenge has been to develop methods for combining these heterogeneous networks to extract useful protein feature representations for function prediction. Most of the existing approaches for network integration use shallow models that encounter difficulty in capturing complex and highly-nonlinear network structures. Thus, we propose deepNF, a network fusion method based on Multimodal Deep Autoencoders to extract high-level features of proteins from multiple heterogeneous interaction networks. We apply this method to combine STRING networks to construct a common low-dimensional representation containing high-level protein features. We use separate layers for different network types in the early stages of the multimodal autoencoder, later connecting all the layers into a single bottleneck layer from which we extract features to predict protein function. We compare the cross-validation and temporal holdout predictive performance of our method with state-of-the-art methods, including the recently proposed method Mashup. Our results show that our method outperforms previous methods for both human and yeast STRING networks. We also show substantial improvement in the performance of our method in predicting GO terms of varying type and specificity. deepNF is freely available at: https://github.com/VGligorijevic/deepNF. vgligorijevic@flatironinstitute.org, rb133@nyu.edu. Supplementary data are available at Bioinformatics online.

The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains

PubMed Central

Benoit, Matthieu P. M. H.; Imbert, Lionel; Palencia, Andrés; Pérard, Julien; Ebel, Christine; Boisbouvier, Jérôme; Plevin, Michael J.

2013-01-01

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer. PMID:23435228

Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum–plasma membrane junctions

PubMed Central

Wu, Minnie M.; Covington, Elizabeth D.; Lewis, Richard S.

2014-01-01

Following endoplasmic reticulum (ER) Ca2+ depletion, STIM1 and Orai1 complexes assemble autonomously at ER–plasma membrane (PM) junctions to trigger store-operated Ca2+ influx. One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions through interactions with the PM, and STIM1 then traps Orai1 in the PM through binding of its calcium release-activated calcium activation domain. We tested this model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian, while Orai1 is slightly subdiffusive. After store depletion, both proteins slow to the same speeds, consistent with complex formation, and are confined to a corral similar in size to ER–PM junctions. While the escape probability at high STIM:Orai expression ratios is <1%, it is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM-Orai complexes are trapped by their physical connections across the junctional gap, but also reveal that the complexes are surprisingly dynamic, suggesting that readily reversible binding reactions generate free STIM1 and Orai1, which engage in constant diffusional exchange with extrajunctional pools. PMID:25057023

Effects of Cavities at the Nicotinamide Binding Site of Liver Alcohol Dehydrogenase on Structure, Dynamics and Catalysis

PubMed Central

2015-01-01

A role for protein dynamics in enzymatic catalysis of hydrogen transfer has received substantial scientific support, but the connections between protein structure and catalysis remain to be established. Valine residues 203 and 207 are at the binding site for the nicotinamide ring of the coenzyme in liver alcohol dehydrogenase and have been suggested to facilitate catalysis with “protein-promoting vibrations” (PPV). We find that the V207A substitution has small effects on steady-state kinetic constants and the rate of hydrogen transfer; the introduced cavity is empty and is tolerated with minimal effects on structure (determined at 1.2 Å for the complex with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol). Thus, no evidence is found to support a role for Val-207 in the dynamics of catalysis. The protein structures and ligand geometries (including donor–acceptor distances) in the V203A enzyme complexed with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or 2,2,2-trifluoroethanol (determined at 1.1 Å) are very similar to those for the wild-type enzyme, except that the introduced cavity accommodates a new water molecule that contacts the nicotinamide ring. The structures of the V203A enzyme complexes suggest, in contrast to previous studies, that the diminished tunneling and decreased rate of hydride transfer (16-fold, relative to that of the wild-type enzyme) are not due to differences in ground-state ligand geometries. The V203A substitution may alter the PPV and the reorganization energy for hydrogen transfer, but the protein scaffold and equilibrium thermal motions within the Michaelis complex may be more significant for enzyme catalysis. PMID:24437493

Werner complex deficiency in cells disrupts the Nuclear Pore Complex and the distribution of lamin B1.

PubMed

Li, Zhi; Zhu, Yizhou; Zhai, Yujia; R Castroagudin, Michelle; Bao, Yifei; White, Tommy E; Glavy, Joseph S

2013-12-01

From the surrounding shell to the inner machinery, nuclear proteins provide the functional plasticity of the nucleus. This study highlights the nuclear association of Pore membrane (POM) protein NDC1 and Werner protein (WRN), a RecQ helicase responsible for the DNA instability progeria disorder, Werner Syndrome. In our previous publication, we connected the DNA damage sensor Werner's Helicase Interacting Protein (WHIP), a binding partner of WRN, to the NPC. Here, we confirm the association of the WRN/WHIP complex and NDC1. In established WRN/WHIP knockout cell lines, we further demonstrate the interdependence of WRN/WHIP and Nucleoporins (Nups). These changes do not completely abrogate the barrier of the Nuclear Envelope (NE) but do affect the distribution of FG Nups and the RAN gradient, which are necessary for nuclear transport. Evidence from WRN/WHIP knockout cell lines demonstrates changes in the processing and nucleolar localization of lamin B1. The appearance of "RAN holes" void of RAN corresponds to regions within the nucleolus filled with condensed pools of lamin B1. From WRN/WHIP knockout cell line extracts, we found three forms of lamin B1 that correspond to mature holoprotein and two potential post-translationally modified forms of the protein. Upon treatment with topoisomerase inhibitors lamin B1 cleavage occurs only in WRN/WHIP knockout cells. Our data suggest the link of the NDC1 and WRN as one facet of the network between the nuclear periphery and genome stability. Loss of WRN complex leads to multiple alterations at the NPC and the nucleolus. © 2013. Published by Elsevier B.V. All rights reserved.

Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

PubMed Central

Anong, William A.; Franco, Taina; Chu, Haiyan; Weis, Tahlia L.; Devlin, Emily E.; Bodine, David M.; An, Xiuli; Mohandas, Narla

2009-01-01

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. PMID:19567882

Folding pathway of a multidomain protein depends on its topology of domain connectivity

PubMed Central

Inanami, Takashi; Terada, Tomoki P.; Sasai, Masaki

2014-01-01

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant. PMID:25267632

Heat Shock Partially Dissociates the Overlapping Modules of the Yeast Protein-Protein Interaction Network: A Systems Level Model of Adaptation

PubMed Central

Mihalik, Ágoston; Csermely, Peter

2011-01-01

Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a ‘stratus-cumulus’ type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle.

PubMed

Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R; Nguyen, Diep M N; Schut, Gerrit J; Adams, Michael W W; Peters, John W; Boyd, Eric S; Bothner, Brian

2018-01-01

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP + oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units. Copyright © 2017 Elsevier B.V. All rights reserved.

Simulation and display of macromolecular complexes

NASA Technical Reports Server (NTRS)

Nir, S.; Garduno, R.; Rein, R.; Macelroy, R. D.

1977-01-01

In association with an investigation of the interaction of proteins with DNA and RNA, an interactive computer program for building, manipulating, and displaying macromolecular complexes has been designed. The system provides perspective, planar, and stereoscopic views on the computer terminal display, as well as views for standard and nonstandard observer locations. The molecule or its parts may be rotated and/or translated in any direction; bond connections may be added or removed by the viewer. Molecular fragments may be juxtaposed in such a way that given bonds are aligned, and given planes and points coincide. Another subroutine provides for the duplication of a given unit such as a DNA or amino-acid base.

Making the Chromosome-Gene-Protein Connection.

ERIC Educational Resources Information Center

Mulvihill, Charlotte

1996-01-01

Presents an exercise that demonstrates the chromosome-gene-protein connection using sickle-cell anemia, a genetic disease with a well-characterized molecular basis. Involves connecting changes in DNA to protein outcomes and tying them into the next generation by meiosis and gamete formation with genetic crosses. Motivates students to integrate…

Self-assembling complexes of quantum dots and scFv antibodies for cancer cell targeting and imaging.

PubMed

Zdobnova, Tatiana A; Stremovskiy, Oleg A; Lebedenko, Ekaterina N; Deyev, Sergey M

2012-01-01

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex.

Self-Assembling Complexes of Quantum Dots and scFv Antibodies for Cancer Cell Targeting and Imaging

PubMed Central

Zdobnova, Tatiana A.; Stremovskiy, Oleg A.; Lebedenko, Ekaterina N.; Deyev, Sergey M.

2012-01-01

Semiconductor quantum dots represent a novel class of fluorophores with unique physical and chemical properties which could enable a remarkable broadening of the current applications of fluorescent imaging and optical diagnostics. Complexes of quantum dots and antibodies are promising visualising agents for fluorescent detection of selective biomarkers overexpressed in tumor tissues. Here we describe the construction of self-assembling fluorescent complexes of quantum dots and anti-HER1 or anti-HER2/neu scFv antibodies and their interactions with cultured tumor cells. A binding strategy based on a very specific non-covalent interaction between two proteins, barnase and barstar, was used to connect quantum dots and the targeting antibodies. Such a strategy allows combining the targeting and visualization functions simply by varying the corresponding modules of the fluorescent complex. PMID:23133578

Decreased cerebellar-cerebral connectivity contributes to complex task performance

PubMed Central

Knops, André

2016-01-01

The cerebellum's role in nonmotor processes is now well accepted, but cerebellar interaction with cerebral targets is not well understood. Complex cognitive tasks activate cerebellar, parietal, and frontal regions, but the effective connectivity between these regions has never been tested. To this end, we used psycho-physiological interactions (PPI) analysis to test connectivity changes of cerebellar and parietal seed regions in complex (2-digit by 1-digit multiplication, e.g., 12 × 3) vs. simple (1-digit by 1-digit multiplication, e.g., 4 × 3) task conditions (“complex − simple”). For cerebellar seed regions (lobule VI, hemisphere and vermis), we found significantly decreased cerebellar-parietal, cerebellar-cingulate, and cerebellar-frontal connectivity in complex multiplication. For parietal seed regions (PFcm, PFop, PFm) we found significantly increased parietal-parietal and parietal-frontal connectivity in complex multiplication. These results suggest that decreased cerebellar-cerebral connectivity contributes to complex task performance. Interestingly, BOLD activity contrasts revealed partially overlapping parietal areas of increased BOLD activity but decreased cerebellar-parietal PPI connectivity. PMID:27334957

Correlations between Community Structure and Link Formation in Complex Networks

PubMed Central

Liu, Zhen; He, Jia-Lin; Kapoor, Komal; Srivastava, Jaideep

2013-01-01

Background Links in complex networks commonly represent specific ties between pairs of nodes, such as protein-protein interactions in biological networks or friendships in social networks. However, understanding the mechanism of link formation in complex networks is a long standing challenge for network analysis and data mining. Methodology/Principal Findings Links in complex networks have a tendency to cluster locally and form so-called communities. This widely existed phenomenon reflects some underlying mechanism of link formation. To study the correlations between community structure and link formation, we present a general computational framework including a theory for network partitioning and link probability estimation. Our approach enables us to accurately identify missing links in partially observed networks in an efficient way. The links having high connection likelihoods in the communities reveal that links are formed preferentially to create cliques and accordingly promote the clustering level of the communities. The experimental results verify that such a mechanism can be well captured by our approach. Conclusions/Significance Our findings provide a new insight into understanding how links are created in the communities. The computational framework opens a wide range of possibilities to develop new approaches and applications, such as community detection and missing link prediction. PMID:24039818

Domain mapping of the Rad51 paralog protein complexes

PubMed Central

Miller, Kristi A.; Sawicka, Dorota; Barsky, Daniel; Albala, Joanna S.

2004-01-01

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes. PMID:14704354

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization.

PubMed

Jinek, Martin; Eulalio, Ana; Lingel, Andreas; Helms, Sigrun; Conti, Elena; Izaurralde, Elisa

2008-10-01

The removal of the 5' cap structure by the DCP1-DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all alpha-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family.

Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategy.

PubMed

Korecká, Lucie; Jankovicová, Barbora; Krenková, Jana; Hernychová, Lenka; Slováková, Marcela; Le-Nell, Anne; Chmelik, Josef; Foret, Frantisek; Viovy, Jean-Louis; Bilková, Zusana

2008-02-01

We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG)-Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach.

Structures of the prefusion form of measles virus fusion protein in complex with inhibitors.

PubMed

Hashiguchi, Takao; Fukuda, Yoshinari; Matsuoka, Rei; Kuroda, Daisuke; Kubota, Marie; Shirogane, Yuta; Watanabe, Shumpei; Tsumoto, Kouhei; Kohda, Daisuke; Plemper, Richard Karl; Yanagi, Yusuke

2018-03-06

Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity. Here we show the crystal structures of the prefusion MeV-F alone and in complex with the small compound AS-48 or a fusion inhibitor peptide. Notably, these independently developed inhibitors bind the same hydrophobic pocket located at the region connecting the head and stalk of MeV-F, where a number of substitutions in MeV isolates from neurodegenerative diseases are also localized. Since these inhibitors could suppress membrane fusion mediated by most of the hyperfusogenic MeV-F mutants, the development of more effective inhibitors based on the structures may be warranted to treat MeV-induced neurodegenerative diseases.

Mechano-sensing and mechano-reaction of soft connective tissue cells

NASA Astrophysics Data System (ADS)

Lambert, Ch. A.; Nusgens, B. V.; Lapière, Ch. M.

One main function of the connective tissues is to provide cells with a mechanically resistant attachment support required for survival, division and differentiation. All cells contain membrane-anchored attachment proteins able to recognize specific chemical motifs in the extracellular macromolecules forming the supporting scaffold, made of various types of collagen, adhesive glycoproteins, elastin, proteoglycans, etc... These cell-matrix interactions are mainly mediated by re ceptors of the integrins family, heterodimeric molecules made of an extracellular domain connected through a transmembrane sequence to an intracytoplasmic tail. Upon recognition of the extracellular ligand, the clustering and activation of the integrins result in the recruitment of a complex of proteins and formation of the focal adhesion plaque, containing both cytoskeletal and catalytic signaling molecules. Activation results in polymerization of actin and formation of stress fibers. These structures establish a physical link between the extracellular matrix components and the cytoskeleton through the integrins providing a continuous path acting as a mechanotransducer. This connection is used by the cells to perform their mechanical functions as adhesion, migration and traction. In vitro experimental models using fibroblasts in a collagen gel demonstrate that cells are in mechanical equilibrium with their support which regulates their replicative and biosynthetic phenotype. The present review discusses the molecular structures operating in the transmission of the mechanical messages from the support to the connective tissue cells, and their effect on the cellular machinery. We present arguments for investigating these mechanisms in understanding the perception of reduced gravity and the resulting reaction leading to microgravity induced pathologies.

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

PubMed

Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

Initial photoinduced dynamics of the photoactive yellow protein.

PubMed

Larsen, Delmar S; van Grondelle, Rienk

2005-05-01

The photoactive yellow protein (PYP) is the photoreceptor protein responsible for initiating the blue-light repellent response of the Halorhodospira halophila bacterium. Optical excitation of the intrinsic chromophore in PYP, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. The dynamical processes responsible for the initiation of the PYP photocycle have been explored with several time-resolved techniques, which include ultrafast electronic and vibrational spectroscopies. Ultrafast electronic spectroscopies, such as pump-visible probe, pump-dump-visible probe, and fluorescence upconversion, are useful in identifying the timescales and connectivity of the transient intermediates, while ultrafast vibrational spectroscopies link these intermediates to dynamic structures. Herein, we present the use of these techniques for exploring the initial dynamics of PYP, and show how these techniques provide the basis for understanding the complex relationship between protein and chromophore, which ultimately results in biological function.

Progress in Top-Down Proteomics and the Analysis of Proteoforms

PubMed Central

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

2017-01-01

From a molecular perspective, enactors of function in biology are intact proteins that can be variably modified at the genetic, transcriptional, or post-translational level. Over the past 30 years, mass spectrometry (MS) has become a powerful method for the analysis of proteomes. Prevailing bottom-up proteomics operates at the level of the peptide, leading to issues with protein inference, connectivity, and incomplete sequence/modification information. Top-down proteomics (TDP), alternatively, applies MS at the proteoform level to analyze intact proteins with diverse sources of intramolecular complexity preserved during analysis. Fortunately, advances in prefractionation workflows, MS instrumentation, and dissociation methods for whole-protein ions have helped TDP emerge as an accessible and potentially disruptive modality with increasingly translational value. In this review, we discuss technical and conceptual advances in TDP, along with the growing power of proteoform-resolved measurements in clinical and translational research. PMID:27306313

Unfolded protein response in filamentous fungi-implications in biotechnology.

PubMed

Heimel, Kai

2015-01-01

The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology.

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

PubMed Central

Borrero-de Acuña, José Manuel; Rohde, Manfred; Wissing, Josef; Jänsch, Lothar; Schobert, Max; Molinari, Gabriella; Timmis, Kenneth N.

2016-01-01

ABSTRACT Oxidative phosphorylation using multiple-component, membrane-associated protein complexes is the most effective way for a cell to generate energy. Here, we systematically investigated the multiple protein-protein interactions of the denitrification apparatus of the pathogenic bacterium Pseudomonas aeruginosa. During denitrification, nitrate (Nar), nitrite (Nir), nitric oxide (Nor), and nitrous oxide (Nos) reductases catalyze the reaction cascade of NO3− → NO2− → NO → N2O → N2. Genetic experiments suggested that the nitric oxide reductase NorBC and the regulatory protein NosR are the nucleus of the denitrification protein network. We utilized membrane interactomics in combination with electron microscopy colocalization studies to elucidate the corresponding protein-protein interactions. The integral membrane proteins NorC, NorB, and NosR form the core assembly platform that binds the nitrate reductase NarGHI and the periplasmic nitrite reductase NirS via its maturation factor NirF. The periplasmic nitrous oxide reductase NosZ is linked via NosR. The nitrate transporter NarK2, the nitrate regulatory system NarXL, various nitrite reductase maturation proteins, NirEJMNQ, and the Nos assembly lipoproteins NosFL were also found to be attached. A number of proteins associated with energy generation, including electron-donating dehydrogenases, the complete ATP synthase, almost all enzymes of the tricarboxylic acid (TCA) cycle, and the Sec system of protein transport, among many other proteins, were found to interact with the denitrification proteins. This deduced nitrate respirasome is presumably only one part of an extensive cytoplasmic membrane-anchored protein network connecting cytoplasmic, inner membrane, and periplasmic proteins to mediate key activities occurring at the barrier/interface between the cytoplasm and the external environment. IMPORTANCE The processes of cellular energy generation are catalyzed by large multiprotein enzyme complexes. The molecular basis for the interaction of these complexes is poorly understood. We employed membrane interactomics and electron microscopy to determine the protein-protein interactions involved. The well-investigated enzyme complexes of denitrification of the pathogenic bacterium Pseudomonas aeruginosa served as a model. Denitrification is one essential step of the universal N cycle and provides the bacterium with an effective alternative to oxygen respiration. This process allows the bacterium to form biofilms, which create low-oxygen habitats and which are a key in the infection mechanism. Our results provide new insights into the molecular basis of respiration, as well as opening a new window into the infection strategies of this pathogen. PMID:26903416

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc{sub 1} function in vivo

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

Ekiert, Robert; Czapla, Monika; Sarewicz, Marcin

2014-08-22

Highlights: • We used hybrid fusion bc{sub 1} complex to test inter-monomer electron transfer in vivo. • Cross-inactivated complexes were able to sustain photoheterotrophic growth. • Inter-monomer electron transfer supports catalytic cycle in vivo. • bc{sub 1} dimer is functional even when cytochrome b subunits come from different species. - Abstract: Electronic connection between Q{sub o} and Q{sub i} quinone catalytic sites of dimeric cytochrome bc{sub 1} is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the lattermore » remains unclear. Here, using a series of mutated hybrid cytochrome bc{sub 1}-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.« less

KOSMOS: a universal morph server for nucleic acids, proteins and their complexes

PubMed Central

Seo, Sangjae; Kim, Moon Ki

2012-01-01

KOSMOS is the first online morph server to be able to address the structural dynamics of DNA/RNA, proteins and even their complexes, such as ribosomes. The key functions of KOSMOS are the harmonic and anharmonic analyses of macromolecules. In the harmonic analysis, normal mode analysis (NMA) based on an elastic network model (ENM) is performed, yielding vibrational modes and B-factor calculations, which provide insight into the potential biological functions of macromolecules based on their structural features. Anharmonic analysis involving elastic network interpolation (ENI) is used to generate plausible transition pathways between two given conformations by optimizing a topology-oriented cost function that guarantees a smooth transition without steric clashes. The quality of the computed pathways is evaluated based on their various facets, including topology, energy cost and compatibility with the NMA results. There are also two unique features of KOSMOS that distinguish it from other morph servers: (i) the versatility in the coarse-graining methods and (ii) the various connection rules in the ENM. The models enable us to analyze macromolecular dynamics with the maximum degrees of freedom by combining a variety of ENMs from full-atom to coarse-grained, backbone and hybrid models with one connection rule, such as distance-cutoff, number-cutoff or chemical-cutoff. KOSMOS is available at http://bioengineering.skku.ac.kr/kosmos. PMID:22669912

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

NASA Astrophysics Data System (ADS)

Zhou, Mowei; Yan, Jing; Romano, Christine A.; Tebo, Bradley M.; Wysocki, Vicki H.; Paša-Tolić, Ljiljana

2018-01-01

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase, Mnx, from Bacillus sp. PL-12 has been successfully overexpressed in active form as a protein complex with a molecular mass of 211 kDa. We have recently used surface induced dissociation (SID) and ion mobility-mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data from the native mass spectrometry experiments led to a plausible structural model of this multicopper oxidase, which has been difficult to study by conventional structural biology methods. It was also revealed that each Mnx subunit binds a variable number of copper ions. Becasue of the heterogeneity of the protein and limited mass resolution, ambiguities in assigning some of the observed peaks remained as a barrier to fully understanding the role of metals and potential unknown ligands in Mnx. In this study, we performed SID in a modified Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer. The high mass accuracy and resolution offered by FTICR unveiled unexpected artificial modifications on the protein that had been previously thought to be iron bound species based on lower resolution spectra. Additionally, isotopically resolved spectra of the released subcomplexes revealed the metal binding stoichiometry at different structural levels. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes. [Figure not available: see fulltext.

Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR.

PubMed

Thompson, Matthew K; Ehlinger, Aaron C; Chazin, Walter J

2017-01-01

Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional dynamics of modular multidomain proteins is a major challenge confronting structural biology today because their structure is not fixed in time. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy have proven particularly useful for the analysis of the structural dynamics of modular multidomain proteins because they provide highly complementary information for characterizing the architectural landscape accessible to these proteins. SAXS provides a global snapshot of all architectural space sampled by a molecule in solution. Furthermore, SAXS is sensitive to conformational changes, organization and oligomeric states of protein assemblies, and the existence of flexibility between globular domains in multiprotein complexes. The power of NMR to characterize dynamics provides uniquely complementary information to the global snapshot of the architectural ensemble provided by SAXS because it can directly measure domain motion. In particular, NMR parameters can be used to define the diffusion of domains within modular multidomain proteins, connecting the amplitude of interdomain motion to the architectural ensemble derived from SAXS. Our laboratory has been studying the roles of modular multidomain proteins involved in human DNA replication using SAXS and NMR. Here, we present the procedure for acquiring and analyzing SAXS and NMR data, using DNA primase and replication protein A as examples. © 2017 Elsevier Inc. All rights reserved.

Changes in some thylakoid membrane proteins and pigments upon desiccation of the resurrection plant Haberlea rhodopensis.

PubMed

Georgieva, Katya; Röding, Anja; Büchel, Claudia

2009-09-15

The changes in some proteins involved in the light reactions of photosynthesis of the resurrection plant Haberlea rhodopensis were examined in connection with desiccation. Fully hydrated (control) and completely desiccated plants (relative water content (RWC) 6.5%) were used for thylakoid preparations. The chlorophyll (Chl) a to Chl b ratios of thylakoids isolated from control and desiccated leaves were very similar, which was also confirmed by measuring their absorption spectra. HPLC analysis revealed that beta-carotene content was only slightly enhanced in desiccated leaves compared with the control, but the zeaxanthin level was strongly increased. Desiccation of H. rhodopensis to an air-dried state at very low light irradiance led to a little decrease in the level of D1, D2, PsbS and PsaA/B proteins in thylakoids, but a relative increase in LHC polypeptides. To further elucidate whether the composition of the protein complexes of the thylakoid membranes had changed, we performed a separation of solubilized thylakoids on sucrose density gradients. In contrast to spinach, Haberlea thylakoids appeared to be much more resistant to the same solubilization procedure, i.e. complexes were not separated completely and complexes of higher density were found. However, the fractions analyzed provided clear evidence for a move of part of the antenna complexes from PSII to PSI when plants became desiccated. This move was also confirmed by low temperature emission spectra of thylakoids. Overall, the photosynthetic proteins remained comparatively stable in dried Haberlea leaves when plants were desiccated under conditions similar to their natural habitat. Low light during desiccation was enough to induce a rise in the xanthophyll zeaxanthin and beta-carotene. Together with the extensive leaf shrinkage and some leaf folding, increased zeaxanthin content and the observed shift in antenna proteins from PSII to PSI during desiccation of Haberlea contributed to the integrity of the photosynthetic apparatus, which is important for rapid recovery after rehydration.

Allosteric Regulation of the Hsp90 Dynamics and Stability by Client Recruiter Cochaperones: Protein Structure Network Modeling

PubMed Central

Blacklock, Kristin; Verkhivker, Gennady M.

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins. PMID:24466147

Allosteric regulation of the Hsp90 dynamics and stability by client recruiter cochaperones: protein structure network modeling.

PubMed

Blacklock, Kristin; Verkhivker, Gennady M

2014-01-01

The fundamental role of the Hsp90 chaperone in supporting functional activity of diverse protein clients is anchored by specific cochaperones. A family of immune sensing client proteins is delivered to the Hsp90 system with the aid of cochaperones Sgt1 and Rar1 that act cooperatively with Hsp90 to form allosterically regulated dynamic complexes. In this work, functional dynamics and protein structure network modeling are combined to dissect molecular mechanisms of Hsp90 regulation by the client recruiter cochaperones. Dynamic signatures of the Hsp90-cochaperone complexes are manifested in differential modulation of the conformational mobility in the Hsp90 lid motif. Consistent with the experiments, we have determined that targeted reorganization of the lid dynamics is a unifying characteristic of the client recruiter cochaperones. Protein network analysis of the essential conformational space of the Hsp90-cochaperone motions has identified structurally stable interaction communities, interfacial hubs and key mediating residues of allosteric communication pathways that act concertedly with the shifts in conformational equilibrium. The results have shown that client recruiter cochaperones can orchestrate global changes in the dynamics and stability of the interaction networks that could enhance the ATPase activity and assist in the client recruitment. The network analysis has recapitulated a broad range of structural and mutagenesis experiments, particularly clarifying the elusive role of Rar1 as a regulator of the Hsp90 interactions and a stability enhancer of the Hsp90-cochaperone complexes. Small-world organization of the interaction networks in the Hsp90 regulatory complexes gives rise to a strong correspondence between highly connected local interfacial hubs, global mediator residues of allosteric interactions and key functional hot spots of the Hsp90 activity. We have found that cochaperone-induced conformational changes in Hsp90 may be determined by specific interaction networks that can inhibit or promote progression of the ATPase cycle and thus control the recruitment of client proteins.

From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

NASA Astrophysics Data System (ADS)

Gruber, J. Michael; Malý, Pavel; Krüger, Tjaart P. J.; Grondelle, Rienk van

2018-01-01

The conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10-20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains.

PubMed

Remis, Jonathan P; Wei, Dongguang; Gorur, Amita; Zemla, Marcin; Haraga, Jessica; Allen, Simon; Witkowska, H Ewa; Costerton, J William; Berleman, James E; Auer, Manfred

2014-02-01

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

NRF2-ome: an integrated web resource to discover protein interaction and regulatory networks of NRF2.

PubMed

Türei, Dénes; Papp, Diána; Fazekas, Dávid; Földvári-Nagy, László; Módos, Dezső; Lenti, Katalin; Csermely, Péter; Korcsmáros, Tamás

2013-01-01

NRF2 is the master transcriptional regulator of oxidative and xenobiotic stress responses. NRF2 has important roles in carcinogenesis, inflammation, and neurodegenerative diseases. We developed an online resource, NRF2-ome, to provide an integrated and systems-level database for NRF2. The database contains manually curated and predicted interactions of NRF2 as well as data from external interaction databases. We integrated NRF2 interactome with NRF2 target genes, NRF2 regulating TFs, and miRNAs. We connected NRF2-ome to signaling pathways to allow mapping upstream NRF2 regulatory components that could directly or indirectly influence NRF2 activity totaling 35,967 protein-protein and signaling interactions. The user-friendly website allows researchers without computational background to search, browse, and download the database. The database can be downloaded in SQL, CSV, BioPAX, SBML, PSI-MI, and in a Cytoscape CYS file formats. We illustrated the applicability of the website by suggesting a posttranscriptional negative feedback of NRF2 by MAFG protein and raised the possibility of a connection between NRF2 and the JAK/STAT pathway through STAT1 and STAT3. NRF2-ome can also be used as an evaluation tool to help researchers and drug developers to understand the hidden regulatory mechanisms in the complex network of NRF2.

Bringing functions together with fusion enzymes--from nature's inventions to biotechnological applications.

PubMed

Elleuche, Skander

2015-02-01

It is a mammoth task to develop a modular protein toolbox enabling the production of posttranslational organized multifunctional enzymes that catalyze reactions in complex pathways. However, nature has always guided scientists to mimic evolutionary inventions in the laboratory and, nowadays, versatile methods have been established to experimentally connect enzymatic activities with multiple advantages. Among the oldest known natural examples is the linkage of two or more juxtaposed proteins catalyzing consecutive, non-consecutive, or opposing reactions by a native peptide bond. There are multiple reasons for the artificial construction of such fusion enzymes including improved catalytic activities, enabled substrate channelling by proximity of biocatalysts, higher stabilities, and cheaper production processes. To produce fused proteins, it is either possible to genetically fuse coding open reading frames or to connect proteins in a posttranslational process. Molecular biology techniques that have been established for the production of end-to-end or insertional fusions include overlap extension polymerase chain reaction, cloning, and recombination approaches. Depending on their flexibility and applicability, these methods offer various advantages to produce fusion genes in high throughput, different orientations, and including linker sequences to maximize the flexibility and performance of fusion partners. In this review, practical techniques to fuse genes are highlighted, enzymatic parameters to choose adequate enzymes for fusion approaches are summarized, and examples with biotechnological relevance are presented including a focus on plant biomass-degrading glycosyl hydrolases.

Offdiagonal complexity: A computationally quick complexity measure for graphs and networks

NASA Astrophysics Data System (ADS)

Claussen, Jens Christian

2007-02-01

A vast variety of biological, social, and economical networks shows topologies drastically differing from random graphs; yet the quantitative characterization remains unsatisfactory from a conceptual point of view. Motivated from the discussion of small scale-free networks, a biased link distribution entropy is defined, which takes an extremum for a power-law distribution. This approach is extended to the node-node link cross-distribution, whose nondiagonal elements characterize the graph structure beyond link distribution, cluster coefficient and average path length. From here a simple (and computationally cheap) complexity measure can be defined. This offdiagonal complexity (OdC) is proposed as a novel measure to characterize the complexity of an undirected graph, or network. While both for regular lattices and fully connected networks OdC is zero, it takes a moderately low value for a random graph and shows high values for apparently complex structures as scale-free networks and hierarchical trees. The OdC approach is applied to the Helicobacter pylori protein interaction network and randomly rewired surrogates.

Pib2 and the EGO complex are both required for activation of TORC1.

PubMed

Varlakhanova, Natalia V; Mihalevic, Michael J; Bernstein, Kara A; Ford, Marijn G J

2017-11-15

The TORC1 complex is a key regulator of cell growth and metabolism in Saccharomyces cerevisiae The vacuole-associated EGO complex couples activation of TORC1 to the availability of amino acids, specifically glutamine and leucine. The EGO complex is also essential for reactivation of TORC1 following rapamycin-induced growth arrest and for its distribution on the vacuolar membrane. Pib2, a FYVE-containing phosphatidylinositol 3-phosphate (PI3P)-binding protein, is a newly discovered and poorly characterized activator of TORC1. Here, we show that Pib2 is required for reactivation of TORC1 following rapamycin-induced growth arrest. Pib2 is required for EGO complex-mediated activation of TORC1 by glutamine and leucine as well as for redistribution of Tor1 on the vacuolar membrane. Therefore, Pib2 and the EGO complex cooperate to activate TORC1 and connect phosphoinositide 3-kinase (PI3K) signaling and TORC1 activity. © 2017. Published by The Company of Biologists Ltd.

Immersion freezing of ice nucleation active protein complexes

NASA Astrophysics Data System (ADS)

Hartmann, S.; Augustin, S.; Clauss, T.; Wex, H.; Šantl-Temkiv, T.; Voigtländer, J.; Niedermeier, D.; Stratmann, F.

2013-06-01

Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS), the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between -5 °C to -38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA) bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about -6 °C to about -10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei) which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a) the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b) the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice nucleation are attached to the outer membrane of intact bacteria or membrane fragments, (c) the temperature range in which heterogeneous droplet freezing occurs, and the fraction of droplets being able to freeze, both depend on the actual number of INA protein complexes present in the droplet ensemble, and (d) possible artifacts suspected to occur in connection with the drop freezing method, i.e., the method frequently used by biologist for quantifying ice nucleation behaviour, are of minor importance, at least for substances such as P. syringae, which induce freezing at comparably high temperatures. The last statement implies that for single ice nucleation entities such as INA protein complexes, it is the number of entities present in the droplet population, and the entities' nucleation rate, which control the freezing behaviour of the droplet population. Quantities such as ice active surface site density are not suitable in this context. The results obtained in this study allow a different perspective on the quantification of the immersion freezing behaviour of bacterial ice nucleation.

The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility

PubMed Central

Yamamoto, Ryosuke; Song, Kangkang; Yanagisawa, Haru-aki; Fox, Laura; Yagi, Toshiki; Wirschell, Maureen; Hirono, Masafumi; Kamiya, Ritsu; Nicastro, Daniela

2013-01-01

Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin–dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility. PMID:23569216

Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction.

PubMed

Rose, Alexander S; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W

2015-01-01

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

Differential accumulation of photosynthetic proteins regulates diurnal photochemical adjustments of PSII in common fig (Ficus carica L.) leaves.

PubMed

Mlinarić, Selma; Antunović Dunić, Jasenka; Skendrović Babojelić, Martina; Cesar, Vera; Lepeduš, Hrvoje

2017-02-01

Molecular processes involved in photosystem II adaptation of woody species to diurnal changes in light and temperature conditions are still not well understood. Regarding this, here we investigated differences between young and mature leaves of common fig (Ficus carica L.) in photosynthetic performance as well as accumulation of the main photosynthetic proteins: light harvesting complex II, D1 protein and Rubisco large subunit. Investigated leaf types revealed different adjustment mechanisms to keep effective photosynthesis. Rather stable diurnal accumulation of light harvesting complex II in mature leaves enabled efficient excitation energy utilization (negative L-band) what triggered faster D1 protein degradation at high light. However, after photoinhibition, greater accumulation of D1 during the night enabled them faster recovery. So, the most photosynthetic parameters, as the maximum quantum yield for primary photochemistry, electron transport and overall photosynthetic efficiency in mature leaves successfully restored to their initial values at 1a.m. Reduced connectivity of light harvesting complexes II to its reaction centers (positive L-band) in young leaves increased dissipation of excess light causing less pressure to D1 and its slower degradation. Decreased electron transport in young leaves, due to reduced transfer beyond primary acceptor Q A - most probably additionally induced degradation of Rubisco large subunit what consequently led to the stronger decrease of overall photosynthetic efficiency in young leaves at noon. Copyright © 2016 Elsevier GmbH. All rights reserved.

Network-Based Disease Module Discovery by a Novel Seed Connector Algorithm with Pathobiological Implications.

PubMed

Wang, Rui-Sheng; Loscalzo, Joseph

2018-05-20

Understanding the genetic basis of complex diseases is challenging. Prior work shows that disease-related proteins do not typically function in isolation. Rather, they often interact with each other to form a network module that underlies dysfunctional mechanistic pathways. Identifying such disease modules will provide insights into a systems-level understanding of molecular mechanisms of diseases. Owing to the incompleteness of our knowledge of disease proteins and limited information on the biological mediators of pathobiological processes, the key proteins (seed proteins) for many diseases appear scattered over the human protein-protein interactome and form a few small branches, rather than coherent network modules. In this paper, we develop a network-based algorithm, called the Seed Connector algorithm (SCA), to pinpoint disease modules by adding as few additional linking proteins (seed connectors) to the seed protein pool as possible. Such seed connectors are hidden disease module elements that are critical for interpreting the functional context of disease proteins. The SCA aims to connect seed disease proteins so that disease mechanisms and pathways can be decoded based on predicted coherent network modules. We validate the algorithm using a large corpus of 70 complex diseases and binding targets of over 200 drugs, and demonstrate the biological relevance of the seed connectors. Lastly, as a specific proof of concept, we apply the SCA to a set of seed proteins for coronary artery disease derived from a meta-analysis of large-scale genome-wide association studies and obtain a coronary artery disease module enriched with important disease-related signaling pathways and drug targets not previously recognized. Copyright © 2018 Elsevier Ltd. All rights reserved.

Striatal Transcriptome and Interactome Analysis of Shank3-overexpressing Mice Reveals the Connectivity between Shank3 and mTORC1 Signaling

PubMed Central

Lee, Yeunkum; Kim, Sun Gyun; Lee, Bokyoung; Zhang, Yinhua; Kim, Yoonhee; Kim, Shinhyun; Kim, Eunjoon; Kang, Hyojin; Han, Kihoon

2017-01-01

Mania causes symptoms of hyperactivity, impulsivity, elevated mood, reduced anxiety and decreased need for sleep, which suggests that the dysfunction of the striatum, a critical component of the brain motor and reward system, can be causally associated with mania. However, detailed molecular pathophysiology underlying the striatal dysfunction in mania remains largely unknown. In this study, we aimed to identify the molecular pathways showing alterations in the striatum of SH3 and multiple ankyrin repeat domains 3 (Shank3)-overexpressing transgenic (TG) mice that display manic-like behaviors. The results of transcriptome analysis suggested that mammalian target of rapamycin complex 1 (mTORC1) signaling may be the primary molecular signature altered in the Shank3 TG striatum. Indeed, we found that striatal mTORC1 activity, as measured by mTOR S2448 phosphorylation, was significantly decreased in the Shank3 TG mice compared to wild-type (WT) mice. To elucidate the potential underlying mechanism, we re-analyzed previously reported protein interactomes, and detected a high connectivity between Shank3 and several upstream regulators of mTORC1, such as tuberous sclerosis 1 (TSC1), TSC2 and Ras homolog enriched in striatum (Rhes), via 94 common interactors that we denominated “Shank3-mTORC1 interactome”. We noticed that, among the 94 common interactors, 11 proteins were related to actin filaments, the level of which was increased in the dorsal striatum of Shank3 TG mice. Furthermore, we could co-immunoprecipitate Shank3, Rhes and Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) proteins from the striatal lysate of Shank3 TG mice. By comparing with the gene sets of psychiatric disorders, we also observed that the 94 proteins of Shank3-mTORC1 interactome were significantly associated with bipolar disorder (BD). Altogether, our results suggest a protein interaction-mediated connectivity between Shank3 and certain upstream regulators of mTORC1 that might contribute to the abnormal striatal mTORC1 activity and to the manic-like behaviors of Shank3 TG mice. PMID:28701918

Cell density signal protein suitable for treatment of connective tissue injuries and defects

DOEpatents

Schwarz, Richard I.

2002-08-13

Identification, isolation and partial sequencing of a cell density protein produced by fibroblastic cells. The cell density signal protein comprising a 14 amino acid peptide or a fragment, variant, mutant or analog thereof, the deduced cDNA sequence from the 14 amino acid peptide, a recombinant protein, protein and peptide-specific antibodies, and the use of the peptide and peptide-specific antibodies as therapeutic agents for regulation of cell differentiation and proliferation. A method for treatment and repair of connective tissue and tendon injuries, collagen deficiency, and connective tissue defects.

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

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

Wu, William Ka Kei, E-mail: wukakei@cuhk.edu.hk; Department of Medicine and Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong; Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong

2009-09-04

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85more » S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.« less

The physics of complex systems in information and biology

NASA Astrophysics Data System (ADS)

Walker, Dylan

Citation networks have re-emerged as a topic intense interest in the complex networks community with the recent availability of large-scale data sets. The ranking of citation networks is a necessary practice as a means to improve information navigability and search. Unlike many information networks, the aging characteristics of citation networks require the development of new ranking methods. To account for strong aging characteristics of citation networks, we modify the PageRank algorithm by initially distributing random surfers exponentially with age, in favor of more recent publications. The output of this algorithm, which we call CiteRank, is interpreted as approximate traffic to individual publications in a simple model of how researchers find new information. We optimize parameters of our algorithm to achieve the best performance. The results are compared for two rather different citation networks: all American Physical Society publications between 1893-2003 and the set of high-energy physics theory (hep-th) preprints. Despite major differences between these two networks, we find that their optimal parameters for the CiteRank algorithm are remarkably similar. The advantages and performance of CiteRank over more conventional methods of ranking publications are discussed. Collaborative voting systems have emerged as an abundant form of real-world, complex information systems that exist in a variety of online applications. These systems are comprised of large populations of users that collectively submit and vote on objects. While the specific properties of these systems vary widely, many of them share a core set of features and dynamical behaviors that govern their evolution. We study a subset of these systems that involve material of a time-critical nature as in the popular example of news items. We consider a general model system in which articles are introduced, voted on by a population of users, and subsequently expire after a proscribed period of time. To study the interaction between popularity and quality, we introduce simple stochastic models of user behavior that approximate differing user quality and susceptibility to the common notion of popularity. We define a metric to quantify user reputation in a manner that is self-consistent, adaptable and content-blind and shows good correlation with the probability that a user behaves in an optimal fashion. We further construct a mechanism for ranking documents that take into account user reputation and provides substantial improvement in the time-critical performance of the system. The structure of complex systems have been well studied in the context of both information and biological systems. More recently, dynamics in complex systems that occur over the background of the underlying network has received a great deal of attention. In particular, the study of fluctuations in complex systems has emerged as an issue central to understanding dynamical behavior. We approach the problem of collective effects of the underlying network on dynamical fluctuations by considering the protein-protein interaction networks for the system of the living cell. We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by relatively slow changes in total concentrations (copy numbers) of interacting proteins. The second type, to which we refer to as spontaneous, is caused by quickly decaying thermodynamic deviations away from the mass-action equilibrium of the system. As such they are amenable to methods of equilibrium statistical mechanics used in our study. We investigate the effects of network connectivity on these fluctuations by comparing them to different scenarios in which the interacting pair is isolated form the rest of the network. Such comparison allows us to analytically derive upper and lower bounds on network fluctuations. The collective effects are shown to sometimes lead to relatively large amplification of spontaneous fluctuations as compared to the expectation for isolated dimers. As a consequence of this, the strength of both types of fluctuations is positively correlated with the overall network connectivity of proteins forming the complex. On the other hand, the relative amplitude of fluctuations is negatively correlated with the equilibrium concentration of the complex. Our general findings are illustrated using a curated network of protein-protein interactions and multi-protein complexes in bakers yeast with experimentally determined protein concentrations.

A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions.

PubMed

Bachmann, André; Kobler, Oliver; Kittel, Robert J; Wichmann, Carolin; Sierralta, Jimena; Sigrist, Stephan J; Gundelfinger, Eckart D; Knust, Elisabeth; Thomas, Ulrich

2010-04-28

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Emerging roles of the neurotrophin receptor TrkC in synapse organization.

PubMed

Naito, Yusuke; Lee, Alfred Kihoon; Takahashi, Hideto

2017-03-01

Tropomyosin-receptor-kinase (Trk) receptors have been extensively studied for their roles in kinase-dependent signaling cascades in nervous system development. Synapse organization is coordinated by trans-synaptic interactions of various cell adhesion proteins, a representative example of which is the neurexin-neuroligin complex. Recently, a novel role for TrkC as a synapse organizing protein has been established. Post-synaptic TrkC binds to pre-synaptic type-IIa receptor-type protein tyrosine phosphatase sigma (PTPσ). TrkC-PTPσ specifically induces excitatory synapses in a kinase domain-independent manner. TrkC has distinct extracellular domains for PTPσ- and NT-3-binding and thus may bind both ligands simultaneously. Indeed, NT-3 enhances the TrkC-PTPσ interaction, thus facilitating synapse induction at the pre-synaptic side and increasing pre-synaptic vesicle recycling in a kinase-independent fashion. A crystal structure study has revealed the detailed structure of the TrkC-PTPσ complex as well as competitive modulation of TrkC-mediated synaptogenesis by heparan sulfate proteoglycans (HSPGs), which bind the same domain of TrkC as PTPσ. Thus, there is strong evidence supporting a role for the TrkC-PTPσ complex in mechanisms underlying the fine turning of neural connectivity. Furthermore, disruption of the TrkC-PTPσ complex may be the underlying cause of certain psychiatric disorders caused by mutations in the gene encoding TrkC (NTRK3), supporting its role in cognitive functions. Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment

NASA Astrophysics Data System (ADS)

Eschweiler, Joseph D.; Frank, Aaron T.; Ruotolo, Brandon T.

2017-10-01

Multiprotein complexes are central to our understanding of cellular biology, as they play critical roles in nearly every biological process. Despite many impressive advances associated with structural characterization techniques, large and highly-dynamic protein complexes are too often refractory to analysis by conventional, high-resolution approaches. To fill this gap, ion mobility-mass spectrometry (IM-MS) methods have emerged as a promising approach for characterizing the structures of challenging assemblies due in large part to the ability of these methods to characterize the composition, connectivity, and topology of large, labile complexes. In this Critical Insight, we present a series of bioinformatics studies aimed at assessing the information content of IM-MS datasets for building models of multiprotein structure. Our computational data highlights the limits of current coarse-graining approaches, and compelled us to develop an improved workflow for multiprotein topology modeling, which we benchmark against a subset of the multiprotein complexes within the PDB. This improved workflow has allowed us to ascertain both the minimal experimental restraint sets required for generation of high-confidence multiprotein topologies, and quantify the ambiguity in models where insufficient IM-MS information is available. We conclude by projecting the future of IM-MS in the context of protein quaternary structure assignment, where we predict that a more complete knowledge of the ultimate information content and ambiguity within such models will undoubtedly lead to applications for a broader array of challenging biomolecular assemblies. [Figure not available: see fulltext.

Actomyosin-dependent formation of the mechanosensitive talin–vinculin complex reinforces actin anchoring

PubMed Central

Ciobanasu, Corina; Faivre, Bruno; Le Clainche, Christophe

2014-01-01

The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin–vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin–talin–vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton. PMID:24452080

Clinical Characteristics of Connective Tissue Nevi in Tuberous Sclerosis Complex With Special Emphasis on Shagreen Patches.

PubMed

Bongiorno, Michelle A; Nathan, Neera; Oyerinde, Oyetewa; Wang, Ji-An; Lee, Chyi-Chia Richard; Brown, G Thomas; Moss, Joel; Darling, Thomas N

2017-07-01

Patients with tuberous sclerosis complex (TSC) frequently develop collagenous connective tissue nevi. The prototypical lesion is a large shagreen patch located on the lower back, but some patients only manifest small collagenomas or have lesions elsewhere on the body. The ability to recognize these variable presentations can be important for the diagnosis of TSC. To describe the clinical characteristics of connective tissue nevi on the trunk and extremities of patients with tuberous sclerosis complex. A retrospective analysis of patient medical records and skin photography was performed; 104 adult patients with TSC were enrolled in an observational cohort study that was enriched for those with pulmonary lymphangioleiomyomatosis, and was therefore composed mostly of women (99 women, 5 men). All patients included were examined at the National Institutes of Health (NIH) in Bethesda, Maryland, from 1998 to 2013. Connective tissue nevi were categorized per anatomic location and size. Lesions less than 1 cm in diameter were termed collagenomas. Shagreen patches were characterized as small (1 to <4 cm), medium (4 to <8 cm), and large (≥8 cm). Frequency, anatomic location, size, and histological appearance of connective tissue nevi in patients with TSC. Overall, 58 of 104 patients (median [range] age, 42 [19-70] years) with TSC (56%) had at least 1 connective tissue nevus on the trunk or thighs; of these, 28 of 58 patients (48%) had a solitary lesion, and 30 of 58 patients (52%) had 2 or more lesions. Overall, 120 lesions from 55 patients were classified by size; 46 lesions (38%) were collagenomas; 39 lesions (32%) were small shagreen patches; 21 lesions (18%), medium shagreen patches; and 14 lesions (12%), large shagreen patches. The distribution of lesions was 9% (n = 11), upper back; 29% (n = 35), middle back; 51% (n = 61), lower back; and 11% (n = 13), other locations. All 26 shagreen patches that were analyzed histopathologically had coarse collagen fibers and 24 of 26 stained with Miller elastic stain had decreased elastic fibers. On immunoblot analysis, fibroblasts grown from shagreen patches expressed higher levels of phosphorylated ribosomal protein S6 than paired fibroblasts from normal-appearing skin. Tuberous sclerosis complex-related connective tissue nevi are not limited to the lower back, and occasionally present on the central or upper back, buttocks, or thighs. Elastic fibers are typically decreased. Recognition of these variable presentations can be important for TSC diagnosis.

Proteomic Analysis of Connexin 43 Reveals Novel Interactors Related to Osteoarthritis*

PubMed Central

Gago-Fuentes, Raquel; Fernández-Puente, Patricia; Megias, Diego; Carpintero-Fernández, Paula; Mateos, Jesus; Acea, Benigno; Fonseca, Eduardo; Blanco, Francisco Javier; Mayan, Maria Dolores

2015-01-01

We have previously reported that articular chondrocytes in tissue contain long cytoplasmic arms that physically connect two distant cells. Cell-to-cell communication occurs through connexin channels termed Gap Junction (GJ) channels, which achieve direct cellular communication by allowing the intercellular exchange of ions, small RNAs, nutrients, and second messengers. The Cx43 protein is overexpressed in several human diseases and inflammation processes and in articular cartilage from patients with osteoarthritis (OA). An increase in the level of Cx43 is known to alter gene expression, cell signaling, growth, and cell proliferation. The interaction of proteins with the C-terminal tail of connexin 43 (Cx43) directly modulates GJ-dependent and -independent functions. Here, we describe the isolation of Cx43 complexes using mild extraction conditions and immunoaffinity purification. Cx43 complexes were extracted from human primary articular chondrocytes isolated from healthy donors and patients with OA. The proteomic content of the native complexes was determined using LC-MS/MS, and protein associations with Cx43 were validated using Western blot and immunolocalization experiments. We identified >100 Cx43-associated proteins including previously uncharacterized proteins related to nucleolar functions, RNA transport, and translation. We also identified several proteins involved in human diseases, cartilage structure, and OA as novel functional Cx43 interactors, which emphasized the importance of Cx43 in the normal physiology and structural and functional integrity of chondrocytes and articular cartilage. Gene Ontology (GO) terms of the proteins identified in the OA samples showed an enrichment of Cx43-interactors related to cell adhesion, calmodulin binding, the nucleolus, and the cytoskeleton in OA samples compared with healthy samples. However, the mitochondrial proteins SOD2 and ATP5J2 were identified only in samples from healthy donors. The identification of Cx43 interactors will provide clues to the functions of Cx43 in human cells and its roles in the development of several diseases, including OA. PMID:25903580

Chromatin boundaries in budding yeast: the nuclear pore connection.

PubMed

Ishii, Kojiro; Arib, Ghislaine; Lin, Clayton; Van Houwe, Griet; Laemmli, Ulrich K

2002-05-31

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Comparison of phosphorylated proteins in intact rat spermatozoa from caput and cauda epididymidis.

PubMed

Chulavatnatol, M; Panyim, S; Wititsuwannakul, D

1982-02-01

Spermatozoa from rat epididymis were incubated with [32P] orthophosphate and the radioactively labeled proteins were solubilized for analysis by electrophoresis in SDS-gels or in two-dimensional gels by isoelectric focusing and SDS electrophoresis. Three major phosphorylated protein bands of Mr 42,700, 56,200, and 76,200 were identified together with several minor phosphorylated proteins. The phosphorylated proteins of Mr 42,700 and 76,200 were more heterogeneous in charge than the one of Mr 56,000. The major phosphorylated proteins were not found in the isolated heads of cytosol derived from sperm sonicate. They were not solubilized by 1% Triton X-100 and 2 mM DTT, which removed the plasma membrane and mitochondria, but they were solubilized by 6 M urea and 5 mM DTT away from the insoluble fibrous sheath which contained no appreciable radioactivity. Most of the major phosphorylated bands were solubilized by 2% SDS and 4 mM DTT, leaving the insoluble outer dense fiber-connecting piece (ODF-CP) complex with some of the proteins. The ODF-CP complex of the spermatozoa from the cauda epididymis contained more of the major phosphorylated bands than did that of the spermatozoa from the caput region. Treatment with 1% SDS alone can solubilize about half of the major phosphorylated bands from the spermatozoa of the caput region and essentially none from the spermatozoa of the caudal part. The latter required 1% SDS and 13 mM DTT to achieve solubilization, suggesting the formation of disulfide bonds holding the three major phosphorylated proteins to some intracellular structure during sperm maturation.

Integrity and Function of the Saccharomyces cerevisiae Spindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

PubMed Central

Casey, Amanda K.; Dawson, T. Renee; Chen, Jingjing; Friederichs, Jennifer M.; Jaspersen, Sue L.; Wente, Susan R.

2012-01-01

The nuclear envelope in Saccharomyces cerevisiae harbors two essential macromolecular protein assemblies: the nuclear pore complexes (NPCs) that enable nucleocytoplasmic transport, and the spindle pole bodies (SPBs) that mediate chromosome segregation. Previously, based on metazoan and budding yeast studies, we reported that reticulons and Yop1/DP1 play a role in the early steps of de novo NPC assembly. Here, we examined if Rtn1 and Yop1 are required for SPB function in S. cerevisiae. Electron microscopy of rtn1Δ yop1Δ cells revealed lobular abnormalities in SPB structure. Using an assay that monitors lateral expansion of the SPB central layer, we found that rtn1Δ yop1Δ SPBs had decreased connections to the NE compared to wild type, suggesting that SPBs are less stable in the NE. Furthermore, large budded rtn1Δ yop1Δ cells exhibited a high incidence of short mitotic spindles, which were frequently misoriented with respect to the mother–daughter axis. This correlated with cytoplasmic microtubule defects. We found that overexpression of the SPB insertion factors NDC1, MPS2, or BBP1 rescued the SPB defects observed in rtn1Δ yop1Δ cells. However, only overexpression of NDC1, which is also required for NPC biogenesis, rescued both the SPB and NPC associated defects. Rtn1 and Yop1 also physically interacted with Ndc1 and other NPC membrane proteins. We propose that NPC and SPB biogenesis are altered in cells lacking Rtn1 and Yop1 due to competition between these complexes for Ndc1, an essential common component of both NPCs and SPBs. PMID:22798490

Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

PubMed Central

Byron, Adam; Humphries, Jonathan D.; Randles, Michael J.; Carisey, Alex; Murphy, Stephanie; Knight, David; Brenchley, Paul E.; Zent, Roy; Humphries, Martin J.

2014-01-01

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456. PMID:24436468

Role of actin in auxin transport and transduction of gravity

NASA Astrophysics Data System (ADS)

Hu, S.; Basu, S.; Brady, S.; Muday, G.

Transport of the plant hormone auxin is polar and the direction of the hormone movement appears to be controlled by asymmetric distribution of auxin transport protein complexes. Changes in the direction of auxin transport are believed to drive asymmetric growth in response to changes in the gravity vector. To test the possibility that asymmetric distribution of the auxin transport protein complex is mediated by attachment to the actin cytoskeleton, a variety of experimental approaches have been used. The most direct demonstration of the role of the actin cytoskeleton in localization of the protein complex is the ability of one protein in this complex to bind to affinity columns containing actin filaments. Additionally, treatments of plant tissues with drugs that fragment the actin c toskeleton reducey polar transport. In order to explore this actin interaction and the affect of gravity on auxin transport and developmental polarity, embryos of the brown alga, Fucus have been examined. Fucus zygotes are initially symmetrical, but develop asymmetry in response to environmental gradients, with light gradients being the best- characterized signal. Gravity will polarize these embryos and gravity-induced polarity is randomized by clinorotation. Auxin transport also appears necessary for environmental controls of polarity, since auxin efflux inhibitors perturb both photo- and gravity-polarization at a very discrete temporal window within six hours after fertilization. The actin cytoskeleton has previously been shown to reorganize after fertilization of Fucus embryos leading to formation of an actin patch at the site of polar outgrowth. These actin patches still form in Fucus embryos treated with auxin efflux inhibitors, yet the position of these patches is randomized. Together, these results suggest that there are connections between the actin cytoskeleton, auxin transport, and gravity oriented growth and development. (Supported by NASA Grant: NAG2-1203)

Osteogenesis imperfecta: recent findings shed new light on this once well-understood condition.

PubMed

Basel, Donald; Steiner, Robert D

2009-06-01

Osteogenesis imperfecta is a systemic heritable disorder of connective tissue whose cardinal manifestation is bone fragility. In approximately 90% of individuals with osteogenesis imperfecta, mutations in either of the genes encoding the pro-alpha1 or pro-alpha2 chains of type I collagen (COL1A1 or COL1A2) can be identified. Of those without collagen mutations, a number of them will have mutations involving the enzyme complex responsible for posttranslational hydroxylation of the position 3 proline residue of COL1A1. Two of the genes encoding proteins involved in that enzyme complex, LEPRE1 and cartilage-associated protein, when mutated have been shown to cause autosomal recessive osteogenesis imperfecta, which has a moderate to severe clinical phenotype, often indistinguishable from osteogenesis imperfecta types II or III. Mutations in COL1A1 or COL1A2 which result in an abnormal protein still capable of forming a triple helix cause a more severe phenotype than mutations that lead to decreased collagen production as a result of the dominant negative effect mediated by continuous protein turnover. The current standard of care includes a multidisciplinary approach with surgical intervention when necessary, proactive physiotherapy, and consideration for the use of bisphosphonates all in attempts to improve quality of life.

A side-effect free method for identifying cancer drug targets.

PubMed

Ashraf, Md Izhar; Ong, Seng-Kai; Mujawar, Shama; Pawar, Shrikant; More, Pallavi; Paul, Somnath; Lahiri, Chandrajit

2018-04-27

Identifying effective drug targets, with little or no side effects, remains an ever challenging task. A potential pitfall of failing to uncover the correct drug targets, due to side effect of pleiotropic genes, might lead the potential drugs to be illicit and withdrawn. Simplifying disease complexity, for the investigation of the mechanistic aspects and identification of effective drug targets, have been done through several approaches of protein interactome analysis. Of these, centrality measures have always gained importance in identifying candidate drug targets. Here, we put forward an integrated method of analysing a complex network of cancer and depict the importance of k-core, functional connectivity and centrality (KFC) for identifying effective drug targets. Essentially, we have extracted the proteins involved in the pathways leading to cancer from the pathway databases which enlist real experimental datasets. The interactions between these proteins were mapped to build an interactome. Integrative analyses of the interactome enabled us to unearth plausible reasons for drugs being rendered withdrawn, thereby giving future scope to pharmaceutical industries to potentially avoid them (e.g. ESR1, HDAC2, F2, PLG, PPARA, RXRA, etc). Based upon our KFC criteria, we have shortlisted ten proteins (GRB2, FYN, PIK3R1, CBL, JAK2, LCK, LYN, SYK, JAK1 and SOCS3) as effective candidates for drug development.

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.

PubMed

Lakbub, Jude C; Shipman, Joshua T; Desaire, Heather

2018-04-01

Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.

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.

mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

PubMed

Sundararaj, Kamala; Pleasant, Dorea L; Moschella, Phillip C; Panneerselvam, Kavin; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

2016-02-01

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

The NG2 Protein Is Not Required for Glutamatergic Neuron-NG2 Cell Synaptic Signaling.

PubMed

Passlick, Stefan; Trotter, Jacqueline; Seifert, Gerald; Steinhäuser, Christian; Jabs, Ronald

2016-01-01

NG2 glial cells (as from now NG2 cells) are unique in receiving synaptic input from neurons. However, the components regulating formation and maintenance of these neuron-glia synapses remain elusive. The transmembrane protein NG2 has been considered a potential mediator of synapse formation and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering, because it contains 2 extracellular Laminin G/Neurexin/Sex Hormone-Binding Globulin domains, which in neurons are crucial for formation of transsynaptic neuroligin-neurexin complexes. NG2 is connected via Glutamate Receptor-Interacting Protein with GluA2/3-containing AMPARs, thereby possibly mediating receptor clustering in glial postsynaptic density. To elucidate the role of NG2 in neuron-glia communication, we investigated glutamatergic synaptic transmission in juvenile and aged hippocampal NG2 cells of heterozygous and homozygous NG2 knockout mice. Neuron-NG2 cell synapses readily formed in the absence of NG2. Short-term plasticity, synaptic connectivity, postsynaptic AMPAR current kinetics, and density were not affected by NG2 deletion. During development, an NG2-independent acceleration of AMPAR current kinetics and decreased synaptic connectivity were observed. Our results indicate that the lack of NG2 does not interfere with genesis and basic properties of neuron-glia synapses. In addition, we demonstrate frequent expression of neuroligins 1-3 in juvenile and aged NG2 cells, suggesting a role of these molecules in synapse formation between NG2 glia and neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Overview of the Muscle Cytoskeleton

PubMed Central

Henderson, Christine A.; Gomez, Christopher G.; Novak, Stefanie M.; Mi-Mi, Lei; Gregorio, Carol C.

2018-01-01

Cardiac and skeletal striated muscles are intricately designed machines responsible for muscle contraction. Coordination of the basic contractile unit, the sarcomere, and the complex cytoskeletal networks are critical for contractile activity. The sarcomere is comprised of precisely organized individual filament systems that include thin (actin), thick (myosin), titin, and nebulin. Connecting the sarcomere to other organelles (e.g., mitochondria and nucleus) and serving as the scaffold to maintain cellular integrity are the intermediate filaments. The costamere, on the other hand, tethers the sarcomere to the cell membrane. Unique structures like the intercalated disc in cardiac muscle and the myotendinous junction in skeletal muscle help synchronize and transmit force. Intense investigation has been done on many of the proteins that make up these cytoskeletal assemblies. Yet the details of their function and how they interconnect have just started to be elucidated. A vast number of human myopathies are contributed to mutations in muscle proteins; thus understanding their basic function provides a mechanistic understanding of muscle disorders. In this review, we highlight the components of striated muscle with respect to their interactions, signaling pathways, functions, and connections to disease. PMID:28640448

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia. I. Physiological relevance and functional connection to photosystems.

PubMed

Kotabová, Eva; Jarešová, Jana; Kaňa, Radek; Sobotka, Roman; Bína, David; Prášil, Ondřej

2014-06-01

Chromera velia is an alveolate alga associated with scleractinian corals. Here we present detailed work on chromatic adaptation in C. velia cultured under either blue or red light. Growth of C. velia under red light induced the accumulation of a light harvesting antenna complex exhibiting unusual spectroscopic properties with red-shifted absorption and atypical 710nm fluorescence emission at room temperature. Due to these characteristic features the complex was designated "Red-shifted Chromera light harvesting complex" (Red-CLH complex). Its detailed biochemical survey is described in the accompanying paper (Bina et al. 2013, this issue). Here, we show that the accumulation of Red-CLH complex under red light represents a slow acclimation process (days) that is reversible with much faster kinetics (hours) under blue light. This chromatic adaptation allows C. velia to maintain all important parameters of photosynthesis constant under both light colors. We further demonstrated that the C. velia Red-CLH complex is assembled from a 17kDa antenna protein and is functionally connected to photosystem II as it shows variability of chlorophyll fluorescence. Red-CLH also serves as an additional locus for non-photochemical quenching. Although overall rates of oxygen evolution and carbon fixation were similar for both blue and red light conditions, the presence of Red-CLH in C. velia cells increases the light harvesting potential of photosystem II, which manifested as a doubled oxygen evolution rate at illumination above 695nm. This data demonstrates a remarkable long-term remodeling of C. velia light-harvesting system according to light quality and suggests physiological significance of 'red' antenna complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

PubMed Central

Rohira, Harsha; Bhat, Ashwini G.; Passi, Anurag; Mukherjee, Keya; Choudhary, Kumari Sonal; Kumar, Vikas; Arora, Anshula; Munusamy, Prabhakaran; Subramanian, Ahalyaa; Venkatachalam, Aparna; S, Gayathri; Raj, Sweety; Chitra, Vijaya; Verma, Kaveri; Zaheer, Salman; J, Balaganesh; Gurusamy, Malarvizhi; Razeeth, Mohammed; Raja, Ilamathi; Thandapani, Madhumohan; Mevada, Vishal; Soni, Raviraj; Rana, Shruti; Ramanna, Girish Muthagadhalli; Raghavan, Swetha; Subramanya, Sunil N.; Kholia, Trupti; Patel, Rajesh; Bhavnani, Varsha; Chiranjeevi, Lakavath; Sengupta, Soumi; Singh, Pankaj Kumar; Atray, Naresh; Gandhi, Swati; Avasthi, Tiruvayipati Suma; Nisthar, Shefin; Anurag, Meenakshi; Sharma, Pratibha; Hasija, Yasha; Dash, Debasis; Sharma, Arun; Scaria, Vinod; Thomas, Zakir; Chandra, Nagasuma; Brahmachari, Samir K.; Bhardwaj, Anshu

2012-01-01

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative ‘Connect to Decode’ (C2D) to generate the first and largest manually curated interactome of Mtb termed ‘interactome pathway’ (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach. PMID:22808064

Dissecting the telomere–inner nuclear membrane interface formed in meiosis

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

Pendlebury, Devon F.; Fujiwara, Yasuhiro; Tesmer, Valerie M.

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere–INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1–TERB1 interface to reveal the structural basis for telomere–INM linkage. Disruption of this interface abrogates binding and compromises telomere–INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1–TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, ourmore » biochemical analysis implicates distinct complexes for telomere–INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere–INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.« less

Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense.

PubMed

Zhang, Mengmeng; Su, Jianbin; Zhang, Yan; Xu, Juan; Zhang, Shuqun

2018-05-09

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive endogenous and exogenous stimuli such as hormones, peptide ligands, and pathogen-derived patterns/effectors. In this review, we summarize recent advances in the establishment of MAPK cascades as unified signaling modules downstream of receptor-like kinases (RLKs) and receptor-like proteins (RLPs) in plant growth and defense, the identification of components connecting the RLK/RLP receptor complexes to the MAPK cascades, and the interactions between MAPK and hormone signaling pathways. We also propose a set of criteria for defining the physiological substrates of plant MAPKs. With only a limited number of MAPK components, multiple functional pathways often share the same MAPK cascade. As a result, understanding the signaling specificity, which requires detailed information about the spatiotemporal expression of the components involved, their complex formation, and the consequence of substrate phosphorylation, is central to our study of MAPK functions. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

DOE PAGES

Huang, Jian -Ping; Tunc-Ozdemir, Meral; Chang, Ying; ...

2015-10-13

HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1more » in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. Lastly, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose.« less

Hub Protein Controversy: Taking a Closer Look at Plant Stress Response Hubs

PubMed Central

Vandereyken, Katy; Van Leene, Jelle; De Coninck, Barbara; Cammue, Bruno P. A.

2018-01-01

Plant stress responses involve numerous changes at the molecular and cellular level and are regulated by highly complex signaling pathways. Studying protein-protein interactions (PPIs) and the resulting networks is therefore becoming increasingly important in understanding these responses. Crucial in PPI networks are the so-called hubs or hub proteins, commonly defined as the most highly connected central proteins in scale-free PPI networks. However, despite their importance, a growing amount of confusion and controversy seems to exist regarding hub protein identification, characterization and classification. In order to highlight these inconsistencies and stimulate further clarification, this review critically analyses the current knowledge on hub proteins in the plant interactome field. We focus on current hub protein definitions, including the properties generally seen as hub-defining, and the challenges and approaches associated with hub protein identification. Furthermore, we give an overview of the most important large-scale plant PPI studies of the last decade that identified hub proteins, pointing out the lack of overlap between different studies. As such, it appears that although major advances are being made in the plant interactome field, defining hub proteins is still heavily dependent on the quality, origin and interpretation of the acquired PPI data. Nevertheless, many hub proteins seem to have a reported role in the plant stress response, including transcription factors, protein kinases and phosphatases, ubiquitin proteasome system related proteins, (co-)chaperones and redox signaling proteins. A significant number of identified plant stress hubs are however still functionally uncharacterized, making them interesting targets for future research. This review clearly shows the ongoing improvements in the plant interactome field but also calls attention to the need for a more comprehensive and precise identification of hub proteins, allowing a more efficient systems biology driven unraveling of complex processes, including those involved in stress responses. PMID:29922309

Genetic analysis of the contribution of LTBP-3 to thoracic aneurysm in Marfan syndrome

PubMed Central

Zilberberg, Lior; Phoon, Colin K. L.; Robertson, Ian; Dabovic, Branka; Ramirez, Francesco; Rifkin, Daniel B.

2015-01-01

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, caused by mutations of the microfibrillar protein fibrillin-1, that predisposes affected individuals to aortic aneurysm and rupture and is associated with increased TGFβ signaling. TGFβ is secreted from cells as a latent complex consisting of TGFβ, the TGFβ propeptide, and a molecule of latent TGFβ binding protein (LTBP). Improper extracellular localization of the latent complex can alter active TGFβ levels, and has been hypothesized as an explanation for enhanced TGFβ signaling observed in MFS. We previously reported the absence of LTBP-3 in matrices lacking fibrillin-1, suggesting that perturbed TGFβ signaling in MFS might be due to defective interaction of latent TGFβ complexes containing LTBP-3 with mutant fibrillin-1 microfibrils. To test this hypothesis, we genetically suppressed Ltbp3 expression in a mouse model of progressively severe MFS. Here, we present evidence that MFS mice lacking LTBP-3 have improved survival, essentially no aneurysms, reduced disruption and fragmentation of medial elastic fibers, and decreased Smad2/3 and Erk1/2 activation in their aortas. These data suggest that, in MFS, improper localization of latent TGFβ complexes composed of LTBP-3 and TGFβ contributes to aortic disease progression. PMID:26494287

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

PubMed Central

Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

2011-01-01

Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses. PMID:21949838

Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

PubMed

Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

2011-01-01

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for specific biological responses.

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

PubMed Central

Hennig, Sven; Kong, Geraldine; Mannen, Taro; Sadowska, Agata; Kobelke, Simon; Blythe, Amanda; Knott, Gavin J.; Iyer, K. Swaminathan; Ho, Diwei; Newcombe, Estella A.; Hosoki, Kana; Goshima, Naoki; Kawaguchi, Tetsuya; Hatters, Danny; Trinkle-Mulcahy, Laura; Hirose, Tetsuro; Bond, Charles S.

2015-01-01

Prion-like domains (PLDs) are low complexity sequences found in RNA binding proteins associated with the neurodegenerative disorder amyotrophic lateral sclerosis. Recently, PLDs have been implicated in mediating gene regulation via liquid-phase transitions that drive ribonucleoprotein granule assembly. In this paper, we report many PLDs in proteins associated with paraspeckles, subnuclear bodies that form around long noncoding RNA. We mapped the interactome network of paraspeckle proteins, finding enrichment of PLDs. We show that one protein, RBM14, connects key paraspeckle subcomplexes via interactions mediated by its PLD. We further show that the RBM14 PLD, as well as the PLD of another essential paraspeckle protein, FUS, is required to rescue paraspeckle formation in cells in which their endogenous counterpart has been knocked down. Similar to FUS, the RBM14 PLD also forms hydrogels with amyloid-like properties. These results suggest a role for PLD-mediated liquid-phase transitions in paraspeckle formation, highlighting this nuclear body as an excellent model system for understanding the perturbation of such processes in neurodegeneration. PMID:26283796

Methods for the visualization and analysis of extracellular matrix protein structure and degradation.

PubMed

Leonard, Annemarie K; Loughran, Elizabeth A; Klymenko, Yuliya; Liu, Yueying; Kim, Oleg; Asem, Marwa; McAbee, Kevin; Ravosa, Matthew J; Stack, M Sharon

2018-01-01

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders. © 2018 Elsevier Inc. All rights reserved.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Computer modeling of Epilepsy

PubMed Central

Lytton, William W.

2009-01-01

Preface Epilepsy is a complex set of disorders that can involve many areas of cortex as well as underlying deep brain systems. The myriad manifestations of seizures, as varied as déjà vu and olfactory hallucination, can thereby give researchers insights into regional functions and relations. Epilepsy is also complex genetically and pathophysiologically, involving microscopic (ion channels, synaptic proteins), macroscopic (brain trauma and rewiring) and intermediate changes in a complex interplay of causality. It has long been recognized that computer modeling will be required to disentangle causality, to better understand seizure spread and to understand and eventually predict treatment efficacy. Over the past few years, substantial progress has been made modeling epilepsy at levels ranging from the molecular to the socioeconomic. We review these efforts and connect them to the medical goals of understanding and treating this disorder. PMID:18594562

Structure–function mapping of a heptameric module in the nuclear pore complex

PubMed Central

Fernandez-Martinez, Javier; Phillips, Jeremy; Sekedat, Matthew D.; Diaz-Avalos, Ruben; Velazquez-Muriel, Javier; Franke, Josef D.; Williams, Rosemary; Stokes, David L.; Chait, Brian T.

2012-01-01

The nuclear pore complex (NPC) is a multiprotein assembly that serves as the sole mediator of nucleocytoplasmic exchange in eukaryotic cells. In this paper, we use an integrative approach to determine the structure of an essential component of the yeast NPC, the ∼600-kD heptameric Nup84 complex, to a precision of ∼1.5 nm. The configuration of the subunit structures was determined by satisfaction of spatial restraints derived from a diverse set of negative-stain electron microscopy and protein domain–mapping data. Phenotypic data were mapped onto the complex, allowing us to identify regions that stabilize the NPC’s interaction with the nuclear envelope membrane and connect the complex to the rest of the NPC. Our data allow us to suggest how the Nup84 complex is assembled into the NPC and propose a scenario for the evolution of the Nup84 complex through a series of gene duplication and loss events. This work demonstrates that integrative approaches based on low-resolution data of sufficient quality can generate functionally informative structures at intermediate resolution. PMID:22331846

The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair

PubMed Central

Plys, Aaron J.; Rogacheva, Maria V.; Greene, Eric C.; Alani, Eric

2012-01-01

DNA mismatch repair (MMR) models have proposed that MSH proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH proteins (primarily Mlh1-Pms1 in baker’s yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH-MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20 – 30 nanometers) unstructured arms that connect two terminal globular domains. These arms can vary between 100 to 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker’s yeast Mlh1-Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1-Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2-Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR. PMID:22659005

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

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

Zhou, Mowei; Yan, Jing; Romano, Christine A.

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase Mnx from Bacillus sp. PL-12 has been successfully overexpressed in active form, unexpectedly, as a protein complex with a molecular weight of 211 kDa with no homology to known proteins in the database. We have recently used surface induced dissociation (SID) and ion mobility – mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data frommore » the native mass spectrometry experiment led to a plausible model of this unknown multicopper oxidase which has been difficult to study by conventional structural biology methods. However, because each subunit of Mnx binds copper ions as cofactor at varying ratios, there were remaining ambiguities in assigning some of the observed peaks to metal-binding species because of the sample heterogeneity and limited mass resolution. In this study, we performed SID in a modified Fourier transform – ion cyclotron resonance (FT-ICR) mass spectrometer for obtaining the ultimate resolution on the released subcomplexes of Mnx. The high mass accuracy and resolution unveiled unexpected artificial modifications in the protein that have been previously thought to be iron bound species based on lower resolution data. Additionally, most released subcomplexes were isotopically resolved for defining metal binding stoichiometry at each structural level. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes.« less

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

PubMed Central

Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

2013-01-01

Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

PubMed

Ciossani, Giuseppe; Overlack, Katharina; Petrovic, Arsen; Huis In 't Veld, Pim J; Koerner, Carolin; Wohlgemuth, Sabine; Maffini, Stefano; Musacchio, Andrea

2018-05-10

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising approximately 2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod-Zwilch-ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E-BUBR1 and CENP-F-BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1-CENP-F and BUBR1-CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A random set scoring model for prioritization of disease candidate genes using protein complexes and data-mining of GeneRIF, OMIM and PubMed records.

PubMed

Jiang, Li; Edwards, Stefan M; Thomsen, Bo; Workman, Christopher T; Guldbrandtsen, Bernt; Sørensen, Peter

2014-09-24

Prioritizing genetic variants is a challenge because disease susceptibility loci are often located in genes of unknown function or the relationship with the corresponding phenotype is unclear. A global data-mining exercise on the biomedical literature can establish the phenotypic profile of genes with respect to their connection to disease phenotypes. The importance of protein-protein interaction networks in the genetic heterogeneity of common diseases or complex traits is becoming increasingly recognized. Thus, the development of a network-based approach combined with phenotypic profiling would be useful for disease gene prioritization. We developed a random-set scoring model and implemented it to quantify phenotype relevance in a network-based disease gene-prioritization approach. We validated our approach based on different gene phenotypic profiles, which were generated from PubMed abstracts, OMIM, and GeneRIF records. We also investigated the validity of several vocabulary filters and different likelihood thresholds for predicted protein-protein interactions in terms of their effect on the network-based gene-prioritization approach, which relies on text-mining of the phenotype data. Our method demonstrated good precision and sensitivity compared with those of two alternative complex-based prioritization approaches. We then conducted a global ranking of all human genes according to their relevance to a range of human diseases. The resulting accurate ranking of known causal genes supported the reliability of our approach. Moreover, these data suggest many promising novel candidate genes for human disorders that have a complex mode of inheritance. We have implemented and validated a network-based approach to prioritize genes for human diseases based on their phenotypic profile. We have devised a powerful and transparent tool to identify and rank candidate genes. Our global gene prioritization provides a unique resource for the biological interpretation of data from genome-wide association studies, and will help in the understanding of how the associated genetic variants influence disease or quantitative phenotypes.

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)*

PubMed Central

Cao-Ehlker, Xiaochun; Zong, Xiangang; Hammelmann, Verena; Gruner, Christian; Fenske, Stefanie; Michalakis, Stylianos; Wahl-Schott, Christian; Biel, Martin

2013-01-01

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes. PMID:23382386

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

PubMed

Yamashita, Kazunari; Suzuki, Atsushi; Satoh, Yoshinori; Ide, Mariko; Amano, Yoshiko; Masuda-Hirata, Maki; Hayashi, Yukiko K; Hamada, Keisuke; Ogata, Kazuhiro; Ohno, Shigeo

2010-01-01

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain. Copyright 2009 Elsevier Inc. All rights reserved.

Evolutionary history and functional implications of protein domains and their combinations in eukaryotes.

PubMed

Itoh, Masumi; Nacher, Jose C; Kuma, Kei-ichi; Goto, Susumu; Kanehisa, Minoru

2007-01-01

In higher multicellular eukaryotes, complex protein domain combinations contribute to various cellular functions such as regulation of intercellular or intracellular signaling and interactions. To elucidate the characteristics and evolutionary mechanisms that underlie such domain combinations, it is essential to examine the different types of domains and their combinations among different groups of eukaryotes. We observed a large number of group-specific domain combinations in animals, especially in vertebrates. Examples include animal-specific combinations in tyrosine phosphorylation systems and vertebrate-specific combinations in complement and coagulation cascades. These systems apparently underwent extensive evolution in the ancestors of these groups. In extant animals, especially in vertebrates, animal-specific domains have greater connectivity than do other domains on average, and contribute to the varying number of combinations in each animal subgroup. In other groups, the connectivities of older domains were greater on average. To observe the global behavior of domain combinations during evolution, we traced the changes in domain combinations among animals and fungi in a network analysis. Our results indicate that there is a correlation between the differences in domain combinations among different phylogenetic groups and different global behaviors. Rapid emergence of animal-specific domains was observed in animals, contributing to specific domain combinations and functional diversification, but no such trends were observed in other clades of eukaryotes. We therefore suggest that the strategy for achieving complex multicellular systems in animals differs from that of other eukaryotes.

Epitope mapping of the U1 small nuclear ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease.

PubMed

Somarelli, J A; Mesa, A; Rodriguez, R; Avellan, R; Martinez, L; Zang, Y J; Greidinger, E L; Herrera, R J

2011-03-01

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of 'self ' antigens. Proteins of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein-protein interactions. These data suggest that while some autoantibodies may target U1 snRNP proteins as monomers or apoptosis-induced, protease-digested fragments, others may recognize epitopes on assembled protein subcomplexes of the U1 snRNP. Although nearly all of the peptides are strong predictors of autoimmune illness, none were successful at distinguishing between SLE and MCTD. The antigenicity of some peptides significantly correlated with several clinical symptoms. This investigation implicitly highlights the complexities of autoimmune epitopes, and autoimmune illnesses in general, and demonstrates the variability of antigens in patient populations, all of which contribute to difficult clinical diagnoses.

Comment on "A dynamic network model of mTOR signaling reveals TSC-independent mTORC2 regulation": building a model of the mTOR signaling network with a potentially faulty tool.

PubMed

Manning, Brendan D

2012-07-10

In their study published in Science Signaling (Research Article, 27 March 2012, DOI: 10.1126/scisignal.2002469), Dalle Pezze et al. tackle the dynamic and complex wiring of the signaling network involving the protein kinase mTOR, which exists within two distinct protein complexes (mTORC1 and mTORC2) that differ in their regulation and function. The authors use a combination of immunoblotting for specific phosphorylation events and computational modeling. The primary experimental tool employed is to monitor the autophosphorylation of mTOR on Ser(2481) in cell lysates as a surrogate for mTOR activity, which the authors conclude is a specific readout for mTORC2. However, Ser(2481) phosphorylation occurs on both mTORC1 and mTORC2 and will dynamically change as the network through which these two complexes are connected is manipulated. Therefore, models of mTOR network regulation built using this tool are inherently imperfect and open to alternative explanations. Specific issues with the main conclusion made in this study, involving the TSC1-TSC2 (tuberous sclerosis complex 1 and 2) complex and its potential regulation of mTORC2, are discussed here. A broader goal of this Letter is to clarify to other investigators the caveats of using mTOR Ser(2481) phosphorylation in cell lysates as a specific readout for either of the two mTOR complexes.

Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology

PubMed Central

Chong, Wai Chin; Shastri, Madhur D.; Eri, Rajaraman

2017-01-01

The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases. PMID:28379196

Endoplasmic Reticulum Stress and Oxidative Stress: A Vicious Nexus Implicated in Bowel Disease Pathophysiology.

PubMed

Chong, Wai Chin; Shastri, Madhur D; Eri, Rajaraman

2017-04-05

The endoplasmic reticulum (ER) is a complex protein folding and trafficking organelle. Alteration and discrepancy in the endoplasmic reticulum environment can affect the protein folding process and hence, can result in the production of misfolded proteins. The accumulation of misfolded proteins causes cellular damage and elicits endoplasmic reticulum stress. Under such stress conditions, cells exhibit reduced functional synthesis, and will undergo apoptosis if the stress is prolonged. To resolve the ER stress, cells trigger an intrinsic mechanism called an unfolded protein response (UPR). UPR is an adaptive signaling process that triggers multiple pathways through the endoplasmic reticulum transmembrane transducers, to reduce and remove misfolded proteins and improve the protein folding mechanism, in order to improve and maintain endoplasmic reticulum homeostasis. An increasing number of studies support the view that oxidative stress has a strong connection with ER stress. During the protein folding process, reactive oxygen species are produced as by-products, leading to impaired reduction-oxidation (redox) balance conferring oxidative stress. As the protein folding process is dependent on redox homeostasis, the oxidative stress can disrupt the protein folding mechanism and enhance the production of misfolded proteins, causing further ER stress. It is proposed that endoplasmic reticulum stress and oxidative stress together play significant roles in the pathophysiology of bowel diseases.

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex.

PubMed

Moise, Nicolae; Moya, Ismaël

2004-06-28

We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified "reversible radical pair" kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean tau-Phi relationship).

Defining the Role of Essential Genes in Human Disease

PubMed Central

Robertson, David L.; Hentges, Kathryn E.

2011-01-01

A greater understanding of the causes of human disease can come from identifying characteristics that are specific to disease genes. However, a full understanding of the contribution of essential genes to human disease is lacking, due to the premise that these genes tend to cause developmental abnormalities rather than adult disease. We tested the hypothesis that human orthologs of mouse essential genes are associated with a variety of human diseases, rather than only those related to miscarriage and birth defects. We segregated human disease genes according to whether the knockout phenotype of their mouse ortholog was lethal or viable, defining those with orthologs producing lethal knockouts as essential disease genes. We show that the human orthologs of mouse essential genes are associated with a wide spectrum of diseases affecting diverse physiological systems. Notably, human disease genes with essential mouse orthologs are over-represented among disease genes associated with cancer, suggesting links between adult cellular abnormalities and developmental functions. The proteins encoded by essential genes are highly connected in protein-protein interaction networks, which we find correlates with an over-representation of nuclear proteins amongst essential disease genes. Disease genes associated with essential orthologs also are more likely than those with non-essential orthologs to contribute to disease through an autosomal dominant inheritance pattern, suggesting that these diseases may actually result from semi-dominant mutant alleles. Overall, we have described attributes found in disease genes according to the essentiality status of their mouse orthologs. These findings demonstrate that disease genes do occupy highly connected positions in protein-protein interaction networks, and that due to the complexity of disease-associated alleles, essential genes cannot be ignored as candidates for causing diverse human diseases. PMID:22096564

Global loss of a nuclear lamina component, lamin A/C, and LINC complex components SUN1, SUN2, and nesprin-2 in breast cancer.

PubMed

Matsumoto, Ayaka; Hieda, Miki; Yokoyama, Yuhki; Nishioka, Yu; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Matsuura, Nariaki

2015-10-01

Cancer cells exhibit a variety of features indicative of atypical nuclei. However, the molecular mechanisms underlying these phenomena remain to be elucidated. The linker of nucleoskeleton and cytoskeleton (LINC) complex, a nuclear envelope protein complex consisting mainly of the SUN and nesprin proteins, connects nuclear lamina and cytoskeletal filaments and helps to regulate the size and shape of the nucleus. Using immunohistology, we found that a nuclear lamina component, lamin A/C and all of the investigated LINC complex components, SUN1, SUN2, and nesprin-2, were downregulated in human breast cancer tissues. In the majority of cases, we observed lower expression levels of these analytes in samples' cancerous regions as compared to their cancer-associated noncancerous regions (in cancerous regions, percentage of tissue samples exhibiting low protein expression: lamin A/C, 85% [n = 73]; SUN1, 88% [n = 43]; SUN2, 74% [n = 43]; and nesprin-2, 79% [n = 53]). Statistical analysis showed that the frequencies of recurrence and HER2 expression were negatively correlated with lamin A/C expression (P < 0.05), and intrinsic subtype and ki-67 level were associated with nesprin-2 expression (P < 0.05). In addition, combinatorial analysis using the above four parameters showed that all patients exhibited reduced expression of at least one of four components despite the tumor's pathological classification. Furthermore, several cultured breast cancer cell lines expressed less SUN1, SUN2, nesprin-2 mRNA, and lamin A/C compared to noncancerous mammary gland cells. Together, these results suggest that the strongly reduced expression of LINC complex and nuclear lamina components may play fundamental pathological functions in breast cancer progression. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Brain Structural Networks in Mouse Exposed to Chronic Maternal Undernutrition.

PubMed

Barbeito-Andrés, Jimena; Gleiser, Pablo M; Bernal, Valeria; Hallgrímsson, Benedikt; Gonzalez, Paula N

2018-06-01

Brain structural connectivity is known to be altered in cases of intrauterine growth restriction and premature birth, although the specific effect of maternal nutritional restriction, a common burden in human populations, has not been assessed yet. Here we analyze the effects of maternal undernutrition during pregnancy and lactation by establishing three experimental groups of female mice divided according to their diet: control (Co), moderate calorie-protein restriction (MCP) and severe protein restriction (SP). Nutritionally restricted dams gained relatively less weight during pregnancy and the body weight of the offspring was also affected by maternal undernutrition, showing global growth restriction. We performed magnetic resonance imaging (MRI) of the offspring's brains after weaning and analyzed their connectivity patterns using complex graph theory. In general, changes observed in the MCP group were more subtle than in SP. Results indicated that brain structures were not homogeneously affected by early nutritional stress. In particular, the growth of central brain regions, such as the temporo-parietal cortex, and long integrative myelinated tracts were relatively preserved, while the frequency of short tracts was relatively reduced. We also found a differential effect on network parameters: network degree, clustering, characteristic path length and small-worldness remained mainly unchanged, while the rich-club index was lower in nutritionally restricted animals. Rich-club decrease reflects an impairment in the structure by which brain regions with large number of connections tend to be more densely linked among themselves. Overall, the findings presented here support the hypothesis that chronic nutritional stress produces long-term changes in brain structural connectivity. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

PubMed

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-02-17

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.

Structural aspects of the solvation shell of lysine and acetylated lysine: A Car-Parrinello and classical molecular dynamics investigation

NASA Astrophysics Data System (ADS)

Carnevale, V.; Raugei, S.

2009-12-01

Lysine acetylation is a post-translational modification, which modulates the affinity of protein-protein and/or protein-DNA complexes. Its crucial role as a switch in signaling pathways highlights the relevance of charged chemical groups in determining the interactions between water and biomolecules. A great effort has been recently devoted to assess the reliability of classical molecular dynamics simulations in describing the solvation properties of charged moieties. In the spirit of these investigations, we performed classical and Car-Parrinello molecular dynamics simulations on lysine and acetylated-lysine in aqueous solution. A comparative analysis between the two computational schemes is presented with a focus on the first solvation shell of the charged groups. An accurate structural analysis unveils subtle, yet statistically significant, differences which are discussed in connection to the significant electronic density charge transfer occurring between the solute and the surrounding water molecules.

Multiscale polar theory of microtubule and motor-protein assemblies

DOE PAGES

Gao, Tong; Blackwell, Robert; Glaser, Matthew A.; ...

2015-01-27

Microtubules and motor proteins are building blocks of self-organized subcellular biological structures such as the mitotic spindle and the centrosomal microtubule array. These same ingredients can form new “bioactive” liquid-crystalline fluids that are intrinsically out of equilibrium and which display complex flows and defect dynamics. It is not yet well understood how microscopic activity, which involves polarity-dependent interactions between motor proteins and microtubules, yields such larger-scale dynamical structures. In our multiscale theory, Brownian dynamics simulations of polar microtubule ensembles driven by cross-linking motors allow us to study microscopic organization and stresses. Polarity sorting and cross-link relaxation emerge as two polar-specificmore » sources of active destabilizing stress. On larger length scales, our continuum Doi-Onsager theory captures the hydrodynamic flows generated by polarity-dependent active stresses. Finally, the results connect local polar structure to flow structures and defect dynamics.« less

Network inference reveals novel connections in pathways regulating growth and defense in the yeast salt response.

PubMed

MacGilvray, Matthew E; Shishkova, Evgenia; Chasman, Deborah; Place, Michael; Gitter, Anthony; Coon, Joshua J; Gasch, Audrey P

2018-05-01

Cells respond to stressful conditions by coordinating a complex, multi-faceted response that spans many levels of physiology. Much of the response is coordinated by changes in protein phosphorylation. Although the regulators of transcriptome changes during stress are well characterized in Saccharomyces cerevisiae, the upstream regulatory network controlling protein phosphorylation is less well dissected. Here, we developed a computational approach to infer the signaling network that regulates phosphorylation changes in response to salt stress. We developed an approach to link predicted regulators to groups of likely co-regulated phospho-peptides responding to stress, thereby creating new edges in a background protein interaction network. We then use integer linear programming (ILP) to integrate wild type and mutant phospho-proteomic data and predict the network controlling stress-activated phospho-proteomic changes. The network we inferred predicted new regulatory connections between stress-activated and growth-regulating pathways and suggested mechanisms coordinating metabolism, cell-cycle progression, and growth during stress. We confirmed several network predictions with co-immunoprecipitations coupled with mass-spectrometry protein identification and mutant phospho-proteomic analysis. Results show that the cAMP-phosphodiesterase Pde2 physically interacts with many stress-regulated transcription factors targeted by PKA, and that reduced phosphorylation of those factors during stress requires the Rck2 kinase that we show physically interacts with Pde2. Together, our work shows how a high-quality computational network model can facilitate discovery of new pathway interactions during osmotic stress.

Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

PubMed

Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

2007-01-01

RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

Joint Analysis of Band-Specific Functional Connectivity and Signal Complexity in Autism

ERIC Educational Resources Information Center

Ghanbari, Yasser; Bloy, Luke; Edgar, J. Christopher; Blaskey, Lisa; Verma, Ragini; Roberts, Timothy P. L.

2015-01-01

Examination of resting state brain activity using electrophysiological measures like complexity as well as functional connectivity is of growing interest in the study of autism spectrum disorders (ASD). The present paper jointly examined complexity and connectivity to obtain a more detailed characterization of resting state brain activity in ASD.…

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

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

Liu, Lingling, E-mail: liulingling2012@163.com; Luo, Qing, E-mail: qing.luo@cqu.edu.cn; Sun, Jinghui, E-mail: sunjhemail@163.com

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and reviewmore » how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from receptors at the cell membrane through the cytoskeletal architecture to the nucleus and into the chromosomes. On a 2D substrate (B), the nucleus can be subjected to tensional forces emanating from the stress fibers and compressive forces due to the actin cap structures and the resistance of the surface. In a 3D environment (C), the migration process requires reshaping of the nucleus and squeezing it through narrow openings in the ECM. During this process the cells may also experience both tension generated by the actomyosin filaments and compression resulting from the high pressure of the anterior compartment. - Highlights: • The influence of nuclear size and stiffness in cell migration is discussed. • We describe molecular components that govern the mechanical properties of the nucleus. • We discuss the roles of chromatin, lamin A/C in nuclear mechanical properties and cell migration. • We review how nuclear dynamics are connected to cytoskeleton. • We discuss the role of nucleo-cytoskeletal coupling in cell migration.« less

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis

PubMed Central

Varshavsky, Alexander

2012-01-01

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca2+ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca2+-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca2+ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca2+ alone or Ca2+ and its ionophore (Erickson et al., Science 1978;199:1219–1221; Harris, Pharmacol Biochem Behav 1979;10:527–534; Erickson et al., Pharmacol Biochem Behav 1980;12:651–656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep. PMID:22930402

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

PubMed

Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

2016-10-01

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Exploring the Sequence-based Prediction of Folding Initiation Sites in Proteins.

PubMed

Raimondi, Daniele; Orlando, Gabriele; Pancsa, Rita; Khan, Taushif; Vranken, Wim F

2017-08-18

Protein folding is a complex process that can lead to disease when it fails. Especially poorly understood are the very early stages of protein folding, which are likely defined by intrinsic local interactions between amino acids close to each other in the protein sequence. We here present EFoldMine, a method that predicts, from the primary amino acid sequence of a protein, which amino acids are likely involved in early folding events. The method is based on early folding data from hydrogen deuterium exchange (HDX) data from NMR pulsed labelling experiments, and uses backbone and sidechain dynamics as well as secondary structure propensities as features. The EFoldMine predictions give insights into the folding process, as illustrated by a qualitative comparison with independent experimental observations. Furthermore, on a quantitative proteome scale, the predicted early folding residues tend to become the residues that interact the most in the folded structure, and they are often residues that display evolutionary covariation. The connection of the EFoldMine predictions with both folding pathway data and the folded protein structure suggests that the initial statistical behavior of the protein chain with respect to local structure formation has a lasting effect on its subsequent states.

Endothelin-1 regulates rat bone sialoprotein gene transcription.

PubMed

Li, Xinyue; Wang, Zhitao; Yang, Li; Li, Zhengyang; Ogata, Yorimasa

2010-06-01

Endothelin-1 (ET-1) was originally discovered as a vasoconstrictor protein excreted by vascular endothelial cells. Recently, tumor-produced ET-1 has been considered to stimulate osteoblasts to form new bone, and to be an important mediator of osteoblastic bone metastasis. ET-1 has high affinity for two different membrane receptors, ET(A)R and ET(B)R, which are expressed by many types of cells including osteoblasts. Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein associated with mineralized connective tissues. To investigate the effects of ET-1 on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. Levels of BSP and osteopontin mRNA were increased at 12 h after treatment with ET-1 (10 ng/ml), and ET-1 at the same concentration induced luciferase activity of a -116 to +60 BSP promoter construct at 6 h. Transcriptional activity of -84BSPLUC, which contains the cAMP response element (CRE), was increased by ET-1. Furthermore, at 6 h, ET-1 (10 ng/ml) increased the binding of nuclear protein to CRE, the FGF2 response element (FRE) and the homeodomain protein-binding site (HOX). Antibodies against CREB1, JunD and Fra2 disrupted the formation of CRE-protein complexes, while antibodies against Runx2 and Dlx5 reduced the formation of FRE- and HOX-protein complexes. These findings indicate that ET-1 increases BSP transcription via the CRE, FRE and HOX sites in the rat BSP gene promoter.

Dynamic interactions of proteins in complex networks

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

Appella, E.; Anderson, C.

2009-10-01

Recent advances in techniques such as NMR and EPR spectroscopy have enabled the elucidation of how proteins undergo structural changes to act in concert in complex networks. The three minireviews in this series highlight current findings and the capabilities of new methodologies for unraveling the dynamic changes controlling diverse cellular functions. They represent a sampling of the cutting-edge research presented at the 17th Meeting of Methods in Protein Structure Analysis, MPSA2008, in Sapporo, Japan, 26-29 August, 2008 (http://www.iapsap.bnl.gov). The first minireview, by Christensen and Klevit, reports on a structure-based yeast two-hybrid method for identifying E2 ubiquitin-conjugating enzymes that interact withmore » the E3 BRCA1/BARD1 heterodimer ligase to generate either mono- or polyubiquitinated products. This method demonstrated for the first time that the BRCA1/BARD1 E3 can interact with 10 different E2 enzymes. Interestingly, the interaction with multiple E2 enzymes displayed unique ubiquitin-transfer properties, a feature expected to be common among other RING and U-box E3s. Further characterization of new E3 ligases and the E2 enzymes that interact with them will greatly enhance our understanding of ubiquitin transfer and facilitate studies of roles of ubiquitin and ubiquitin-like proteins in protein processing and trafficking. Stein et al., in the second minireview, describe recent progress in defining the binding specificity of different peptide-binding domains. The authors clearly point out that transient peptide interactions mediated by both post-translational modifications and disordered regions ensure a high level of specificity. They postulate that a regulatory code may dictate the number of combinations of domains and post-translational modifications needed to achieve the required level of interaction specificity. Moreover, recognition alone is not enough to obtain a stable complex, especially in a complex cellular environment. Increasing evidence indicates that disordered domains can acquire structural features that modulate the binding and strength of the signaling cascade. Whereas the first two minireviews describe ways in which protein interactions are modulated, the third, by Tompa, focuses on the importance of protein disorder in a subset of amyloid proteins. It is apparent that within this group, part of the polypeptide chain remains disordered during amyloid formation. Moreover, the disordered segments have different amino acid composition and physicochemical characteristics, which suggests that they may play a role in amyloid stability. The disordered region may serve as a linker to connect the ordered core and a globular domain, maintaining the stability and structure of the globular domain and minimizing protein refolding upon amyloid formation. As techniques in protein chemistry advance, we are learning more and more about the mechanisms that regulate and are regulated by protein interactions. The three minireviews in this series offer a glimpse of the complex dynamics fundamental to protein-protein interactions. In the future, we expect that the knowledge gained will help to augment our ability to control complex pathologies and treat diverse diseases states.« less

Predicting disulfide connectivity from protein sequence using multiple sequence feature vectors and secondary structure.

PubMed

Song, Jiangning; Yuan, Zheng; Tan, Hao; Huber, Thomas; Burrage, Kevin

2007-12-01

Disulfide bonds are primary covalent crosslinks between two cysteine residues in proteins that play critical roles in stabilizing the protein structures and are commonly found in extracy-toplasmatic or secreted proteins. In protein folding prediction, the localization of disulfide bonds can greatly reduce the search in conformational space. Therefore, there is a great need to develop computational methods capable of accurately predicting disulfide connectivity patterns in proteins that could have potentially important applications. We have developed a novel method to predict disulfide connectivity patterns from protein primary sequence, using a support vector regression (SVR) approach based on multiple sequence feature vectors and predicted secondary structure by the PSIPRED program. The results indicate that our method could achieve a prediction accuracy of 74.4% and 77.9%, respectively, when averaged on proteins with two to five disulfide bridges using 4-fold cross-validation, measured on the protein and cysteine pair on a well-defined non-homologous dataset. We assessed the effects of different sequence encoding schemes on the prediction performance of disulfide connectivity. It has been shown that the sequence encoding scheme based on multiple sequence feature vectors coupled with predicted secondary structure can significantly improve the prediction accuracy, thus enabling our method to outperform most of other currently available predictors. Our work provides a complementary approach to the current algorithms that should be useful in computationally assigning disulfide connectivity patterns and helps in the annotation of protein sequences generated by large-scale whole-genome projects. The prediction web server and Supplementary Material are accessible at http://foo.maths.uq.edu.au/~huber/disulfide

Comparing multi-module connections in membrane chromatography scale-up.

PubMed

Yu, Zhou; Karkaria, Tishtar; Espina, Marianela; Hunjun, Manjeet; Surendran, Abera; Luu, Tina; Telychko, Julia; Yang, Yan-Ping

2015-07-20

Membrane chromatography is increasingly used for protein purification in the biopharmaceutical industry. Membrane adsorbers are often pre-assembled by manufacturers as ready-to-use modules. In large-scale protein manufacturing settings, the use of multiple membrane modules for a single batch is often required due to the large quantity of feed material. The question as to how multiple modules can be connected to achieve optimum separation and productivity has been previously approached using model proteins and mass transport theories. In this study, we compare the performance of multiple membrane modules in series and in parallel in the production of a protein antigen. Series connection was shown to provide superior separation compared to parallel connection in the context of competitive adsorption. Copyright © 2015 Elsevier B.V. All rights reserved.

The COPD Knowledge Base: enabling data analysis and computational simulation in translational COPD research.

PubMed

Cano, Isaac; Tényi, Ákos; Schueller, Christine; Wolff, Martin; Huertas Migueláñez, M Mercedes; Gomez-Cabrero, David; Antczak, Philipp; Roca, Josep; Cascante, Marta; Falciani, Francesco; Maier, Dieter

2014-11-28

Previously we generated a chronic obstructive pulmonary disease (COPD) specific knowledge base (http://www.copdknowledgebase.eu) from clinical and experimental data, text-mining results and public databases. This knowledge base allowed the retrieval of specific molecular networks together with integrated clinical and experimental data. The COPDKB has now been extended to integrate over 40 public data sources on functional interaction (e.g. signal transduction, transcriptional regulation, protein-protein interaction, gene-disease association). In addition we integrated COPD-specific expression and co-morbidity networks connecting over 6 000 genes/proteins with physiological parameters and disease states. Three mathematical models describing different aspects of systemic effects of COPD were connected to clinical and experimental data. We have completely redesigned the technical architecture of the user interface and now provide html and web browser-based access and form-based searches. A network search enables the use of interconnecting information and the generation of disease-specific sub-networks from general knowledge. Integration with the Synergy-COPD Simulation Environment enables multi-scale integrated simulation of individual computational models while integration with a Clinical Decision Support System allows delivery into clinical practice. The COPD Knowledge Base is the only publicly available knowledge resource dedicated to COPD and combining genetic information with molecular, physiological and clinical data as well as mathematical modelling. Its integrated analysis functions provide overviews about clinical trends and connections while its semantically mapped content enables complex analysis approaches. We plan to further extend the COPDKB by offering it as a repository to publish and semantically integrate data from relevant clinical trials. The COPDKB is freely available after registration at http://www.copdknowledgebase.eu.

Evolutionary tradeoffs in cellular composition across diverse bacteria

PubMed Central

Kempes, Christopher P; Wang, Lawrence; Amend, Jan P; Doyle, John; Hoehler, Tori

2016-01-01

One of the most important classic and contemporary interests in biology is the connection between cellular composition and physiological function. Decades of research have allowed us to understand the detailed relationship between various cellular components and processes for individual species, and have uncovered common functionality across diverse species. However, there still remains the need for frameworks that can mechanistically predict the tradeoffs between cellular functions and elucidate and interpret average trends across species. Here we provide a comprehensive analysis of how cellular composition changes across the diversity of bacteria as connected with physiological function and metabolism, spanning five orders of magnitude in body size. We present an analysis of the trends with cell volume that covers shifts in genomic, protein, cellular envelope, RNA and ribosomal content. We show that trends in protein content are more complex than a simple proportionality with the overall genome size, and that the number of ribosomes is simply explained by cross-species shifts in biosynthesis requirements. Furthermore, we show that the largest and smallest bacteria are limited by physical space requirements. At the lower end of size, cell volume is dominated by DNA and protein content—the requirement for which predicts a lower limit on cell size that is in good agreement with the smallest observed bacteria. At the upper end of bacterial size, we have identified a point at which the number of ribosomes required for biosynthesis exceeds available cell volume. Between these limits we are able to discuss systematic and dramatic shifts in cellular composition. Much of our analysis is connected with the basic energetics of cells where we show that the scaling of metabolic rate is surprisingly superlinear with all cellular components. PMID:27046336

Centrioles, centrosomes, and cilia in health and disease.

PubMed

Nigg, Erich A; Raff, Jordan W

2009-11-13

Centrioles are barrel-shaped structures that are essential for the formation of centrosomes, cilia, and flagella. Here we review recent advances in our understanding of the function and biogenesis of these organelles, and we emphasize their connection to human disease. Deregulation of centrosome numbers has long been proposed to contribute to genome instability and tumor formation, whereas mutations in centrosomal proteins have recently been genetically linked to microcephaly and dwarfism. Finally, structural or functional centriole aberrations contribute to ciliopathies, a variety of complex diseases that stem from the absence or dysfunction of cilia.

Neuroligins Provide Molecular Links Between Syndromic and Non-Syndromic Autism

PubMed Central

Singh, Sandeep K.; Eroglu, Cagla

2014-01-01

Autism is a common and heritable neuropsychiatric disorder that can be categorized into two types: syndromic and non-syndromic, the former of which are associated with other neurological disorders or syndromes. Molecular and functional links between syndromic and non-syndromic autism genes were lacking until studies aimed at understanding role of trans-synaptic adhesion molecule neuroligin, which is associated with non-syndromic autism, provided important connections. Here, we integrate data from these studies into a model of how neuroligin functions to control synaptic connectivity in the central nervous system and how neuroligin dysfunction may participate in the pathophysiology of autism. Understanding the complex functional interactions between neuroligins and other autism-associated proteins at the synapse is crucial to understand the pathology of autism. This understanding might bring us closer to development of therapeutic approaches for autism. PMID:23838185

Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.

PubMed Central

Campagnola, Paul J; Millard, Andrew C; Terasaki, Mark; Hoppe, Pamela E; Malone, Christian J; Mohler, William A

2002-01-01

We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices. PMID:11751336

Computer modelling of epilepsy.

PubMed

Lytton, William W

2008-08-01

Epilepsy is a complex set of disorders that can involve many areas of the cortex, as well as underlying deep-brain systems. The myriad manifestations of seizures, which can be as varied as déjà vu and olfactory hallucination, can therefore give researchers insights into regional functions and relations. Epilepsy is also complex genetically and pathophysiologically: it involves microscopic (on the scale of ion channels and synaptic proteins), macroscopic (on the scale of brain trauma and rewiring) and intermediate changes in a complex interplay of causality. It has long been recognized that computer modelling will be required to disentangle causality, to better understand seizure spread and to understand and eventually predict treatment efficacy. Over the past few years, substantial progress has been made in modelling epilepsy at levels ranging from the molecular to the socioeconomic. We review these efforts and connect them to the medical goals of understanding and treating the disorder.

The structure of plant photosystem I super-complex at 2.8 Å resolution

PubMed Central

Mazor, Yuval; Borovikova, Anna; Nelson, Nathan

2015-01-01

Most life forms on Earth are supported by solar energy harnessed by oxygenic photosynthesis. In eukaryotes, photosynthesis is achieved by large membrane-embedded super-complexes, containing reaction centers and connected antennae. Here, we report the structure of the higher plant PSI-LHCI super-complex determined at 2.8 Å resolution. The structure includes 16 subunits and more than 200 prosthetic groups, which are mostly light harvesting pigments. The complete structures of the four LhcA subunits of LHCI include 52 chlorophyll a and 9 chlorophyll b molecules, as well as 10 carotenoids and 4 lipids. The structure of PSI-LHCI includes detailed protein pigments and pigment–pigment interactions, essential for the mechanism of excitation energy transfer and its modulation in one of nature's most efficient photochemical machines. DOI: http://dx.doi.org/10.7554/eLife.07433.001 PMID:26076232

Partial connectivity increases cultural accumulation within groups.

PubMed

Derex, Maxime; Boyd, Robert

2016-03-15

Complex technologies used in most human societies are beyond the inventive capacities of individuals. Instead, they result from a cumulative process in which innovations are gradually added to existing cultural traits across many generations. Recent work suggests that a population's ability to develop complex technologies is positively affected by its size and connectedness. Here, we present a simple computer-based experiment that compares the accumulation of innovations by fully and partially connected groups of the same size in a complex fitness landscape. We find that the propensity to learn from successful individuals drastically reduces cultural diversity within fully connected groups. In comparison, partially connected groups produce more diverse solutions, and this diversity allows them to develop complex solutions that are never produced in fully connected groups. These results suggest that explanations of ancestral patterns of cultural complexity may need to consider levels of population fragmentation and interaction patterns between partially isolated groups.

Partial connectivity increases cultural accumulation within groups

PubMed Central

Boyd, Robert

2016-01-01

Complex technologies used in most human societies are beyond the inventive capacities of individuals. Instead, they result from a cumulative process in which innovations are gradually added to existing cultural traits across many generations. Recent work suggests that a population’s ability to develop complex technologies is positively affected by its size and connectedness. Here, we present a simple computer-based experiment that compares the accumulation of innovations by fully and partially connected groups of the same size in a complex fitness landscape. We find that the propensity to learn from successful individuals drastically reduces cultural diversity within fully connected groups. In comparison, partially connected groups produce more diverse solutions, and this diversity allows them to develop complex solutions that are never produced in fully connected groups. These results suggest that explanations of ancestral patterns of cultural complexity may need to consider levels of population fragmentation and interaction patterns between partially isolated groups. PMID:26929364

Making connections for life: an in vivo map of the yeast interactome.

PubMed

Kast, Juergen

2008-10-01

Proteins are the true workhorses of any cell. To carry out specific tasks, they frequently bind other molecules in their surroundings. Due to their structural complexity and flexibility, the most diverse array of interactions is seen with other proteins. The different geometries and affinities available for such interactions typically bestow specific functions on proteins. Having available a map of protein-protein interactions is therefore of enormous importance for any researcher interested in gaining insight into biological systems at the level of cells and organisms. In a recent report, a novel approach has been employed that relies on the spontaneous folding of complementary enzyme fragments fused to two different proteins to test whether these interact in their actual cellular context [Tarassov et al., Science 320, 1465-1470 (2008)]. Genome-wide application of this protein-fragment complementation assay has resulted in the first map of the in vivo interactome of Saccharomyces cerevisiae. The current data show striking similarities but also significant differences to those obtained using other large-scale approaches for the same task. This warrants a general discussion of the current state of affairs of protein-protein interaction studies and foreseeable future trends, highlighting their significance for a variety of applications and their potential to revolutionize our understanding of the architecture and dynamics of biological systems.

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

PubMed Central

Kudalkar, Emily M.; Scarborough, Emily A.; Umbreit, Neil T.; Zelter, Alex; Gestaut, Daniel R.; Riffle, Michael; Johnson, Richard S.; MacCoss, Michael J.; Asbury, Charles L.; Davis, Trisha N.

2015-01-01

Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore–microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex. PMID:26430240

Identifying essential proteins based on sub-network partition and prioritization by integrating subcellular localization information.

PubMed

Li, Min; Li, Wenkai; Wu, Fang-Xiang; Pan, Yi; Wang, Jianxin

2018-06-14

Essential proteins are important participants in various life activities and play a vital role in the survival and reproduction of living organisms. Identification of essential proteins from protein-protein interaction (PPI) networks has great significance to facilitate the study of human complex diseases, the design of drugs and the development of bioinformatics and computational science. Studies have shown that highly connected proteins in a PPI network tend to be essential. A series of computational methods have been proposed to identify essential proteins by analyzing topological structures of PPI networks. However, the high noise in the PPI data can degrade the accuracy of essential protein prediction. Moreover, proteins must be located in the appropriate subcellular localization to perform their functions, and only when the proteins are located in the same subcellular localization, it is possible that they can interact with each other. In this paper, we propose a new network-based essential protein discovery method based on sub-network partition and prioritization by integrating subcellular localization information, named SPP. The proposed method SPP was tested on two different yeast PPI networks obtained from DIP database and BioGRID database. The experimental results show that SPP can effectively reduce the effect of false positives in PPI networks and predict essential proteins more accurately compared with other existing computational methods DC, BC, CC, SC, EC, IC, NC. Copyright © 2018 Elsevier Ltd. All rights reserved.

Electron cryo-tomographic structure of cystovirus phi 12.

PubMed

Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul

2008-03-01

Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed "the donut" and appears to inhibit BHT-induced viral envelope fusion.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli.

PubMed

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-12-24

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

HPr antagonizes the anti-σ70 activity of Rsd in Escherichia coli

PubMed Central

Park, Young-Ha; Lee, Chang-Ro; Choe, Mangyu; Seok, Yeong-Jae

2013-01-01

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIAGlc (EIIAGlc) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIAGlc in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ70 in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ70. Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ70-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ70 and σS for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ70 activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd. PMID:24324139

Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli

PubMed Central

Matsumoto, Ayaka; Sakamoto, Chiyomi; Matsumori, Haruka; Katahira, Jun; Yasuda, Yoko; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Goldberg, Ilya G; Matsuura, Nariaki; Nakao, Mitsuyoshi; Saitoh, Noriko; Hieda, Miki

2016-01-01

ABSTRACT A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells. PMID:26962703

Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli.

PubMed

Matsumoto, Ayaka; Sakamoto, Chiyomi; Matsumori, Haruka; Katahira, Jun; Yasuda, Yoko; Yoshidome, Katsuhide; Tsujimoto, Masahiko; Goldberg, Ilya G; Matsuura, Nariaki; Nakao, Mitsuyoshi; Saitoh, Noriko; Hieda, Miki

2016-01-01

A supervised machine learning algorithm, which is qualified for image classification and analyzing similarities, is based on multiple discriminative morphological features that are automatically assembled during the learning processes. The algorithm is suitable for population-based analysis of images of biological materials that are generally complex and heterogeneous. Here we used the algorithm wndchrm to quantify the effects on nucleolar morphology of the loss of the components of nuclear envelope in a human mammary epithelial cell line. The linker of nucleoskeleton and cytoskeleton (LINC) complex, an assembly of nuclear envelope proteins comprising mainly members of the SUN and nesprin families, connects the nuclear lamina and cytoskeletal filaments. The components of the LINC complex are markedly deficient in breast cancer tissues. We found that a reduction in the levels of SUN1, SUN2, and lamin A/C led to significant changes in morphologies that were computationally classified using wndchrm with approximately 100% accuracy. In particular, depletion of SUN1 caused nucleolar hypertrophy and reduced rRNA synthesis. Further, wndchrm revealed a consistent negative correlation between SUN1 expression and the size of nucleoli in human breast cancer tissues. Our unbiased morphological quantitation strategies using wndchrm revealed an unexpected link between the components of the LINC complex and the morphologies of nucleoli that serves as an indicator of the malignant phenotype of breast cancer cells.

End-binding protein 1 controls signal propagation from the T cell receptor

PubMed Central

Martín-Cófreces, Noa B; Baixauli, Francesc; López, María J; Gil, Diana; Monjas, Alicia; Alarcón, Balbino; Sánchez-Madrid, Francisco

2012-01-01

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome. PMID:22922463

Evaluation of the collaborative network of highly correlating skin proteins and its change following treatment with glucocorticoids

PubMed Central

2010-01-01

Background Glucocorticoids (GC) represent the core treatment modality for many inflammatory diseases. Its mode of action is difficult to grasp, not least because it includes direct modulation of many components of the extracellular matrix as well as complex anti-inflammatory effects. Protein expression profile of skin proteins is being changed with topical application of GC, however, the knowledge about singular markers in this regard is only patchy and collaboration is ill defined. Material/Methods Scar formation was observed under different doses of GC, which were locally applied on the back skin of mice (1 to 3 weeks). After euthanasia we analyzed protein expression of collagen I and III (picrosirius) in scar tissue together with 16 additional protein markers, which are involved in wound healing, with immunhistochemistry. For assessing GC's effect on co-expression we compared our results with a model of random figures to estimate how many significant correlations should be expected by chance. Results GC altered collagen and protein expression with distinct results in different areas of investigation. Most often we observed a reduced expression after application of low dose GC. In the scar infiltrate a multivariate analysis confirmed the significant impact of both GC concentrations. Calculation of Spearman's correlation coefficient similarly resulted in a significant impact of GC, and furthermore, offered the possibility to grasp the entire interactive profile in between all variables studied. The biological markers, which were connected by significant correlations could be arranged in a highly cross-linked network that involved most of the markers measured. A marker highly cross-linked with more than 3 significant correlations was indicated by a higher variation of all its correlations to the other variables, resulting in a standard deviation of > 0.2. Conclusion In addition to immunohistochemical analysis of single protein markers multivariate analysis of co-expressions by use of correlation coefficients reveals the complexity of biological relationships and identifies complex biological effects of GC on skin scarring. Depiction of collaborative clusters will help to understand functional pathways. The functional importance of highly cross-linked proteins will have to be proven in subsequent studies. PMID:20509951

Dissecting the herpesvirus architecture by targeted proteolysis.

PubMed

Daniel, Gina R; Pegg, Caitlin E; Smith, Gregory A

2018-06-13

Herpesvirus particles have a complex architecture consisting of an icosahedral capsid that is surrounded by a lipid envelope. Connecting these two components is a layer of tegument that consists of varying amounts of twenty or more proteins. The arrangement of proteins within the tegument cannot easily be assessed and instead is inferred from tegument interactions identified in reductionist models. To better understand the tegument architecture, we have developed an approach to probe capsid-tegument interactions of extracellular viral particles by encoding tobacco etch virus (TEV) protease sites in viral structural proteins, along with distinct fluorescent tags in capsid and tegument components. In this study, TEV sites were engineered within the pUL36 large tegument protein: a critical structural element that is anchored directly on the capsid surface. Purified pseudorabies virus extracellular particles were permeabilized and TEV protease was added to selectively cleave the exposed pUL36 backbone. Interactions with the capsid were assessed in situ by monitoring the fate of the fluorescent signals following cleavage. Although several regions of pUL36 are proposed to bind capsids, pUL36 was found stably anchored to the capsid exclusively at its carboxyl terminus. Two additional tegument proteins, pUL37 and pUS3, were tethered to the capsid via pUL36 whereas the pUL16, pUL47, pUL48, and pUL49 tegument proteins were not stably bound to the capsid. IMPORTANCE: Neuroinvasive alphaherpesviruses produce diseases of clinical and economic significance in humans and veterinary animals, but are predominantly associated with less serious recurrent disease. Like all viruses, herpesviruses assemble a metastable particle that selectively dismantles during initial infection. This process is made more complex by the presence of a tegument layer that resides between the capsid surface and envelope. Components of the tegument are essential for particle assembly and also serve as critical effectors that promote infection upon entry into cells. How this dynamic network of protein interactions is arranged within virions is largely unknown. We present a molecular approach to dissect the tegument and with it, begin to tease apart the protein interactions that underlie this complex layer of the virion architecture. Copyright © 2018 American Society for Microbiology.

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

PubMed

Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

2017-06-05

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1 fl/fl ), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria*

PubMed Central

Xu, Yongbin; Moeller, Arne; Jun, So-Young; Le, Minho; Yoon, Bo-Young; Kim, Jin-Sik; Lee, Kangseok; Ha, Nam-Chul

2012-01-01

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria. PMID:22308040

Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

PubMed Central

Oldham, William M.; Van Eps, Ned; Preininger, Anita M.; Hubbell, Wayne L.; Hamm, Heidi E.

2007-01-01

Heterotrimeric G proteins function as molecular relays that mediate signal transduction from heptahelical receptors in the cell membrane to intracellular effector proteins. Crystallographic studies have demonstrated that guanine nucleotide exchange on the Gα subunit causes specific conformational changes in three key “switch” regions of the protein, which regulate binding to Gβγ subunits, receptors, and effector proteins. In the present study, nitroxide side chains were introduced at sites within the switch I region of Gαi to explore the structure and dynamics of this region throughout the G protein cycle. EPR spectra obtained for each of the Gα(GDP), Gα(GDP)βγ heterotrimer and Gα(GTPγS) conformations are consistent with the local environment observed in the corresponding crystal structures. Binding of the heterotrimer to activated rhodopsin to form the nucleotide-free (empty) complex, for which there is no crystal structure, causes prominent changes relative to the heterotrimer in the structure of switch I and contiguous sequences. The data identify a putative pathway of allosteric changes triggered by receptor binding and, together with previously published data, suggest elements of a mechanism for receptor-catalyzed nucleotide exchange. PMID:17463080

RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton.

PubMed

Mamon, L A; Ginanova, V R; Kliver, S F; Yakimova, A O; Atsapkina, A A; Golubkova, E V

2017-04-01

The mutual relationship between mRNA and the cytoskeleton can be seen from two points of view. On the one hand, the cytoskeleton is necessary for mRNA trafficking and anchoring to subcellular domains. On the other hand, cytoskeletal growth and rearrangement require the translation of mRNAs that are connected to the cytoskeleton. β-actin mRNA localization may influence dynamic changes in the actin cytoskeleton. In the cytoplasm, long-lived mRNAs exist in the form of RNP (ribonucleoprotein) complexes, where they interact with RNA-binding proteins, including NXF (Nuclear eXport Factor). Dm NXF1 is an evolutionarily conserved protein in Drosophila melanogaster that has orthologs in different animals. The universal function of nxf1 genes is the nuclear export of different mRNAs in various organisms. In this mini-review, we briefly discuss the evidence demonstrating that Dm NXF1 fulfils not only universal but also specialized cytoplasmic functions. This protein is detected not only in the nucleus but also in the cytoplasm. It is a component of neuronal granules. Dm NXF1 marks nuclear division spindles during early embryogenesis and the dense body on one side of the elongated spermatid nuclei. The characteristic features of sbr mutants (sbr 10 and sbr 5 ) are impairment of chromosome segregation and spindle formation anomalies during female meiosis. sbr 12 mutant sterile males with immobile spermatozoa exhibit disturbances in the axoneme, mitochondrial derivatives and cytokinesis. These data allow us to propose that the Dm NXF1 proteins transport certain mRNAs in neurites and interact with localized mRNAs that are necessary for dynamic changes of the cytoskeleton. © 2017 Wiley Periodicals, Inc.

Connective tissue-activating peptide III: a novel blood biomarker for early lung cancer detection.

PubMed

Yee, John; Sadar, Marianne D; Sin, Don D; Kuzyk, Michael; Xing, Li; Kondra, Jennifer; McWilliams, Annette; Man, S F Paul; Lam, Stephen

2009-06-10

There are no reliable blood biomarkers to detect early lung cancer. We used a novel strategy that allows discovery of differentially present proteins against a complex and variable background. Mass spectrometry analyses of paired pulmonary venous-radial arterial blood from 16 lung cancer patients were applied to identify plasma proteins potentially derived from the tumor microenvironment. Two differentially expressed proteins were confirmed in 64 paired venous-arterial blood samples using an immunoassay. Twenty-eight pre- and postsurgical resection peripheral blood samples and two independent, blinded sets of plasma from 149 participants in a lung cancer screening study (49 lung cancers and 100 controls) and 266 participants from the National Heart Lung and Blood Institute Lung Health Study (45 lung cancer and 221 matched controls) determined the accuracy of the two protein markers to detect subclinical lung cancer. Connective tissue-activating peptide III (CTAP III)/ neutrophil activating protein-2 (NAP-2) and haptoglobin were identified to be significantly higher in venous than in arterial blood. CTAP III/NAP-2 levels decreased after tumor resection (P = .01). In two independent population cohorts, CTAP III/NAP-2 was significantly associated with lung cancer and improved the accuracy of a lung cancer risk prediction model that included age, smoking, lung function (FEV(1)), and an interaction term between FEV(1) and CTAP III/NAP-2 (area under the curve, 0.84; 95% CI, 0.77 to 0.91) compared to CAPIII/NAP-2 alone. We identified CTAP III/NAP-2 as a novel biomarker to detect preclinical lung cancer. The study underscores the importance of applying blood biomarkers as part of a multimodal lung cancer risk prediction model instead of as stand-alone tests.

A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes

PubMed Central

Zhang, Bei; Rapolu, Madhusudhan; Liang, Zhibin; Han, Zhenlin; Williams, Philip G.; Su, Wei Wen

2015-01-01

Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine. PMID:25712612

Systems Biology Graphical Notation: Entity Relationship language Level 1 Version 2.

PubMed

Sorokin, Anatoly; Le Novère, Nicolas; Luna, Augustin; Czauderna, Tobias; Demir, Emek; Haw, Robin; Mi, Huaiyu; Moodie, Stuart; Schreiber, Falk; Villéger, Alice

2015-09-04

The Systems Biological Graphical Notation (SBGN) is an international community effort for standardized graphical representations of biological pathways and networks. The goal of SBGN is to provide unambiguous pathway and network maps for readers with different scientific backgrounds as well as to support efficient and accurate exchange of biological knowledge between different research communities, industry, and other players in systems biology. Three SBGN languages, Process Description (PD), Entity Relationship (ER) and Activity Flow (AF), allow for the representation of different aspects of biological and biochemical systems at different levels of detail. The SBGN Entity Relationship language (ER) represents biological entities and their interactions and relationships within a network. SBGN ER focuses on all potential relationships between entities without considering temporal aspects. The nodes (elements) describe biological entities, such as proteins and complexes. The edges (connections) provide descriptions of interactions and relationships (or influences), e.g., complex formation, stimulation and inhibition. Among all three languages of SBGN, ER is the closest to protein interaction networks in biological literature and textbooks, but its well-defined semantics offer a superior precision in expressing biological knowledge.

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

PubMed Central

Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

2011-01-01

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

Stress granules at the intersection of autophagy and ALS

PubMed Central

Monahan, Zachary; Shewmaker, Frank; Pandey, Udai Bhan

2016-01-01

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease caused by loss of upper and lower motor neurons. The majority of ALS cases are classified as sporadic (80-90%), with the remaining considered familial based on patient history. The last decade has seen a surge in the identification of ALS-causing genes – including TARDBP (TDP-43), FUS, MATR3 (Matrin-3), C9ORF72 and several others – providing important insights into the molecular pathways involved in pathogenesis. Most of the protein products of ALS-linked genes fall into two functional categories: RNA-binding/homeostasis and protein-quality control (i.e. autophagy and proteasome). The RNA-binding proteins tend to be aggregation-prone with low-complexity domains similar to the prion-forming domains of yeast. Many also incorporate into stress granules (SGs), which are cytoplasmic ribonucleoprotein complexes that form in response to cellular stress. Mutant forms of TDP-43 and FUS perturb SG dynamics, lengthening their cytoplasmic persistence. Recent evidence suggests that SGs are regulated by the autophagy pathway, suggesting a unifying connection between many of the ALS-linked genes. Persistent SGs may give rise to intractable aggregates that disrupt neuronal homeostasis, thus failure to clear SGs by autophagic processes may promote ALS pathogenesis. PMID:27181519

TNFα- and IKKβ-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKγ and NF-κB activation

PubMed Central

Bonif, Marianne; Meuwis, Marie-Alice; Close, Pierre; Benoit, Valérie; Heyninck, Karen; Chapelle, Jean-Paul; Bours, Vincent; Merville, Marie-Paule; Piette, Jacques; Beyaert, Rudi; Chariot, Alain

2005-01-01

Pro-inflammatory cytokines trigger signalling cascades leading to NF-κB (nuclear factor-κB)-dependent gene expression through IKK [IκB (inhibitory κB) kinase]-dependent phosphorylation and subsequent degradation of the IκB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFα (tumour necrosis factor α) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-κB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKβ upon TNFα stimulation and that this modification negatively regulates TANK binding to NEMO (NF-κB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFα-mediated induction of a subset of NF-κB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFα by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKβ-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-κB activation. PMID:16336209

Tracking the Resolution of Student Misconceptions about the Central Dogma of Molecular Biology†

PubMed Central

Briggs, Amy G.; Morgan, Stephanie K.; Sanderson, Seth K.; Schulting, Molly C.; Wieseman, Laramie J.

2016-01-01

The goal of our study was to track changes in student understanding of the central dogma of molecular biology before and after taking a genetics course. Concept maps require the ability to synthesize new information into existing knowledge frameworks, and so the hypothesis guiding this study was that student performance on concept maps reveals specific central dogma misconceptions gained, lost, and retained by students. Students in a genetics course completed pre- and posttest concept mapping tasks using terms related to the central dogma. Student maps increased in complexity and validity, indicating learning gains in both content and complexity of understanding. Changes in each of the 351 possible connections in the mapping task were tracked for each student. Our students did not retain much about the central dogma from their introductory biology courses, but they did move to more advanced levels of understanding by the end of the genetics course. The information they retained from their introductory courses focused on structural components (e.g., protein is made of amino acids) and not on overall mechanistic components (e.g., DNA comes before RNA, the ribosome makes protein). Students made the greatest gains in connections related to transcription, and they resolved the most prior misconceptions about translation. These concept-mapping tasks revealed that students are able to correct prior misconceptions about the central dogma during an intermediate-level genetics course. From these results, educators can design new classroom interventions to target those aspects of this foundational principle with which students have the most trouble. PMID:28101260

Tracking the Resolution of Student Misconceptions about the Central Dogma of Molecular Biology.

PubMed

Briggs, Amy G; Morgan, Stephanie K; Sanderson, Seth K; Schulting, Molly C; Wieseman, Laramie J

2016-12-01

The goal of our study was to track changes in student understanding of the central dogma of molecular biology before and after taking a genetics course. Concept maps require the ability to synthesize new information into existing knowledge frameworks, and so the hypothesis guiding this study was that student performance on concept maps reveals specific central dogma misconceptions gained, lost, and retained by students. Students in a genetics course completed pre- and posttest concept mapping tasks using terms related to the central dogma. Student maps increased in complexity and validity, indicating learning gains in both content and complexity of understanding. Changes in each of the 351 possible connections in the mapping task were tracked for each student. Our students did not retain much about the central dogma from their introductory biology courses, but they did move to more advanced levels of understanding by the end of the genetics course. The information they retained from their introductory courses focused on structural components (e.g., protein is made of amino acids) and not on overall mechanistic components (e.g., DNA comes before RNA, the ribosome makes protein). Students made the greatest gains in connections related to transcription, and they resolved the most prior misconceptions about translation. These concept-mapping tasks revealed that students are able to correct prior misconceptions about the central dogma during an intermediate-level genetics course. From these results, educators can design new classroom interventions to target those aspects of this foundational principle with which students have the most trouble.

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

PubMed Central

2011-01-01

Background One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. Methods The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. Results We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. Conclusions These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development. PMID:21329510

Mapping of the binding sites involved in PSP94-CRISP-3 interaction by molecular dissection of the complex.

PubMed

Breed, Ananya A; Gomes, Amanda; Roy, Binita Sur; Mahale, Smita D; Pathak, Bhakti R

2013-04-01

Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known. In order to identify the residues and/or regions involved in PSP94-CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP). For PSP94, amino acids Y(3), F(4), P(56) and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C(37)A-C(73)A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94-CRISP-3 complex has been proposed. The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3. Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity. Copyright © 2013 Elsevier B.V. All rights reserved.

Catalytic effects of glycine on prebiotic divaline and diproline formation.

PubMed

Plankensteiner, Kristof; Reiner, Hannes; Rode, Bernd M

2005-07-01

The catalytic effects of the simple amino acid glycine on the formation of diproline and divaline in the prebiotically relevant salt-induced peptide formation (SIPF) reaction was investigated in systems of different amino acid starting concentrations and using the two enantiomeric forms of the respective amino acid. Results show an improved applicability of the SIPF reaction to prebiotic conditions, especially at low amino acid concentrations, as presumably present in a primordial scenario, and indicate excellent conditions and resources for chemical evolution of peptides and proteins on the early earth. For valine, furthermore differences in catalytic yield increase are found indicating a chiral selectivity of the active copper complex of the reaction and showing a connection to previously found enantiomeric differences in complex formation constants with amino acids.

Human Prostate Cancer Hallmarks Map

PubMed Central

Datta, Dipamoy; Aftabuddin, Md.; Gupta, Dinesh Kumar; Raha, Sanghamitra; Sen, Prosenjit

2016-01-01

Human prostate cancer is a complex heterogeneous disease that mainly affects elder male population of the western world with a high rate of mortality. Acquisitions of diverse sets of hallmark capabilities along with an aberrant functioning of androgen receptor signaling are the central driving forces behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. These hallmark capabilities arise due to an intense orchestration of several crucial factors, including deregulation of vital cell physiological processes, inactivation of tumor suppressive activity and disruption of prostate gland specific cellular homeostasis. The molecular complexity and redundancy of oncoproteins signaling in prostate cancer demands for concurrent inhibition of multiple hallmark associated pathways. By an extensive manual curation of the published biomedical literature, we have developed Human Prostate Cancer Hallmarks Map (HPCHM), an onco-functional atlas of human prostate cancer associated signaling and events. It explores molecular architecture of prostate cancer signaling at various levels, namely key protein components, molecular connectivity map, oncogenic signaling pathway map, pathway based functional connectivity map etc. Here, we briefly represent the systems level understanding of the molecular mechanisms associated with prostate tumorigenesis by considering each and individual molecular and cell biological events of this disease process. PMID:27476486

Prokaryotic ancestry of eukaryotic protein networks mediating innate immunity and apoptosis.

PubMed

Dunin-Horkawicz, Stanislaw; Kopec, Klaus O; Lupas, Andrei N

2014-04-03

Protein domains characteristic of eukaryotic innate immunity and apoptosis have many prokaryotic counterparts of unknown function. By reconstructing interactomes computationally, we found that bacterial proteins containing these domains are part of a network that also includes other domains not hitherto associated with immunity. This network is connected to the network of prokaryotic signal transduction proteins, such as histidine kinases and chemoreceptors. The network varies considerably in domain composition and degree of paralogy, even between strains of the same species, and its repetitive domains are often amplified recently, with individual repeats sharing up to 100% sequence identity. Both phenomena are evidence of considerable evolutionary pressure and thus compatible with a role in the "arms race" between host and pathogen. In order to investigate the relationship of this network to its eukaryotic counterparts, we performed a cluster analysis of organisms based on a census of its constituent domains across all fully sequenced genomes. We obtained a large central cluster of mainly unicellular organisms, from which multicellular organisms radiate out in two main directions. One is taken by multicellular bacteria, primarily cyanobacteria and actinomycetes, and plants form an extension of this direction, connected via the basal, unicellular cyanobacteria. The second main direction is taken by animals and fungi, which form separate branches with a common root in the α-proteobacteria of the central cluster. This analysis supports the notion that the innate immunity networks of eukaryotes originated from their endosymbionts and that increases in the complexity of these networks accompanied the emergence of multicellularity. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Robustness, efficiency, and optimality in the Fenna-Matthews-Olson photosynthetic pigment-protein complex

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

Baker, Lewis A.; Habershon, Scott, E-mail: S.Habershon@warwick.ac.uk

Pigment-protein complexes (PPCs) play a central role in facilitating excitation energy transfer (EET) from light-harvesting antenna complexes to reaction centres in photosynthetic systems; understanding molecular organisation in these biological networks is key to developing better artificial light-harvesting systems. In this article, we combine quantum-mechanical simulations and a network-based picture of transport to investigate how chromophore organization and protein environment in PPCs impacts on EET efficiency and robustness. In a prototypical PPC model, the Fenna-Matthews-Olson (FMO) complex, we consider the impact on EET efficiency of both disrupting the chromophore network and changing the influence of (local and global) environmental dephasing. Surprisingly,more » we find a large degree of resilience to changes in both chromophore network and protein environmental dephasing, the extent of which is greater than previously observed; for example, FMO maintains EET when 50% of the constituent chromophores are removed, or when environmental dephasing fluctuations vary over two orders-of-magnitude relative to the in vivo system. We also highlight the fact that the influence of local dephasing can be strongly dependent on the characteristics of the EET network and the initial excitation; for example, initial excitations resulting in rapid coherent decay are generally insensitive to the environment, whereas the incoherent population decay observed following excitation at weakly coupled chromophores demonstrates a more pronounced dependence on dephasing rate as a result of the greater possibility of local exciton trapping. Finally, we show that the FMO electronic Hamiltonian is not particularly optimised for EET; instead, it is just one of many possible chromophore organisations which demonstrate a good level of EET transport efficiency following excitation at different chromophores. Overall, these robustness and efficiency characteristics are attributed to the highly connected nature of the chromophore network and the presence of multiple EET pathways, features which might easily be built into artificial photosynthetic systems.« less

Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures.

PubMed

Testa, Claudia M; Sherer, Todd B; Greenamyre, J Timothy

2005-03-24

Rotenone, a pesticide and complex I inhibitor, causes nigrostriatal degeneration similar to Parkinson disease pathology in a chronic, systemic, in vivo rodent model [M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317-324; R. Betarbet, T.B. Sherer, G. MacKenzie, M. Garcia-Osuna, A.V. Panov, J.T. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat. Neurosci. 3 (2000) 1301-1306; S.M. Fleming, C. Zhu, P.O. Fernagut, A. Mehta, C.D. DiCarlo, R.L. Seaman, M.F. Chesselet, Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone, Exp. Neurol. 187 (2004) 418-429; T.B. Sherer, J.H. Kim, R. Betarbet, J.T. Greenamyre, Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation, Exp. Neurol. 179 (2003) 9-16.]. To better investigate the role of mitochondria and complex I inhibition in chronic, progressive neurodegenerative disease, we developed methods for long-term culture of rodent postnatal midbrain organotypic slices. Chronic complex I inhibition over weeks by low dose (10-50 nM) rotenone in this system lead to dose- and time-dependent destruction of substantia nigra pars compacta neuron processes, morphologic changes, some neuronal loss, and decreased tyrosine hydroxylase (TH) protein levels. Chronic complex I inhibition also caused oxidative damage to proteins, measured by protein carbonyl levels. This oxidative damage was blocked by the antioxidant alpha-tocopherol (vitamin E). At the same time, alpha-tocopherol also blocked rotenone-induced reductions in TH protein and TH immunohistochemical changes. Thus, oxidative damage is a primary mechanism of mitochondrial toxicity in intact dopaminergic neurons. The organotypic culture system allows close study of this and other interacting mechanisms over a prolonged time period in mature dopaminergic neurons with intact processes, surrounding glia, and synaptic connections.

Detergent-Induced Stabilization and Improved 3D Map of the Human Heteromeric Amino Acid Transporter 4F2hc-LAT2

PubMed Central

Harder, Daniel; Stauffer, Mirko; Jeckelmann, Jean-Marc; Brühlmann, Béla; Rosell, Albert; Ilgü, Hüseyin; Kovar, Karin; Palacín, Manuel; Fotiadis, Dimitrios

2014-01-01

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level. PMID:25299125

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

PubMed

Meury, Marcel; Costa, Meritxell; Harder, Daniel; Stauffer, Mirko; Jeckelmann, Jean-Marc; Brühlmann, Béla; Rosell, Albert; Ilgü, Hüseyin; Kovar, Karin; Palacín, Manuel; Fotiadis, Dimitrios

2014-01-01

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

PubMed

Cioci, Gianluca; Mitchell, Edward P; Chazalet, Valerie; Debray, Henri; Oscarson, Stefan; Lahmann, Martina; Gautier, Catherine; Breton, Christelle; Perez, Serge; Imberty, Anne

2006-04-14

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation

PubMed Central

Iida, Shinji; Nakamura, Haruki; Higo, Junichi

2016-01-01

We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein–protein or protein–ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. PMID:27288028

Drosophila cell cycle under arrest: uncapped telomeres plead guilty.

PubMed

Cenci, Giovanni

2009-04-01

Telomeres are specialized structures that protect chromosome ends from degradation and fusion events. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences preventing chromosome ends from being recognized as DNA double strand breaks (DSBs). Telomeres that lose their cap activate the DNA damage response (DDR) likewise DSBs and, if inappropriately repaired, generate telomeric fusions, which eventually lead to genome instability. In Drosophila there is not telomerase, and telomere length is maintained by transposition of three specialized retroelements. However, fly telomeres are protected by multi protein complexes like their yeast and vertebrate counterparts; these complexes bind chromosome ends in a sequence-independent fashion and are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres elicit a DDR just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. BubR1 accumulations at chromosome ends trigger the SAC that inhibits the metaphase-to-anaphase transition. These findings, reviewed here, highlight an intriguing and unsuspected connection between telomeres and cell cycle regulation, providing a clue to understand human telomere function.

Pro- and Antioxidant Functions of the Peroxisome-Mitochondria Connection and Its Impact on Aging and Disease

PubMed Central

Pascual-Ahuir, Amparo; Manzanares-Estreder, Sara

2017-01-01

Peroxisomes and mitochondria are the main intracellular sources for reactive oxygen species. At the same time, both organelles are critical for the maintenance of a healthy redox balance in the cell. Consequently, failure in the function of both organelles is causally linked to oxidative stress and accelerated aging. However, it has become clear that peroxisomes and mitochondria are much more intimately connected both physiologically and structurally. Both organelles share common fission components to dynamically respond to environmental cues, and the autophagic turnover of both peroxisomes and mitochondria is decisive for cellular homeostasis. Moreover, peroxisomes can physically associate with mitochondria via specific protein complexes. Therefore, the structural and functional connection of both organelles is a critical and dynamic feature in the regulation of oxidative metabolism, whose dynamic nature will be revealed in the future. In this review, we will focus on fundamental aspects of the peroxisome-mitochondria interplay derived from simple models such as yeast and move onto discussing the impact of an impaired peroxisomal and mitochondrial homeostasis on ROS production, aging, and disease in humans. PMID:28811869

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

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Advances in synapse formation: forging connections in the worm.

PubMed

Cherra, Salvatore J; Jin, Yishi

2015-01-01

Synapse formation is the quintessential process by which neurons form specific connections with their targets to enable the development of functional circuits. Over the past few decades, intense research efforts have identified thousands of proteins that localize to the pre- and postsynaptic compartments. Genetic dissection has provided important insights into the nexus of the molecular and cellular network, and has greatly advanced our knowledge about how synapses form and function physiologically. Moreover, recent studies have highlighted the complex regulation of synapse formation with the identification of novel mechanisms involving cell interactions from non-neuronal sources. In this review, we cover the conserved pathways required for synaptogenesis and place specific focus on new themes of synapse modulation arising from studies in Caenorhabditis elegans. For further resources related to this article, please visit the WIREs website. The authors have declared no conflicts of interest for this article. © 2014 Wiley Periodicals, Inc.

Nestedness across biological scales

PubMed Central

Marquitti, Flavia M. D.; Raimundo, Rafael L. G.; Sebastián-González, Esther; Coltri, Patricia P.; Perez, S. Ivan; Brandt, Débora Y. C.; Nunes, Kelly; Daura-Jorge, Fábio G.; Floeter, Sergio R.; Guimarães, Paulo R.

2017-01-01

Biological networks pervade nature. They describe systems throughout all levels of biological organization, from molecules regulating metabolism to species interactions that shape ecosystem dynamics. The network thinking revealed recurrent organizational patterns in complex biological systems, such as the formation of semi-independent groups of connected elements (modularity) and non-random distributions of interactions among elements. Other structural patterns, such as nestedness, have been primarily assessed in ecological networks formed by two non-overlapping sets of elements; information on its occurrence on other levels of organization is lacking. Nestedness occurs when interactions of less connected elements form proper subsets of the interactions of more connected elements. Only recently these properties began to be appreciated in one-mode networks (where all elements can interact) which describe a much wider variety of biological phenomena. Here, we compute nestedness in a diverse collection of one-mode networked systems from six different levels of biological organization depicting gene and protein interactions, complex phenotypes, animal societies, metapopulations, food webs and vertebrate metacommunities. Our findings suggest that nestedness emerge independently of interaction type or biological scale and reveal that disparate systems can share nested organization features characterized by inclusive subsets of interacting elements with decreasing connectedness. We primarily explore the implications of a nested structure for each of these studied systems, then theorize on how nested networks are assembled. We hypothesize that nestedness emerges across scales due to processes that, although system-dependent, may share a general compromise between two features: specificity (the number of interactions the elements of the system can have) and affinity (how these elements can be connected to each other). Our findings suggesting occurrence of nestedness throughout biological scales can stimulate the debate on how pervasive nestedness may be in nature, while the theoretical emergent principles can aid further research on commonalities of biological networks. PMID:28166284

Structure-Function of the Cytochrome b 6f Complex of Oxygenic Photosynthesis

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

Cramer, W. A.; Yamashita, E.; Baniulis, D.

2014-03-20

Structure–function of the major integral membrane cytochrome b 6f complex that functions in cyanobacteria, algae, and green plants to transfer electrons between the two reaction center complexes in the electron transport chain of oxygenic photosynthesis is discussed in the context of recently obtained crystal structures of the complex and soluble domains of cytochrome f and the Rieske iron–sulfur protein. The energy-transducing function of the complex, generation of the proton trans-membrane electrochemical potential gradient, centers on the oxidation/reduction pathways of the plastoquinol/plastoquinone (QH 2/Q), the proton donor/acceptor within the complex. These redox reactions are carried out by five redox prosthetic groupsmore » embedded in each monomer, the high potential two iron–two sulfur cluster and the heme of cytochrome f on the electropositive side (p) of the complex, two noncovalently bound b-type hemes that cross the complex and the membrane, and a covalently bound c-type heme (c n) on the electronegative side (n). These five redox-active groups are organized in high- (cyt f/[2Fe–2S] and low-potential (hemes b p, b n, c n) electron transport pathways that oxidize and reduce the quinol and quinone on the p- and n-sides in a Q-cycle-type mechanism, while translocating as many as 2 H + to the p-side aqueous side for every electron transferred through the high potential chain to the photosystem I reaction center. The presence of heme c n and the connection of the n-side of the membrane and b 6f complex to the cyclic electron transport chain indicate that the Q cycle in the oxygenic photosynthetic electron transport chain differs from those connected to the bc 1 complex in the mitochondrial respiratory chain and the chain in photosynthetic bacteria. Inferences from the structure and C2 symmetry of the complex for the pathway of QH 2/Q transfer within the complex, problems posed by the presence of lipid in the inter-monomer cavity, and the narrow portal for QH2 passage through the p-side oxidation site proximal to the [2Fe–2S] cluster are discussed.« less

Making connections for life: an in vivo map of the yeast interactome

PubMed Central

Kast, Juergen

2008-01-01

Proteins are the true workhorses of any cell. To carry out specific tasks, they frequently bind other molecules in their surroundings. Due to their structural complexity and flexibility, the most diverse array of interactions is seen with other proteins. The different geometries and affinities available for such interactions typically bestow specific functions on proteins. Having available a map of protein–protein interactions is therefore of enormous importance for any researcher interested in gaining insight into biological systems at the level of cells and organisms. In a recent report, a novel approach has been employed that relies on the spontaneous folding of complementary enzyme fragments fused to two different proteins to test whether these interact in their actual cellular context [Tarassov et al., Science 320, 1465–1470 (2008)]. Genome-wide application of this protein-fragment complementation assay has resulted in the first map of the in vivo interactome of Saccharomyces cerevisiae. The current data show striking similarities but also significant differences to those obtained using other large-scale approaches for the same task. This warrants a general discussion of the current state of affairs of protein–protein interaction studies and foreseeable future trends, highlighting their significance for a variety of applications and their potential to revolutionize our understanding of the architecture and dynamics of biological systems. PMID:19404434

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome

PubMed Central

Ciregia, F; Kollipara, L; Giusti, L; Zahedi, R P; Giacomelli, C; Mazzoni, M R; Giannaccini, G; Scarpellini, P; Urbani, A; Sickmann, A; Lucacchini, A; Bazzichi, L

2016-01-01

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments. PMID:27676445

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: a hypothesis.

PubMed

Varshavsky, Alexander

2012-11-01

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca²⁺ fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca²⁺-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca²⁺ transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca²⁺ alone or Ca²⁺ and its ionophore (Erickson et al., Science 1978;199:1219-1221; Harris, Pharmacol Biochem Behav 1979;10:527-534; Erickson et al., Pharmacol Biochem Behav 1980;12:651-656). These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep. Copyright © 2012 The Protein Society.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

On the Effect of Sphere-Overlap on Super Coarse-Grained Models of Protein Assemblies

NASA Astrophysics Data System (ADS)

Degiacomi, Matteo T.

2018-05-01

Ion mobility mass spectrometry (IM/MS) can provide structural information on intact protein complexes. Such data, including connectivity and collision cross sections (CCS) of assemblies' subunits, can in turn be used as a guide to produce representative super coarse-grained models. These models are constituted by ensembles of overlapping spheres, each representing a protein subunit. A model is considered plausible if the CCS and sphere-overlap levels of its subunits fall within predetermined confidence intervals. While the first is determined by experimental error, the latter is based on a statistical analysis on a range of protein dimers. Here, we first propose a new expression to describe the overlap between two spheres. Then we analyze the effect of specific overlap cutoff choices on the precision and accuracy of super coarse-grained models. Finally, we propose a method to determine overlap cutoff levels on a per-case scenario, based on collected CCS data, and show that it can be applied to the characterization of the assembly topology of symmetrical homo-multimers. [Figure not available: see fulltext.

SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

PubMed Central

Nogueira, Cristina; Erlmann, Patrik; Villeneuve, Julien; Santos, António JM; Martínez-Alonso, Emma; Martínez-Menárguez, José Ángel; Malhotra, Vivek

2014-01-01

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18. DOI: http://dx.doi.org/10.7554/eLife.02784.001 PMID:24842878

Modeling Non-homologous End Joining

NASA Technical Reports Server (NTRS)

Li, Yongfeng

2013-01-01

Non-homologous end joining (NHEJ) is the dominant DNA double strand break (DSB) repair pathway and involves several NHEJ proteins such as Ku, DNA-PKcs, XRCC4, Ligase IV and so on. Once DSBs are generated, Ku is first recruited to the DNA end, followed by other NHEJ proteins for DNA end processing and ligation. Because of the direct ligation of break ends without the need for a homologous template, NHEJ turns out to be an error-prone but efficient repair pathway. Some mechanisms have been proposed of how the efficiency of NHEJ repair is affected. The type of DNA damage is an important factor of NHEJ repair. For instance, the length of DNA fragment may determine the recruitment efficiency of NHEJ protein such as Ku [1], or the complexity of the DNA breaks [2] is accounted for the choice of NHEJ proteins and subpathway of NHEJ repair. On the other hand, the chromatin structure also plays a role of the accessibility of NHEJ protein to the DNA damage site. In this talk, some mathematical models of NHEJ, that consist of series of biochemical reactions complying with the laws of chemical reaction (e.g. mass action, etc.), will be introduced. By mathematical and numerical analysis and parameter estimation, the models are able to capture the qualitative biological features and show good agreement with experimental data. As conclusions, from the viewpoint of modeling, how the NHEJ proteins are recruited will be first discussed for connection between the classical sequential model [4] and recently proposed two-phase model [5]. Then how the NHEJ repair pathway is affected, by the length of DNA fragment [6], the complexity of DNA damage [7] and the chromatin structure [8], will be addressed

Reducing eIF4E-eIF4G Interactions Restores the Balance Between Protein Synthesis and Actin Dynamics in Fragile X Syndrome Model Mice*

PubMed Central

Santini, Emanuela; Huynh, Thu N.; Longo, Francesco; Koo, So Yeon; Mojica, Edward; D’Andrea, Laura; Bagni, Claudia; Klann, Eric

2018-01-01

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism spectrum disorder. FXS is caused by silencing of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP), an mRNA-binding protein that represses the translation of its target mRNAs. One mechanism by which FMRP represses translation is through its association with cytoplasmic FMRP-interacting protein 1 (CYFIP1), which binds to and sequesters eukaryotic initiation factor 4E (eIF4E). CYFIP1 shuttles between the FMRP–eIF4E complex and the Rac1–Wave regulatory complex, thereby connecting translation regulation to actin dynamics and dendritic spine morphology, which are dysregulated in FXS model mice that lack FMRP. Treating FXS mice with 4EGI-1, which blocks interactions between eIF4E and eukaryotic factor 4G (eIF4G), a critical interacting partner for protein synthesis, reversed defects in hippocampus-dependent memory and spine morphology. We also found that 4EGI-1 normalized the phenotypes of enhanced metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), upregulated Rac1–p21-activated kinase (PAK)–cofilin signaling, altered actin dynamics, and dysregulated CYFIP1/eIF4E and CYFIP1/Rac1 interactions in FXS mice. Our findings are consistent with the idea that an imbalance of protein synthesis and actin dynamics contributes to pathophysiology in FXS mice, and suggest that targeting eIF4E may be a strategy for treating FXS. PMID:29114037

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

PubMed Central

Pelassa, Ilaria; Fiumara, Ferdinando

2015-01-01

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping the interaction networks of the human proteome, and define proteome-wide knowledge that may guide the informed biological exploration of the role of AARs in protein interactions. PMID:26734058

Protein localization as a principal feature of the etiology and comorbidity of genetic diseases

PubMed Central

Park, Solip; Yang, Jae-Seong; Shin, Young-Eun; Park, Juyong; Jang, Sung Key; Kim, Sanguk

2011-01-01

Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression. PMID:21613983

CORUM: the comprehensive resource of mammalian protein complexes

PubMed Central

Ruepp, Andreas; Brauner, Barbara; Dunger-Kaltenbach, Irmtraud; Frishman, Goar; Montrone, Corinna; Stransky, Michael; Waegele, Brigitte; Schmidt, Thorsten; Doudieu, Octave Noubibou; Stümpflen, Volker; Mewes, H. Werner

2008-01-01

Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. The CORUM (http://mips.gsf.de/genre/proj/corum/index.html) database is a collection of experimentally verified mammalian protein complexes. Information is manually derived by critical reading of the scientific literature from expert annotators. Information about protein complexes includes protein complex names, subunits, literature references as well as the function of the complexes. For functional annotation, we use the FunCat catalogue that enables to organize the protein complex space into biologically meaningful subsets. The database contains more than 1750 protein complexes that are built from 2400 different genes, thus representing 12% of the protein-coding genes in human. A web-based system is available to query, view and download the data. CORUM provides a comprehensive dataset of protein complexes for discoveries in systems biology, analyses of protein networks and protein complex-associated diseases. Comparable to the MIPS reference dataset of protein complexes from yeast, CORUM intends to serve as a reference for mammalian protein complexes. PMID:17965090

An Autoimmune Myositis-Overlap Syndrome Associated With Autoantibodies to Nuclear Pore Complexes

PubMed Central

Senécal, Jean-Luc; Isabelle, Catherine; Fritzler, Marvin J.; Targoff, Ira N.; Goldstein, Rose; Gagné, Michel; Raynauld, Jean-Pierre; Joyal, France; Troyanov, Yves; Dabauvalle, Marie-Christine

2014-01-01

Abstract Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immunomodulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100% long-term survival (mean follow-up 19.5 yr). By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults. Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1∗0501 allele associated with an increased risk for autoimmune myositis. In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an “anti-nup syndrome.” PMID:25500708

Plakins: a family of versatile cytolinker proteins.

PubMed

Leung, Conrad L; Green, Kathleen J; Liem, Ronald K H

2002-01-01

By connecting cytoskeletal elements to each other and to junctional complexes, the plakin family of cytolinkers plays a crucial role in orchestrating cellular development and maintaining tissue integrity. Plakins are built from combinations of interacting domains that bind to microfilaments, microtubules, intermediate filaments, cell-adhesion molecules and members of the armadillo family. Plakins are involved in both inherited and autoimmune diseases that affect the skin, neuronal tissue, and cardiac and skeletal muscle. Here, we describe the members of the plakin family and their interaction partners, and give examples of the cellular defects that result from their dysfunction.

A Structural Biology and Protein Engineering Approach to the Development of Antidotes Against the Inhibition of Human Acetylcholinesterase by OP-based Nerve Agents

DTIC Science & Technology

2015-05-01

structures that we reported earlier (Kryger et al [2000] Acta Crystallogr D Biol Crystallogr 56:1385-1394) were of complexes with the snake venom...interactions between conserved residues in the loop connecting α13 to α14 and residues from helices α18’-α19’, and, conversely, between residues in the...residues in the 4-helix bundle, including Glu376, Thr383, Asp384, Trp385, Gln508, Gln527, Phe535 and Lys538 (hAChE numbering), are strictly conserved in

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation.

PubMed

Miguel-Arribas, Andrés; Hao, Jian-An; Luque-Ortega, Juan R; Ramachandran, Gayetri; Val-Calvo, Jorge; Gago-Córdoba, César; González-Álvarez, Daniel; Abia, David; Alfonso, Carlos; Wu, Ling J; Meijer, Wilfried J J

2017-01-01

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation.

PubMed

Roque, Helio; Saurya, Saroj; Pratt, Metta B; Johnson, Errin; Raff, Jordan W

2018-02-01

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells.

Using high-throughput barcode sequencing to efficiently map connectomes

PubMed Central

Peikon, Ian D.; Kebschull, Justus M.; Vagin, Vasily V.; Ravens, Diana I.; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R.; Bressan, Dario

2017-01-01

Abstract The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision—a ‘connectome’—is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence—an RNA ‘barcode’—which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. PMID:28449067

Phloem unloading in Arabidopsis roots is convective and regulated by the phloem-pole pericycle

PubMed Central

Ross-Elliott, Timothy J; Jensen, Kaare H; Haaning, Katrine S; Wager, Brittney M; Knoblauch, Jan; Howell, Alexander H; Mullendore, Daniel L; Monteith, Alexander G; Paultre, Danae; Yan, Dawei; Otero, Sofia; Bourdon, Matthieu; Sager, Ross; Lee, Jung-Youn; Helariutta, Ykä; Knoblauch, Michael; Oparka, Karl J

2017-01-01

In plants, a complex mixture of solutes and macromolecules is transported by the phloem. Here, we examined how solutes and macromolecules are separated when they exit the phloem during the unloading process. We used a combination of approaches (non-invasive imaging, 3D-electron microscopy, and mathematical modelling) to show that phloem unloading of solutes in Arabidopsis roots occurs through plasmodesmata by a combination of mass flow and diffusion (convective phloem unloading). During unloading, solutes and proteins are diverted into the phloem-pole pericycle, a tissue connected to the protophloem by a unique class of ‘funnel plasmodesmata’. While solutes are unloaded without restriction, large proteins are released through funnel plasmodesmata in discrete pulses, a phenomenon we refer to as ‘batch unloading’. Unlike solutes, these proteins remain restricted to the phloem-pole pericycle. Our data demonstrate a major role for the phloem-pole pericycle in regulating phloem unloading in roots. DOI: http://dx.doi.org/10.7554/eLife.24125.001 PMID:28230527

Arabidopsis thaliana BTB/ POZ-MATH proteins interact with members of the ERF/AP2 transcription factor family.

PubMed

Weber, Henriette; Hellmann, Hanjo

2009-11-01

In Arabidopsis thaliana, the BTB/POZ-MATH (BPM) proteins comprise a small family of six members. They have been described previously to use their broad complex, tram track, bric-a-brac/POX virus and zinc finger (BTB/POZ) domain to assemble with CUL3a and CUL3b and potentially to serve as substrate adaptors to cullin-based E3-ligases in plants. In this article, we show that BPMs can also assemble with members of the ethylene response factor/Apetala2 transcription factor family, and that this is mediated by their meprin and TRAF (tumor necrosis factor receptor-associated factor) homology (MATH) domain. In addition, we provide a detailed description of BPM gene expression patterns in different tissues and on abiotic stress treatments, as well as their subcellular localization. This work connects, for the first time, BPM proteins with ethylene response factor/Apetala2 family members, which is likely to represent a novel regulatory mechanism of transcriptional control.

Proteomic Study Related to Vascular Connections in Watermelon Scions Grafted onto Bottle-Gourd Rootstock under Different Light Intensities

PubMed Central

Muneer, Sowbiya; Ko, Chung Ho; Soundararajan, Prabhakaran; Manivnnan, Abinaya; Park, Yoo Gyeong; Jeong, Byoung Ryong

2015-01-01

Although grafting is broadly used in the production of crops, no information is available about the proteins involved in vascular connections between rootstock and scion. Similarly, proteome changes under the light intensities widely used for grafted seedlings are of practical use. The objective of this study was to determine the proteome of vascular connections using watermelon (Citrullus vulgaris Schrad.) ‘Sambok Honey’ and ‘Speed’ as the scion and bottle gourd (Lagenaria siceraria Stanld.) ‘RS Dongjanggun’ as the rootstock grown under different light intensities (25, 50, 75 and 100 μmol m−2 s−1). Our proteomic analysis revealed 24 and 27 differentially expressed proteins in ‘Sambok Honey’ and ‘Speed’, respectively, under different light intensities. The identified proteins were largely involved in ion binding, amino acid metabolism, transcriptional regulation and defense response. The enhancement of ion-binding, transcriptional regulation, amino acid metabolism, and defense response proteins suggests a strengthening of the connection between the rootstock and scion under high light intensity. Indeed, the accumulation of key enzymes in the biological processes described above appears to play an important role in the vascular connections of grafted seedlings. Moreover, it appears that 100 μmol m−2 s−1 results in better protein expression responses in grafted seedlings. PMID:25789769

Proteomic study related to vascular connections in watermelon scions grafted onto bottle-gourd rootstock under different light intensities.

PubMed

Muneer, Sowbiya; Ko, Chung Ho; Soundararajan, Prabhakaran; Manivnnan, Abinaya; Park, Yoo Gyeong; Jeong, Byoung Ryong

2015-01-01

Although grafting is broadly used in the production of crops, no information is available about the proteins involved in vascular connections between rootstock and scion. Similarly, proteome changes under the light intensities widely used for grafted seedlings are of practical use. The objective of this study was to determine the proteome of vascular connections using watermelon (Citrullus vulgaris Schrad.) 'Sambok Honey' and 'Speed' as the scion and bottle gourd (Lagenaria siceraria Stanld.) 'RS Dongjanggun' as the rootstock grown under different light intensities (25, 50, 75 and 100 μmol m-2 s-1). Our proteomic analysis revealed 24 and 27 differentially expressed proteins in 'Sambok Honey' and 'Speed', respectively, under different light intensities. The identified proteins were largely involved in ion binding, amino acid metabolism, transcriptional regulation and defense response. The enhancement of ion-binding, transcriptional regulation, amino acid metabolism, and defense response proteins suggests a strengthening of the connection between the rootstock and scion under high light intensity. Indeed, the accumulation of key enzymes in the biological processes described above appears to play an important role in the vascular connections of grafted seedlings. Moreover, it appears that 100 μmol m-2 s-1 results in better protein expression responses in grafted seedlings.

The Fanconi anaemia pathway: new players and new functions.

PubMed

Ceccaldi, Raphael; Sarangi, Prabha; D'Andrea, Alan D

2016-06-01

The Fanconi anaemia pathway repairs DNA interstrand crosslinks (ICLs) in the genome. Our understanding of this complex pathway is still evolving, as new components continue to be identified and new biochemical systems are used to elucidate the molecular steps of repair. The Fanconi anaemia pathway uses components of other known DNA repair processes to achieve proper repair of ICLs. Moreover, Fanconi anaemia proteins have functions in genome maintenance beyond their canonical roles of repairing ICLs. Such functions include the stabilization of replication forks and the regulation of cytokinesis. Thus, Fanconi anaemia proteins are emerging as master regulators of genomic integrity that coordinate several repair processes. Here, we summarize our current understanding of the functions of the Fanconi anaemia pathway in ICL repair, together with an overview of its connections with other repair pathways and its emerging roles in genome maintenance.

The driving regulators of the connectivity protein network of brain malignancies

NASA Astrophysics Data System (ADS)

Tahmassebi, Amirhessam; Pinker-Domenig, Katja; Wengert, Georg; Lobbes, Marc; Stadlbauer, Andreas; Wildburger, Norelle C.; Romero, Francisco J.; Morales, Diego P.; Castillo, Encarnacion; Garcia, Antonio; Botella, Guillermo; Meyer-Bäse, Anke

2017-05-01

An important problem in modern therapeutics at the proteomic level remains to identify therapeutic targets in a plentitude of high-throughput data from experiments relevant to a variety of diseases. This paper presents the application of novel modern control concepts, such as pinning controllability and observability applied to the glioma cancer stem cells (GSCs) protein graph network with known and novel association to glioblastoma (GBM). The theoretical frameworks provides us with the minimal number of "driver nodes", which are necessary, and their location to determine the full control over the obtained graph network in order to provide a change in the network's dynamics from an initial state (disease) to a desired state (non-disease). The achieved results will provide biochemists with techniques to identify more metabolic regions and biological pathways for complex diseases, to design and test novel therapeutic solutions.

3DScapeCS: application of three dimensional, parallel, dynamic network visualization in Cytoscape

PubMed Central

2013-01-01

Background The exponential growth of gigantic biological data from various sources, such as protein-protein interaction (PPI), genome sequences scaffolding, Mass spectrometry (MS) molecular networking and metabolic flux, demands an efficient way for better visualization and interpretation beyond the conventional, two-dimensional visualization tools. Results We developed a 3D Cytoscape Client/Server (3DScapeCS) plugin, which adopted Cytoscape in interpreting different types of data, and UbiGraph for three-dimensional visualization. The extra dimension is useful in accommodating, visualizing, and distinguishing large-scale networks with multiple crossed connections in five case studies. Conclusions Evaluation on several experimental data using 3DScapeCS and its special features, including multilevel graph layout, time-course data animation, and parallel visualization has proven its usefulness in visualizing complex data and help to make insightful conclusions. PMID:24225050

Metabolic networks in motion: 13C-based flux analysis

PubMed Central

Sauer, Uwe

2006-01-01

Many properties of complex networks cannot be understood from monitoring the components—not even when comprehensively monitoring all protein or metabolite concentrations—unless such information is connected and integrated through mathematical models. The reason is that static component concentrations, albeit extremely informative, do not contain functional information per se. The functional behavior of a network emerges only through the nonlinear gene, protein, and metabolite interactions across multiple metabolic and regulatory layers. I argue here that intracellular reaction rates are the functional end points of these interactions in metabolic networks, hence are highly relevant for systems biology. Methods for experimental determination of metabolic fluxes differ fundamentally from component concentration measurements; that is, intracellular reaction rates cannot be detected directly, but must be estimated through computer model-based interpretation of stable isotope patterns in products of metabolism. PMID:17102807

Identifying the adaptive mechanism in globular proteins: Fluctuations in densely packed regions manipulate flexible parts

NASA Astrophysics Data System (ADS)

Yilmaz, Lutfu Safak; Atilgan, Ali Rana

2000-09-01

A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier-Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuations that are widely distributed over the native structure. These positional arrangements do not appear in a reciprocal relation between a perturbation and the associated response; the effect of a movement of residue i on residue j is not equal to that of the same movement of residue j on residue i.

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

PubMed Central

Nicosia, Aldo; Maggio, Teresa; Costa, Salvatore; Salamone, Monica; Tagliavia, Marcello; Mazzola, Salvatore; Gianguzza, Fabrizio; Cuttitta, Angela

2016-01-01

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations. PMID:26957029

DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

PubMed

Kunig, Verena; Potowski, Marco; Gohla, Anne; Brunschweiger, Andreas

2018-06-27

DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

Earthquake Complex Network Analysis Before and After the Mw 8.2 Earthquake in Iquique, Chile

NASA Astrophysics Data System (ADS)

Pasten, D.

2017-12-01

The earthquake complex networks have shown that they are abble to find specific features in seismic data set. In space, this networkshave shown a scale-free behavior for the probability distribution of connectivity, in directed networks and theyhave shown a small-world behavior, for the undirected networks.In this work, we present an earthquake complex network analysis for the large earthquake Mw 8.2 in the north ofChile (near to Iquique) in April, 2014. An earthquake complex network is made dividing the three dimensional space intocubic cells, if one of this cells contain an hypocenter, we name this cell like a node. The connections between nodes aregenerated in time. We follow the time sequence of seismic events and we are making the connections betweennodes. Now, we have two different networks: a directed and an undirected network. Thedirected network takes in consideration the time-direction of the connections, that is very important for the connectivityof the network: we are considering the connectivity, ki of the i-th node, like the number of connections going out ofthe node i plus the self-connections (if two seismic events occurred successive in time in the same cubic cell, we havea self-connection). The undirected network is made removing the direction of the connections and the self-connectionsfrom the directed network. For undirected networks, we are considering only if two nodes are or not connected.We have built a directed complex network and an undirected complex network, before and after the large earthquake in Iquique. We have used magnitudes greater than Mw = 1.0 and Mw = 3.0. We found that this method can recognize the influence of thissmall seismic events in the behavior of the network and we found that the size of the cell used to build the network isanother important factor to recognize the influence of the large earthquake in this complex system. This method alsoshows a difference in the values of the critical exponent γ (for the probability distribution of connectivity in the directednetwork) before and after the large earthquake, but this method does not show a change in the clustering behavior ofthe undirected network, before and after the large earthquake, showing a small-world behavior for the network beforeand after of this large seismic event.

An analytical method for computing atomic contact areas in biomolecules.

PubMed

Mach, Paul; Koehl, Patrice

2013-01-15

We propose a new analytical method for detecting and computing contacts between atoms in biomolecules. It is based on the alpha shape theory and proceeds in three steps. First, we compute the weighted Delaunay triangulation of the union of spheres representing the molecule. In the second step, the Delaunay complex is filtered to derive the dual complex. Finally, contacts between spheres are collected. In this approach, two atoms i and j are defined to be in contact if their centers are connected by an edge in the dual complex. The contact areas between atom i and its neighbors are computed based on the caps formed by these neighbors on the surface of i; the total area of all these caps is partitioned according to their spherical Laguerre Voronoi diagram on the surface of i. This method is analytical and its implementation in a new program BallContact is fast and robust. We have used BallContact to study contacts in a database of 1551 high resolution protein structures. We show that with this new definition of atomic contacts, we generate realistic representations of the environments of atoms and residues within a protein. In particular, we establish the importance of nonpolar contact areas that complement the information represented by the accessible surface areas. This new method bears similarity to the tessellation methods used to quantify atomic volumes and contacts, with the advantage that it does not require the presence of explicit solvent molecules if the surface of the protein is to be considered. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

PubMed

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-03-22

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

Plakins in development and disease.

PubMed

Sonnenberg, Arnoud; Liem, Ronald K H

2007-06-10

Plakins are large multi-domain molecules that have various functions to link cytoskeletal elements together and to connect them to junctional complexes. Plakins were first identified in epithelial cells where they were found to connect the intermediate filaments to desmosomes and hemidesmosomes [Ruhrberg, C., and Watt, F.M. (1997). The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 7, 392-397.]. They were subsequently found to be important for the integrity of muscle cells. Most recently, they have been found in the nervous system, where their functions appear to be more complex, including cross-linking of microtubules (MTs) and actin filaments [Leung, C.L., Zheng, M., Prater, S.M., and Liem, R.K. (2001). The BPAG1 locus: Alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 154, 691-697., Leung, C.L., Sun, D., Zheng, M., Knowles, D.R., and Liem, R.K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147, 1275-1286.]. These plakins have also indicated their relationship to the spectrin superfamily of proteins and the plakins appear to be evolutionarily related to the spectrins, but have diverged to perform different specialized functions. In invertebrates, a single plakin is present in both Drosophila melanogaster and Caenorhabditis elegans, which resemble the more complex plakins found in mammals [Roper, K., Gregory, S.L., and Brown, N.H. (2002). The 'spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families. J Cell Sci 115, 4215-4225.]. In contrast, there are seven plakins found in mammals and most of them have alternatively spliced forms leading to a very complex group of proteins with potential tissue specific functions [Jefferson, J.J., Leung, C.L., and Liem, R.K. (2004). Plakins: goliaths that link cell junctions and the cytoskeleton. Nat Rev Mol Cell Biol 5, 542-553.]. In this review, we will first describe the plakins, desmoplakin, plectin, envoplakin and periplakin and then describe two other mammalian plakins, Bullous pemphigoid antigen 1 (BPAG1) and microtubule actin cross-linking factor 1 (MACF1), that are expressed in multiple isoforms in different tissues. We will also describe the relationship of these two proteins to the invertebrate plakins, shortstop (shot) in Drosophila and VAB-10 in C. elegans. Finally, we will describe an unusual mammalian plakin, called epiplakin.

The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit Vps25.

PubMed

Emerman, Amy B; Blower, Michael

2018-06-14

RNA-binding proteins (RBPs) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. Endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II previously has been identified as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV-crosslinking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit Vps25. UV-crosslinking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro. Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNAs and also suggest an unexpected link between endosome biology and RNA regulation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Multiscale multiphysics and multidomain models—Flexibility and rigidity

PubMed Central

Xia, Kelin; Opron, Kristopher; Wei, Guo-Wei

2013-01-01

The emerging complexity of large macromolecules has led to challenges in their full scale theoretical description and computer simulation. Multiscale multiphysics and multidomain models have been introduced to reduce the number of degrees of freedom while maintaining modeling accuracy and achieving computational efficiency. A total energy functional is constructed to put energies for polar and nonpolar solvation, chemical potential, fluid flow, molecular mechanics, and elastic dynamics on an equal footing. The variational principle is utilized to derive coupled governing equations for the above mentioned multiphysical descriptions. Among these governing equations is the Poisson-Boltzmann equation which describes continuum electrostatics with atomic charges. The present work introduces the theory of continuum elasticity with atomic rigidity (CEWAR). The essence of CEWAR is to formulate the shear modulus as a continuous function of atomic rigidity. As a result, the dynamics complexity of a macromolecular system is separated from its static complexity so that the more time-consuming dynamics is handled with continuum elasticity theory, while the less time-consuming static analysis is pursued with atomic approaches. We propose a simple method, flexibility-rigidity index (FRI), to analyze macromolecular flexibility and rigidity in atomic detail. The construction of FRI relies on the fundamental assumption that protein functions, such as flexibility, rigidity, and energy, are entirely determined by the structure of the protein and its environment, although the structure is in turn determined by all the interactions. As such, the FRI measures the topological connectivity of protein atoms or residues and characterizes the geometric compactness of the protein structure. As a consequence, the FRI does not resort to the interaction Hamiltonian and bypasses matrix diagonalization, which underpins most other flexibility analysis methods. FRI's computational complexity is of \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N^2)$\\end{document}O(N2) at most, where N is the number of atoms or residues, in contrast to \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N^3)$\\end{document}O(N3) for Hamiltonian based methods. We demonstrate that the proposed FRI gives rise to accurate prediction of protein B-Factor for a set of 263 proteins. We show that a parameter free FRI is able to achieve about 95% accuracy of the parameter optimized FRI. An interpolation algorithm is developed to construct continuous atomic flexibility functions for visualization and use with CEWAR. PMID:24320318

Multiscale multiphysics and multidomain models—Flexibility and rigidity

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

Xia, Kelin; Opron, Kristopher; Wei, Guo-Wei, E-mail: wei@math.msu.edu

The emerging complexity of large macromolecules has led to challenges in their full scale theoretical description and computer simulation. Multiscale multiphysics and multidomain models have been introduced to reduce the number of degrees of freedom while maintaining modeling accuracy and achieving computational efficiency. A total energy functional is constructed to put energies for polar and nonpolar solvation, chemical potential, fluid flow, molecular mechanics, and elastic dynamics on an equal footing. The variational principle is utilized to derive coupled governing equations for the above mentioned multiphysical descriptions. Among these governing equations is the Poisson-Boltzmann equation which describes continuum electrostatics with atomicmore » charges. The present work introduces the theory of continuum elasticity with atomic rigidity (CEWAR). The essence of CEWAR is to formulate the shear modulus as a continuous function of atomic rigidity. As a result, the dynamics complexity of a macromolecular system is separated from its static complexity so that the more time-consuming dynamics is handled with continuum elasticity theory, while the less time-consuming static analysis is pursued with atomic approaches. We propose a simple method, flexibility-rigidity index (FRI), to analyze macromolecular flexibility and rigidity in atomic detail. The construction of FRI relies on the fundamental assumption that protein functions, such as flexibility, rigidity, and energy, are entirely determined by the structure of the protein and its environment, although the structure is in turn determined by all the interactions. As such, the FRI measures the topological connectivity of protein atoms or residues and characterizes the geometric compactness of the protein structure. As a consequence, the FRI does not resort to the interaction Hamiltonian and bypasses matrix diagonalization, which underpins most other flexibility analysis methods. FRI's computational complexity is of O(N{sup 2}) at most, where N is the number of atoms or residues, in contrast to O(N{sup 3}) for Hamiltonian based methods. We demonstrate that the proposed FRI gives rise to accurate prediction of protein B-Factor for a set of 263 proteins. We show that a parameter free FRI is able to achieve about 95% accuracy of the parameter optimized FRI. An interpolation algorithm is developed to construct continuous atomic flexibility functions for visualization and use with CEWAR.« less

Network-based prediction and knowledge mining of disease genes.

PubMed

Carson, Matthew B; Lu, Hui

2015-01-01

In recent years, high-throughput protein interaction identification methods have generated a large amount of data. When combined with the results from other in vivo and in vitro experiments, a complex set of relationships between biological molecules emerges. The growing popularity of network analysis and data mining has allowed researchers to recognize indirect connections between these molecules. Due to the interdependent nature of network entities, evaluating proteins in this context can reveal relationships that may not otherwise be evident. We examined the human protein interaction network as it relates to human illness using the Disease Ontology. After calculating several topological metrics, we trained an alternating decision tree (ADTree) classifier to identify disease-associated proteins. Using a bootstrapping method, we created a tree to highlight conserved characteristics shared by many of these proteins. Subsequently, we reviewed a set of non-disease-associated proteins that were misclassified by the algorithm with high confidence and searched for evidence of a disease relationship. Our classifier was able to predict disease-related genes with 79% area under the receiver operating characteristic (ROC) curve (AUC), which indicates the tradeoff between sensitivity and specificity and is a good predictor of how a classifier will perform on future data sets. We found that a combination of several network characteristics including degree centrality, disease neighbor ratio, eccentricity, and neighborhood connectivity help to distinguish between disease- and non-disease-related proteins. Furthermore, the ADTree allowed us to understand which combinations of strongly predictive attributes contributed most to protein-disease classification. In our post-processing evaluation, we found several examples of potential novel disease-related proteins and corresponding literature evidence. In addition, we showed that first- and second-order neighbors in the PPI network could be used to identify likely disease associations. We analyzed the human protein interaction network and its relationship to disease and found that both the number of interactions with other proteins and the disease relationship of neighboring proteins helped to determine whether a protein had a relationship to disease. Our classifier predicted many proteins with no annotated disease association to be disease-related, which indicated that these proteins have network characteristics that are similar to disease-related proteins and may therefore have disease associations not previously identified. By performing a post-processing step after the prediction, we were able to identify evidence in literature supporting this possibility. This method could provide a useful filter for experimentalists searching for new candidate protein targets for drug repositioning and could also be extended to include other network and data types in order to refine these predictions.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

PubMed Central

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-01-01

A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines

PubMed Central

Hsu, Hwei-Jan; Drummond-Barbosa, Daniela

2017-01-01

The effect of diet on reproduction is well documented in a large number of organisms; however, much remains to be learned about the molecular mechanisms underlying this connection. The Drosophila ovary has a well described, fast and largely reversible response to diet. Ovarian stem cells and their progeny proliferate and grow faster on a yeast-rich diet than on a yeast-free (poor) diet, and death of early germline cysts, degeneration of early vitellogenic follicles and partial block in ovulation further contribute to the ~60-fold decrease in egg laying observed on a poor diet. Multiple diet-dependent factors, including insulin-like peptides, the steroid ecdysone, the nutrient sensor Target of Rapamycin, AMP-dependent kinase, and adipocyte factors mediate this complex response. Here, we describe the results of a visual screen using a library of green fluorescent protein (GFP) protein trap lines to identify additional factors potentially involved in this response. In each GFP protein trap line, an artificial GFP exon is fused in frame to an endogenous protein, such that the GFP fusion pattern parallels the levels and subcellular localization of the corresponding native protein. We identified 53 GFP-tagged proteins that exhibit changes in levels and/or subcellular localization in the ovary at 12-16 hours after switching females from rich to poor diets, suggesting them as potential candidates for future functional studies. PMID:28093350

A linear framework for time-scale separation in nonlinear biochemical systems.

PubMed

Gunawardena, Jeremy

2012-01-01

Cellular physiology is implemented by formidably complex biochemical systems with highly nonlinear dynamics, presenting a challenge for both experiment and theory. Time-scale separation has been one of the few theoretical methods for distilling general principles from such complexity. It has provided essential insights in areas such as enzyme kinetics, allosteric enzymes, G-protein coupled receptors, ion channels, gene regulation and post-translational modification. In each case, internal molecular complexity has been eliminated, leading to rational algebraic expressions among the remaining components. This has yielded familiar formulas such as those of Michaelis-Menten in enzyme kinetics, Monod-Wyman-Changeux in allostery and Ackers-Johnson-Shea in gene regulation. Here we show that these calculations are all instances of a single graph-theoretic framework. Despite the biochemical nonlinearity to which it is applied, this framework is entirely linear, yet requires no approximation. We show that elimination of internal complexity is feasible when the relevant graph is strongly connected. The framework provides a new methodology with the potential to subdue combinatorial explosion at the molecular level.

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

PubMed

Poulet, Axel; Duc, Céline; Voisin, Maxime; Desset, Sophie; Tutois, Sylvie; Vanrobays, Emmanuel; Benoit, Matthias; Evans, David E; Probst, Aline V; Tatout, Christophe

2017-02-01

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. © 2017. Published by The Company of Biologists Ltd.

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

PubMed Central

Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

2009-01-01

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

Animal Models of Congenital Cardiomyopathies Associated With Mutations in Z-Line Proteins.

PubMed

Bang, Marie-Louise

2017-01-01

The cardiac Z-line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z-line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z-line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z-line proteins and the pathways leading from mutations in Z-line proteins to cardiomyopathy, although genotype-phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z-line and Z-line associated proteins involved in human cardiomyopathies with special emphasis on knock-in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z-line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 232: 38-52, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Defining protein expression in the urothelium: a problem of more than transitional interest

PubMed Central

Yu, Weiqun

2011-01-01

The transitional epithelium of the bladder, the urothelium, is a challenging tissue to study due to its fragility, complex cellular makeup, stratified composition, and intimate connections to both neural and connective tissue elements. With the increasing focus on the urothelium as a mechanosensory tissue with complex autocrine and paracrine signaling activities, there have arisen a number of unresolved controversies in the urothelial literature regarding whether certain important sensory and signaling proteins are expressed by the urothelium. Prominent examples of this include the transient receptor potential (TRP) family member TRPV1 and the purinergic receptor P2X3. The problem is more than one of scientific bookkeeping since studies utilizing genetic models (primarily knockout mice) claim additional credibility for urothelial functions when phenotypes are discovered. Furthermore, both of the above-mentioned receptors are important therapeutic targets for various bladder disorders including inflammatory and neuropathic pain. The reasons for the confusion about urothelial expression are manifold, but they likely include low expression levels in some cases, poor specificity of antibodies (sometimes lacking adequate controls), the presence of nonurothelial cells resident within the urothelium, and the fact that the urothelium is particularly prone to aspecific adsorption of antibodies. In this review, we attempt to summarize some of the pitfalls with currently accepted practices in this regard, as well as to describe a set of guidelines which will improve the reliability of conclusions related to urothelial expression. It is hoped that this will be of value to investigators studying the urothelium, to those attempting to interpret conflicts in the literature, and hopefully also those charged with reviewing unpublished work. These recommendations will outline a set of “baseline” and “best practice” guidelines by which both researchers and reviewers will be able to evaluate the evidence presented. PMID:21880838

Defining protein expression in the urothelium: a problem of more than transitional interest.

PubMed

Yu, Weiqun; Hill, Warren G

2011-11-01

The transitional epithelium of the bladder, the urothelium, is a challenging tissue to study due to its fragility, complex cellular makeup, stratified composition, and intimate connections to both neural and connective tissue elements. With the increasing focus on the urothelium as a mechanosensory tissue with complex autocrine and paracrine signaling activities, there have arisen a number of unresolved controversies in the urothelial literature regarding whether certain important sensory and signaling proteins are expressed by the urothelium. Prominent examples of this include the transient receptor potential (TRP) family member TRPV1 and the purinergic receptor P2X(3). The problem is more than one of scientific bookkeeping since studies utilizing genetic models (primarily knockout mice) claim additional credibility for urothelial functions when phenotypes are discovered. Furthermore, both of the above-mentioned receptors are important therapeutic targets for various bladder disorders including inflammatory and neuropathic pain. The reasons for the confusion about urothelial expression are manifold, but they likely include low expression levels in some cases, poor specificity of antibodies (sometimes lacking adequate controls), the presence of nonurothelial cells resident within the urothelium, and the fact that the urothelium is particularly prone to aspecific adsorption of antibodies. In this review, we attempt to summarize some of the pitfalls with currently accepted practices in this regard, as well as to describe a set of guidelines which will improve the reliability of conclusions related to urothelial expression. It is hoped that this will be of value to investigators studying the urothelium, to those attempting to interpret conflicts in the literature, and hopefully also those charged with reviewing unpublished work. These recommendations will outline a set of "baseline" and "best practice" guidelines by which both researchers and reviewers will be able to evaluate the evidence presented.

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

PubMed Central

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei

2015-01-01

ABSTRACT Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. PMID:26559827

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation.

PubMed

Guo, Yu; Wang, Wenming; Sun, Yuna; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Shen, Shu; Li, Baobin; Lin, Jianping; Deng, Fei; Wang, Hualin; Lou, Zhiyong

2016-01-15

Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusive target for the serological diagnoses. This work reveals the structure of hantavirus NP, furthering the knowledge of hantavirus RNP formation, revealing the relationship between hantavirus NP and serological specificity and raising the potential for the development of new diagnosis and therapeutics targeting hantavirus infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans.

PubMed

Jang, Heeun; Levy, Sagi; Flavell, Steven W; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I

2017-02-14

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9-containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9 -based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits.

Dissection of neuronal gap junction circuits that regulate social behavior in Caenorhabditis elegans

PubMed Central

Jang, Heeun; Levy, Sagi; Flavell, Steven W.; Mende, Fanny; Latham, Richard; Zimmer, Manuel; Bargmann, Cornelia I.

2017-01-01

A hub-and-spoke circuit of neurons connected by gap junctions controls aggregation behavior and related behavioral responses to oxygen, pheromones, and food in Caenorhabditis elegans. The molecular composition of the gap junctions connecting RMG hub neurons with sensory spoke neurons is unknown. We show here that the innexin gene unc-9 is required in RMG hub neurons to drive aggregation and related behaviors, indicating that UNC-9–containing gap junctions mediate RMG signaling. To dissect the circuit in detail, we developed methods to inhibit unc-9–based gap junctions with dominant-negative unc-1 transgenes. unc-1(dn) alters a stomatin-like protein that regulates unc-9 electrical signaling; its disruptive effects can be rescued by a constitutively active UNC-9::GFP protein, demonstrating specificity. Expression of unc-1(dn) in RMG hub neurons, ADL or ASK pheromone-sensing neurons, or URX oxygen-sensing neurons disrupts specific elements of aggregation-related behaviors. In ADL, unc-1(dn) has effects opposite to those of tetanus toxin light chain, separating the roles of ADL electrical and chemical synapses. These results reveal roles of gap junctions in a complex behavior at cellular resolution and provide a tool for similar exploration of other gap junction circuits. PMID:28143932

Stress granules at the intersection of autophagy and ALS.

PubMed

Monahan, Zachary; Shewmaker, Frank; Pandey, Udai Bhan

2016-10-15

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease caused by loss of upper and lower motor neurons. The majority of ALS cases are classified as sporadic (80-90%), with the remaining considered familial based on patient history. The last decade has seen a surge in the identification of ALS-causing genes - including TARDBP (TDP-43), FUS, MATR3 (Matrin-3), C9ORF72 and several others - providing important insights into the molecular pathways involved in pathogenesis. Most of the protein products of ALS-linked genes fall into two functional categories: RNA-binding/homeostasis and protein-quality control (i.e. autophagy and proteasome). The RNA-binding proteins tend to be aggregation-prone with low-complexity domains similar to the prion-forming domains of yeast. Many also incorporate into stress granules (SGs), which are cytoplasmic ribonucleoprotein complexes that form in response to cellular stress. Mutant forms of TDP-43 and FUS perturb SG dynamics, lengthening their cytoplasmic persistence. Recent evidence suggests that SGs are regulated by the autophagy pathway, suggesting a unifying connection between many of the ALS-linked genes. Persistent SGs may give rise to intractable aggregates that disrupt neuronal homeostasis, thus failure to clear SGs by autophagic processes may promote ALS pathogenesis. This article is part of a Special Issue entitled SI:Autophagy. Copyright © 2016 Elsevier B.V. All rights reserved.

CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers

PubMed Central

Thakur, Jitendra; Henikoff, Steven

2016-01-01

Nucleosomes containing the CenH3 (CENPA or CENP-A) histone variant replace H3 nucleosomes at centromeres to provide a foundation for kinetochore assembly. CENPA nucleosomes are part of the constitutive centromere associated network (CCAN) that forms the inner kinetochore on which outer kinetochore proteins assemble. Two components of the CCAN, CENPC and the histone-fold protein CENPT, provide independent connections from the ∼171-bp centromeric α-satellite repeat units to the outer kinetochore. However, the spatial relationship between CENPA nucleosomes and these two branches remains unclear. To address this issue, we use a base-pair resolution genomic readout of protein–protein interactions, comparative chromatin immunoprecipitation (ChIP) with sequencing, together with sequential ChIP, to infer the in vivo molecular architecture of the human CCAN. In contrast to the currently accepted model in which CENPT associates with H3 nucleosomes, we find that CENPT is centered over the CENPB box between two well-positioned CENPA nucleosomes on the most abundant centromeric young α-satellite dimers and interacts with the CENPB/CENPC complex. Upon cross-linking, the entire CENPA/CENPB/CENPC/CENPT complex is nuclease-protected over an α-satellite dimer that comprises the fundamental unit of centromeric chromatin. We conclude that CENPA/CENPC and CENPT pathways for kinetochore assembly are physically integrated over young α-satellite dimers. PMID:27384170

Structure of a force-conveying cadherin bond essential for inner-ear mechanotransduction.

PubMed

Sotomayor, Marcos; Weihofen, Wilhelm A; Gaudet, Rachelle; Corey, David P

2012-12-06

Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals. Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links, fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4, 5). These two proteins are involved in inherited deafness and feature long extracellular domains that interact tip-to-tip in a Ca(2+)-dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15-cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca(2+) removal owing to increased flexure of Ca(2+)-free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis, neural connectivity and cancer.

Hydropathic self-organized criticality: a magic wand for protein physics.

PubMed

Phillips, J C

2012-10-01

Self-organized criticality (SOC) is a popular concept that has been the subject of more than 3000 articles in the last 25 years. The characteristic signature of SOC is the appearance of self-similarity (power-law scaling) in observable properties. A characteristic observable protein property that describes protein-water interactions is the water-accessible (hydropathic) interfacial area of compacted globular protein networks. Here we show that hydropathic power-law (size- or length-scale-dependent) exponents derived from SOC enable theory to connect standard Web-based (BLAST) short-range amino acid (aa) sequence similarities to long-range aa sequence hydropathic roughening form factors that hierarchically describe evolutionary trends in water - membrane protein interactions. Our method utilizes hydropathic aa exponents that define a non-Euclidean metric realistically rooted in the atomic coordinates of 5526 protein segments. These hydropathic aa exponents thereby encapsulate universal (but previously only implicit) non-Euclidean long-range differential geometrical features of the Protein Data Bank. These hydropathic aa exponents easily organize small mutated aa sequence differences between human and proximate species proteins. For rhodopsin, the most studied transmembrane signaling protein associated with night vision, analysis shows that this approach separates Euclidean short- and non-Euclidean long-range aa sequence properties, and shows that they correlate with 96% success for humans, monkeys, cats, mice and rabbits. Proper application of SOC using hydropathic aa exponents promises unprecedented simplifications of exponentially complex protein sequence-structure-function problems, both conceptual and practical.

Food web complexity and stability across habitat connectivity gradients.

PubMed

LeCraw, Robin M; Kratina, Pavel; Srivastava, Diane S

2014-12-01

The effects of habitat connectivity on food webs have been studied both empirically and theoretically, yet the question of whether empirical results support theoretical predictions for any food web metric other than species richness has received little attention. Our synthesis brings together theory and empirical evidence for how habitat connectivity affects both food web stability and complexity. Food web stability is often predicted to be greatest at intermediate levels of connectivity, representing a compromise between the stabilizing effects of dispersal via rescue effects and prey switching, and the destabilizing effects of dispersal via regional synchronization of population dynamics. Empirical studies of food web stability generally support both this pattern and underlying mechanisms. Food chain length has been predicted to have both increasing and unimodal relationships with connectivity as a result of predators being constrained by the patch occupancy of their prey. Although both patterns have been documented empirically, the underlying mechanisms may differ from those predicted by models. In terms of other measures of food web complexity, habitat connectivity has been empirically found to generally increase link density but either reduce or have no effect on connectance, whereas a unimodal relationship is expected. In general, there is growing concordance between empirical patterns and theoretical predictions for some effects of habitat connectivity on food webs, but many predictions remain to be tested over a full connectivity gradient, and empirical metrics of complexity are rarely modeled. Closing these gaps will allow a deeper understanding of how natural and anthropogenic changes in connectivity can affect real food webs.

Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

PubMed

Farach-Carson, Mary C; Warren, Curtis R; Harrington, Daniel A; Carson, Daniel D

2014-02-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. © 2013.

Border Patrol: Insights into the Unique Role of Perlecan/Heparan Sulfate Proteoglycan2 at Cell and Tissue Borders

PubMed Central

Farach-Carson, Mary C.; Warren, Curtis R.; Harrington, Daniel A.; Carson, Daniel D.

2013-01-01

The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550M years) extracellular matrix molecules. In vertebrates, perlecan’s five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously suggested, to that of a critical agent for establishing and patrolling tissue borders in complex tissues in metazoans. We also propose that understanding these unique functions of the individual portions of the perlecan molecule can provide new insights and tools for engineering of complex multi-layered tissues including providing the necessary cues for establishing neotissue borders. PMID:24001398

Soft Computing Methods for Disulfide Connectivity Prediction.

PubMed

Márquez-Chamorro, Alfonso E; Aguilar-Ruiz, Jesús S

2015-01-01

The problem of protein structure prediction (PSP) is one of the main challenges in structural bioinformatics. To tackle this problem, PSP can be divided into several subproblems. One of these subproblems is the prediction of disulfide bonds. The disulfide connectivity prediction problem consists in identifying which nonadjacent cysteines would be cross-linked from all possible candidates. Determining the disulfide bond connectivity between the cysteines of a protein is desirable as a previous step of the 3D PSP, as the protein conformational search space is highly reduced. The most representative soft computing approaches for the disulfide bonds connectivity prediction problem of the last decade are summarized in this paper. Certain aspects, such as the different methodologies based on soft computing approaches (artificial neural network or support vector machine) or features of the algorithms, are used for the classification of these methods.

Signalling networks and dynamics of allosteric transitions in bacterial chaperonin GroEL: implications for iterative annealing of misfolded proteins.

PubMed

Thirumalai, D; Hyeon, Changbong

2018-06-19

Signal transmission at the molecular level in many biological complexes occurs through allosteric transitions. Allostery describes the responses of a complex to binding of ligands at sites that are spatially well separated from the binding region. We describe the structural perturbation method, based on phonon propagation in solids, which can be used to determine the signal-transmitting allostery wiring diagram (AWD) in large but finite-sized biological complexes. Application to the bacterial chaperonin GroEL-GroES complex shows that the AWD determined from structures also drives the allosteric transitions dynamically. From both a structural and dynamical perspective these transitions are largely determined by formation and rupture of salt-bridges. The molecular description of allostery in GroEL provides insights into its function, which is quantitatively described by the iterative annealing mechanism. Remarkably, in this complex molecular machine, a deep connection is established between the structures, reaction cycle during which GroEL undergoes a sequence of allosteric transitions, and function, in a self-consistent manner.This article is part of a discussion meeting issue 'Allostery and molecular machines'. © 2018 The Author(s).

Simulating evolution of protein complexes through gene duplication and co-option.

PubMed

Haarsma, Loren; Nelesen, Serita; VanAndel, Ethan; Lamine, James; VandeHaar, Peter

2016-06-21

We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2-5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome

PubMed Central

Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Ritelli, Marco

2018-01-01

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues. PMID:29346445

Transcriptome analysis of skin fibroblasts with dominant negative COL3A1 mutations provides molecular insights into the etiopathology of vascular Ehlers-Danlos syndrome.

PubMed

Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Ritelli, Marco; Colombi, Marina

2018-01-01

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues.

Lateral assembly of the immunoglobulin protein SynCAM 1 controls its adhesive function and instructs synapse formation.

PubMed

Fogel, Adam I; Stagi, Massimiliano; Perez de Arce, Karen; Biederer, Thomas

2011-09-16

Synapses are specialized adhesion sites between neurons that are connected by protein complexes spanning the synaptic cleft. These trans-synaptic interactions can organize synapse formation, but their macromolecular properties and effects on synaptic morphology remain incompletely understood. Here, we demonstrate that the synaptic cell adhesion molecule SynCAM 1 self-assembles laterally via its extracellular, membrane-proximal immunoglobulin (Ig) domains 2 and 3. This cis oligomerization generates SynCAM oligomers with increased adhesive capacity and instructs the interactions of this molecule across the nascent and mature synaptic cleft. In immature neurons, cis assembly promotes the adhesive clustering of SynCAM 1 at new axo-dendritic contacts. Interfering with the lateral self-assembly of SynCAM 1 in differentiating neurons strongly impairs its synaptogenic activity. At later stages, the lateral oligomerization of SynCAM 1 restricts synaptic size, indicating that this adhesion molecule contributes to the structural organization of synapses. These results support that lateral interactions assemble SynCAM complexes within the synaptic cleft to promote synapse induction and modulate their structure. These findings provide novel insights into synapse development and the adhesive mechanisms of Ig superfamily members.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

PubMed

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria

PubMed Central

Jans, Daniel C.; Wurm, Christian A.; Riedel, Dietmar; Wenzel, Dirk; Stagge, Franziska; Deckers, Markus; Rehling, Peter; Jakobs, Stefan

2013-01-01

The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle. PMID:23676277

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

PubMed

Choe, Jin; Sasaki, Yoko; Zhou, Liming; Takai, Hideki; Nakayama, Yohei; Ogata, Yorimasa

2016-09-01

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Reducing eIF4E-eIF4G interactions restores the balance between protein synthesis and actin dynamics in fragile X syndrome model mice.

PubMed

Santini, Emanuela; Huynh, Thu N; Longo, Francesco; Koo, So Yeon; Mojica, Edward; D'Andrea, Laura; Bagni, Claudia; Klann, Eric

2017-11-07

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism spectrum disorder. FXS is caused by silencing of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP), an mRNA-binding protein that represses the translation of its target mRNAs. One mechanism by which FMRP represses translation is through its association with cytoplasmic FMRP-interacting protein 1 (CYFIP1), which subsequently sequesters and inhibits eukaryotic initiation factor 4E (eIF4E). CYFIP1 shuttles between the FMRP-eIF4E complex and the Rac1-Wave regulatory complex, thereby connecting translational regulation to actin dynamics and dendritic spine morphology, which are dysregulated in FXS model mice that lack FMRP. Treating FXS mice with 4EGI-1, which blocks interactions between eIF4E and eIF4G, a critical interaction partner for translational initiation, reversed defects in hippocampus-dependent memory and spine morphology. We also found that 4EGI-1 normalized the phenotypes of enhanced metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD), enhanced Rac1-p21-activated kinase (PAK)-cofilin signaling, altered actin dynamics, and dysregulated CYFIP1/eIF4E and CYFIP1/Rac1 interactions in FXS mice. Our findings are consistent with the idea that an imbalance in protein synthesis and actin dynamics contributes to pathophysiology in FXS mice, and suggest that targeting eIF4E may be a strategy for treating FXS. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Immunization of complex networks

NASA Astrophysics Data System (ADS)

Pastor-Satorras, Romualdo; Vespignani, Alessandro

2002-03-01

Complex networks such as the sexual partnership web or the Internet often show a high degree of redundancy and heterogeneity in their connectivity properties. This peculiar connectivity provides an ideal environment for the spreading of infective agents. Here we show that the random uniform immunization of individuals does not lead to the eradication of infections in all complex networks. Namely, networks with scale-free properties do not acquire global immunity from major epidemic outbreaks even in the presence of unrealistically high densities of randomly immunized individuals. The absence of any critical immunization threshold is due to the unbounded connectivity fluctuations of scale-free networks. Successful immunization strategies can be developed only by taking into account the inhomogeneous connectivity properties of scale-free networks. In particular, targeted immunization schemes, based on the nodes' connectivity hierarchy, sharply lower the network's vulnerability to epidemic attacks.

Clique-based data mining for related genes in a biomedical database.

PubMed

Matsunaga, Tsutomu; Yonemori, Chikara; Tomita, Etsuji; Muramatsu, Masaaki

2009-07-01

Progress in the life sciences cannot be made without integrating biomedical knowledge on numerous genes in order to help formulate hypotheses on the genetic mechanisms behind various biological phenomena, including diseases. There is thus a strong need for a way to automatically and comprehensively search from biomedical databases for related genes, such as genes in the same families and genes encoding components of the same pathways. Here we address the extraction of related genes by searching for densely-connected subgraphs, which are modeled as cliques, in a biomedical relational graph. We constructed a graph whose nodes were gene or disease pages, and edges were the hyperlink connections between those pages in the Online Mendelian Inheritance in Man (OMIM) database. We obtained over 20,000 sets of related genes (called 'gene modules') by enumerating cliques computationally. The modules included genes in the same family, genes for proteins that form a complex, and genes for components of the same signaling pathway. The results of experiments using 'metabolic syndrome'-related gene modules show that the gene modules can be used to get a coherent holistic picture helpful for interpreting relations among genes. We presented a data mining approach extracting related genes by enumerating cliques. The extracted gene sets provide a holistic picture useful for comprehending complex disease mechanisms.

INTERDISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Synchronization in Complex Networks with Multiple Connections

NASA Astrophysics Data System (ADS)

Wu, Qing-Chu; Fu, Xin-Chu; Sun, Wei-Gang

2010-01-01

In this paper a class of networks with multiple connections are discussed. The multiple connections include two different types of links between nodes in complex networks. For this new model, we give a simple generating procedure. Furthermore, we investigate dynamical synchronization behavior in a delayed two-layer network, giving corresponding theoretical analysis and numerical examples.

Connective tissue growth factor is a substrate of ADAM28

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

Mochizuki, Satsuki; Tanaka, Rena; Shimoda, Masayuki

2010-11-26

Research highlights: {yields} The hyper-variable region in the cysteine-rich domain of ADAM28 binds to C-terminal domain of CTGF. {yields} ADAM28 cleaves CTGF alone and CTGF in the CTGF/VEGF{sub 165} complex. {yields} CTGF digestion by ADAM28 releases biologically active VEGF{sub 165} from the complex. {yields} ADAM28, CTGF and VEGF{sub 165} are commonly co-expressed by carcinoma cells in human breast carcinoma tissues. {yields} These suggest that ADAM28 promotes VEGF{sub 165}-induced angiogenesis in the breast carcinomas by selective CTGF digestion in the CTGF/VEGF{sub 165} complex. -- Abstract: ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinomamore » cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala{sup 181}-Tyr{sup 182} and Asp{sup 191}-Pro{sup 192} bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor{sub 165} (VEGF{sub 165}), releasing biologically active VEGF{sub 165} from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF{sub 165}-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF{sub 165} complex.« less

Identifying Dynamic Protein Complexes Based on Gene Expression Profiles and PPI Networks

PubMed Central

Li, Min; Chen, Weijie; Wang, Jianxin; Pan, Yi

2014-01-01

Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of “closeness” and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

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

NASA Astrophysics Data System (ADS)

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

2017-05-01

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

Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein

PubMed Central

Azevedo, Jacinthe; Garcia, Damien; Pontier, Dominique; Ohnesorge, Stephanie; Yu, Agnes; Garcia, Shahinez; Braun, Laurence; Bergdoll, Marc; Hakimi, Mohamed Ali; Lagrange, Thierry; Voinnet, Olivier

2010-01-01

In plants and invertebrates, viral-derived siRNAs processed by the RNaseIII Dicer guide Argonaute (AGO) proteins as part of antiviral RNA-induced silencing complexes (RISC). As a counterdefense, viruses produce suppressor proteins (VSRs) that inhibit the host silencing machinery, but their mechanisms of action and cellular targets remain largely unknown. Here, we show that the Turnip crinckle virus (TCV) capsid, the P38 protein, acts as a homodimer, or multiples thereof, to mimic host-encoded glycine/tryptophane (GW)-containing proteins normally required for RISC assembly/function in diverse organisms. The P38 GW residues bind directly and specifically to Arabidopsis AGO1, which, in addition to its role in endogenous microRNA-mediated silencing, is identified as a major effector of TCV-derived siRNAs. Point mutations in the P38 GW residues are sufficient to abolish TCV virulence, which is restored in Arabidopsis ago1 hypomorphic mutants, uncovering both physical and genetic interactions between the two proteins. We further show how AGO1 quenching by P38 profoundly impacts the cellular availability of the four Arabidopsis Dicers, uncovering an AGO1-dependent, homeostatic network that functionally connects these factors together. The likely widespread occurrence and expected consequences of GW protein mimicry on host silencing pathways are discussed in the context of innate and adaptive immunity in plants and metazoans. PMID:20439431

Regulation of the basement membrane by epithelia generated forces

NASA Astrophysics Data System (ADS)

Tanner, Kandice

2012-12-01

Tumor metastasis involves a progressive loss of tissue architecture and dissolution of structural boundaries between the epithelium and connective tissue. The basement membrane (BM), a specialized network of extracellular matrix proteins forms a barrier that physically restricts pre-invasive lesions such that they remain as local insults. The BM is not a static structure, but one that is constantly regenerated and remodeled in the adult organism. Matrix organization also regulates cell function. Thus alterations in the balance of synthesis, remodeling and proteolytic degradation of the extracellular matrix proteins may contribute to a loss of structural integrity. However, the de novo assembly and maintenance of the complex structural properties of in vivo basement membranes remain elusive. Here, this paper highlights the current understanding on the structural properties and the establishment of the BM, and discusses the potential role of self-generated forces in adult tissue remodeling and the maintenance of the BM as a malignancy suppressor.

Calcium signaling and cell proliferation.

PubMed

Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

2015-11-01

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

PubMed Central

Borel, Christelle; Mudge, Jonathan M.; Howald, Cédric; Foissac, Sylvain; Ucla, Catherine; Chrast, Jacqueline; Ribeca, Paolo; Martin, David; Murray, Ryan R.; Yang, Xinping; Ghamsari, Lila; Lin, Chenwei; Bell, Ian; Dumais, Erica; Drenkow, Jorg; Tress, Michael L.; Gelpí, Josep Lluís; Orozco, Modesto; Valencia, Alfonso; van Berkum, Nynke L.; Lajoie, Bryan R.; Vidal, Marc; Stamatoyannopoulos, John; Batut, Philippe; Dobin, Alex; Harrow, Jennifer; Hubbard, Tim; Dekker, Job; Frankish, Adam; Salehi-Ashtiani, Kourosh; Reymond, Alexandre; Antonarakis, Stylianos E.; Guigó, Roderic; Gingeras, Thomas R.

2012-01-01

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network. PMID:22238572

Buckling Instabilities and Complex Dynamics in a Model of Uniflagellar Bacterial Locomotion

NASA Astrophysics Data System (ADS)

Nguyen, Frank; Graham, Michael

2015-11-01

Locomotion of microorganisms at low Reynolds number is a long studied problem. Of particular interest are organisms using a single flagellum to undergo a wide range of motions: pushing, pulling, and tumbling or flicking. Recent experiments have connected the stability of the hook protein, connecting cell motor and flagellum, to deviations from typical straight swimming trajectories. We seek physical explanations to these phenomena by developing a computationally inexpensive, rigid-body dynamic model of a uniflagellated organism with a flexible hook connection that captures the fundamental dynamics, kinematics, and configurations. Furthermore, the model addresses the effects of hook loading and geometry on the stability of the system. Simulations with low hook flexibility produce the classic straight trajectory, but a large flexibility produces helical trajectories, leading to directional changes when coupled with transient hook stiffening. Minima for critical flexibilities are found in certain subsets of parameter space, implying preferred geometries for certain swimming dynamics. The model verifies proposed mechanisms for swimming in various modes and highlights the role of flexibility in the biology of real organisms and the engineering of artificial microswimmers. This work was supported by NSF grant PHY-1304942.

Effects of disulfide bridges and backbone connectivity on water sorption by protein matrices.

PubMed

Kim, Sang Beom; Singh, Rakesh S; Paul, Prem K C; Debenedetti, Pablo G

2017-08-11

Understanding the water sorption behavior of protein powders is important in applications such as the preservation of protein-based pharmaceuticals. Most globular proteins exhibit a characteristic sigmoidal water adsorption isotherm at ambient conditions. However, it is not well understood how water sorption behavior is influenced by intrinsic factors that are related to structural properties of proteins. We investigate computationally how structural constraints on proteins influence the water sorption isotherms of amorphous protein powders. Specifically, we study the effects of non-local disulfide linkages and backbone connectivity using pheromone ER-23 and lysozyme as model proteins. We find that non-local disulfide linkages can significantly restrict structural changes during hydration and dehydration, and this in turn greatly reduces the extent of hysteresis between the adsorption and desorption branches. Upon removing the backbone connectivity by breaking all peptide bonds in lysozyme, we find that the hysteresis shifts towards the lower humidity regime, and the water uptake capacity is significantly enhanced. We attribute these changes to the higher aggregation propensity of the constraint-free amino acids in dehydrated condition, and the formation of a spanning water network at high hydration levels.

Connecting genomic alterations to cancer biology with proteomics: the NCI Clinical Proteomic Tumor Analysis Consortium.

PubMed

Ellis, Matthew J; Gillette, Michael; Carr, Steven A; Paulovich, Amanda G; Smith, Richard D; Rodland, Karin K; Townsend, R Reid; Kinsinger, Christopher; Mesri, Mehdi; Rodriguez, Henry; Liebler, Daniel C

2013-10-01

The National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium is applying the latest generation of proteomic technologies to genomically annotated tumors from The Cancer Genome Atlas (TCGA) program, a joint initiative of the NCI and the National Human Genome Research Institute. By providing a fully integrated accounting of DNA, RNA, and protein abnormalities in individual tumors, these datasets will illuminate the complex relationship between genomic abnormalities and cancer phenotypes, thus producing biologic insights as well as a wave of novel candidate biomarkers and therapeutic targets amenable to verification using targeted mass spectrometry methods. ©2013 AACR.

ChloroKB: A Web Application for the Integration of Knowledge Related to Chloroplast Metabolic Network1[OPEN

PubMed Central

Gloaguen, Pauline; Alban, Claude; Ravanel, Stéphane; Seigneurin-Berny, Daphné; Matringe, Michel; Ferro, Myriam; Bruley, Christophe; Rolland, Norbert; Vandenbrouck, Yves

2017-01-01

Higher plants, as autotrophic organisms, are effective sources of molecules. They hold great promise for metabolic engineering, but the behavior of plant metabolism at the network level is still incompletely described. Although structural models (stoichiometry matrices) and pathway databases are extremely useful, they cannot describe the complexity of the metabolic context, and new tools are required to visually represent integrated biocurated knowledge for use by both humans and computers. Here, we describe ChloroKB, a Web application (http://chlorokb.fr/) for visual exploration and analysis of the Arabidopsis (Arabidopsis thaliana) metabolic network in the chloroplast and related cellular pathways. The network was manually reconstructed through extensive biocuration to provide transparent traceability of experimental data. Proteins and metabolites were placed in their biological context (spatial distribution within cells, connectivity in the network, participation in supramolecular complexes, and regulatory interactions) using CellDesigner software. The network contains 1,147 reviewed proteins (559 localized exclusively in plastids, 68 in at least one additional compartment, and 520 outside the plastid), 122 proteins awaiting biochemical/genetic characterization, and 228 proteins for which genes have not yet been identified. The visual presentation is intuitive and browsing is fluid, providing instant access to the graphical representation of integrated processes and to a wealth of refined qualitative and quantitative data. ChloroKB will be a significant support for structural and quantitative kinetic modeling, for biological reasoning, when comparing novel data with established knowledge, for computer analyses, and for educational purposes. ChloroKB will be enhanced by continuous updates following contributions from plant researchers. PMID:28442501

Prediction of Heterodimeric Protein Complexes from Weighted Protein-Protein Interaction Networks Using Novel Features and Kernel Functions

PubMed Central

Ruan, Peiying; Hayashida, Morihiro; Maruyama, Osamu; Akutsu, Tatsuya

2013-01-01

Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes. PMID:23776458

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

The connection Between Plasma Protein Binding and Acute Toxicity as Determined by the LD50 Value.

PubMed

Svennebring, Andreas

2016-02-01

Preclinical Research A dataset of three drug classes (acids, bases, and neutrals) with LD50 values in mice was analysed to investigate a possible connection between high plasma protein binding and acute toxicity. Initially, it was found that high plasma protein binding was associated with toxicity for acids and neutrals, but after compensating for differences in lipophilicity, plasma protein binding was found not to be associated with toxicity. The therapeutic index established by the quotient between mouse LD50 and the defined daily dose was unaffected by both lipophilicity and plasma protein binding. © 2015 Wiley Periodicals, Inc.

Surface Sites for Engineering Allosteric Control in Proteins

PubMed Central

Lee, Jeeyeon; Natarajan, Madhusudan; Nashine, Vishal C.; Socolich, Michael; Vo, Tina; Russ, William P.; Benkovic, Stephen J.; Ranganathan, Rama

2010-01-01

Statistical analyses of protein families reveal networks of coevolving amino acids that functionally link distantly positioned functional surfaces. Such linkages suggest a concept for engineering allosteric control into proteins: The intramolecular networks of two proteins could be joined across their surface sites such that the activity of one protein might control the activity of the other. We tested this idea by creating PAS-DHFR, a designed chimeric protein that connects a light-sensing signaling domain from a plant member of the Per/Arnt/Sim (PAS) family of proteins with Escherichia coli dihydrofolate reductase (DHFR). With no optimization, PAS-DHFR exhibited light-dependent catalytic activity that depended on the site of connection and on known signaling mechanisms in both proteins. PAS-DHFR serves as a proof of concept for engineering regulatory activities into proteins through interface design at conserved allosteric sites. PMID:18927392

Silica-gelatin hybrid sol-gel coatings: a proteomic study with biocompatibility implications.

PubMed

Araújo-Gomes, N; Romero-Gavilán, F; Lara-Sáez, I; Elortza, F; Azkargorta, M; Iloro, I; Martínez-Ibañez, M; Martín de Llano, J J; Gurruchaga, M; Goñi, I; Suay, J; Sánchez-Pérez, A M

2018-05-21

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin, were applied as coatings on sandblasted, acid-etched titanium (SAE-Ti) substrates and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings. This article is protected by copyright. All rights reserved.

Interfacing cellular networks of S. cerevisiae and E. coli: Connecting dynamic and genetic information

PubMed Central

2013-01-01

Background In recent years, various types of cellular networks have penetrated biology and are nowadays used omnipresently for studying eukaryote and prokaryote organisms. Still, the relation and the biological overlap among phenomenological and inferential gene networks, e.g., between the protein interaction network and the gene regulatory network inferred from large-scale transcriptomic data, is largely unexplored. Results We provide in this study an in-depth analysis of the structural, functional and chromosomal relationship between a protein-protein network, a transcriptional regulatory network and an inferred gene regulatory network, for S. cerevisiae and E. coli. Further, we study global and local aspects of these networks and their biological information overlap by comparing, e.g., the functional co-occurrence of Gene Ontology terms by exploiting the available interaction structure among the genes. Conclusions Although the individual networks represent different levels of cellular interactions with global structural and functional dissimilarities, we observe crucial functions of their network interfaces for the assembly of protein complexes, proteolysis, transcription, translation, metabolic and regulatory interactions. Overall, our results shed light on the integrability of these networks and their interfacing biological processes. PMID:23663484

Molecular Interaction Map of the Mammalian Cell Cycle Control and DNA Repair Systems

PubMed Central

Kohn, Kurt W.

1999-01-01

Eventually to understand the integrated function of the cell cycle regulatory network, we must organize the known interactions in the form of a diagram, map, and/or database. A diagram convention was designed capable of unambiguous representation of networks containing multiprotein complexes, protein modifications, and enzymes that are substrates of other enzymes. To facilitate linkage to a database, each molecular species is symbolically represented only once in each diagram. Molecular species can be located on the map by means of indexed grid coordinates. Each interaction is referenced to an annotation list where pertinent information and references can be found. Parts of the network are grouped into functional subsystems. The map shows how multiprotein complexes could assemble and function at gene promoter sites and at sites of DNA damage. It also portrays the richness of connections between the p53-Mdm2 subsystem and other parts of the network. PMID:10436023

Knowledge Discovery in Spectral Data by Means of Complex Networks

PubMed Central

Zanin, Massimiliano; Papo, David; Solís, José Luis González; Espinosa, Juan Carlos Martínez; Frausto-Reyes, Claudio; Anda, Pascual Palomares; Sevilla-Escoboza, Ricardo; Boccaletti, Stefano; Menasalvas, Ernestina; Sousa, Pedro

2013-01-01

In the last decade, complex networks have widely been applied to the study of many natural and man-made systems, and to the extraction of meaningful information from the interaction structures created by genes and proteins. Nevertheless, less attention has been devoted to metabonomics, due to the lack of a natural network representation of spectral data. Here we define a technique for reconstructing networks from spectral data sets, where nodes represent spectral bins, and pairs of them are connected when their intensities follow a pattern associated with a disease. The structural analysis of the resulting network can then be used to feed standard data-mining algorithms, for instance for the classification of new (unlabeled) subjects. Furthermore, we show how the structure of the network is resilient to the presence of external additive noise, and how it can be used to extract relevant knowledge about the development of the disease. PMID:24957895

Knowledge discovery in spectral data by means of complex networks.

PubMed

Zanin, Massimiliano; Papo, David; Solís, José Luis González; Espinosa, Juan Carlos Martínez; Frausto-Reyes, Claudio; Anda, Pascual Palomares; Sevilla-Escoboza, Ricardo; Jaimes-Reategui, Rider; Boccaletti, Stefano; Menasalvas, Ernestina; Sousa, Pedro

2013-03-11

In the last decade, complex networks have widely been applied to the study of many natural and man-made systems, and to the extraction of meaningful information from the interaction structures created by genes and proteins. Nevertheless, less attention has been devoted to metabonomics, due to the lack of a natural network representation of spectral data. Here we define a technique for reconstructing networks from spectral data sets, where nodes represent spectral bins, and pairs of them are connected when their intensities follow a pattern associated with a disease. The structural analysis of the resulting network can then be used to feed standard data-mining algorithms, for instance for the classification of new (unlabeled) subjects. Furthermore, we show how the structure of the network is resilient to the presence of external additive noise, and how it can be used to extract relevant knowledge about the development of the disease.

Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

An autoimmune myositis-overlap syndrome associated with autoantibodies to nuclear pore complexes: description and long-term follow-up of the anti-Nup syndrome.

PubMed

Senécal, Jean-Luc; Isabelle, Catherine; Fritzler, Marvin J; Targoff, Ira N; Goldstein, Rose; Gagné, Michel; Raynauld, Jean-Pierre; Joyal, France; Troyanov, Yves; Dabauvalle, Marie-Christine

2014-11-01

Autoimmune myositis encompasses various myositis-overlap syndromes, each being identified by the presence of serum marker autoantibodies. We describe a novel myositis-overlap syndrome in 4 patients characterized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes. The clinical phenotype was characterized by prominent myositis in association with erosive, anti-CCP, and rheumatoid factor-positive arthritis, trigeminal neuralgia, mild interstitial lung disease, Raynaud phenomenon, and weight loss. The myositis was typically chronic, relapsing, and refractory to corticosteroids alone, but remitted with the addition of a second immunomodulating drug. There was no clinical or laboratory evidence for liver disease. The prognosis was good with 100% long-term survival (mean follow-up 19.5 yr).By indirect immunofluorescence on HEp-2 cells, sera from all 4 patients displayed a high titer of antinuclear autoantibodies (ANA) with a distinct punctate peripheral (rim) fluorescent pattern of the nuclear envelope characteristic of nuclear pore complexes. Reactivity with nuclear pore complexes was confirmed by immunoelectron microscopy. In a cohort of 100 French Canadian patients with autoimmune myositis, the nuclear pore complex fluorescent ANA pattern was restricted to these 4 patients (4%). It was not observed in sera from 393 adult patients with systemic sclerosis (n = 112), mixed connective tissue disease (n = 35), systemic lupus (n = 94), rheumatoid arthritis (n = 45), or other rheumatic diseases (n = 107), nor was it observed in 62 normal adults.Autoantibodies to nuclear pore complexes were predominantly of IgG isotype. No other IgG autoantibody markers for defined connective tissue diseases or overlap syndromes were present, indicating a selective and highly focused immune response. In 3 patients, anti-nuclear pore complex autoantibody titers varied in parallel with myositis activity, suggesting a pathogenic link to pathophysiology. The nuclear pore complex proteins, that is, nucleoporins (nup), recognized by these sera were heterogeneous and included Nup358/RanBP2 (n = 2 patients), Nup90 (n = 1), Nup62 (n = 1), and gp210 (n = 1). Taken together the data suggest that nup autoantigens themselves drive the anti-nup autoimmune response. Immunogenetically, the 4 patients shared the DQA1*0501 allele associated with an increased risk for autoimmune myositis.In conclusion, we report an apparent novel subset of autoimmune myositis in our population of French Canadian patients with connective tissue diseases. This syndrome is recognized by the presence of a unique immunologic marker, autoantibodies to nuclear pore complexes that react with nups, consistent with an "anti-nup syndrome."

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

Ratto, T V; Rudd, R E; Langry, K C

We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, theymore » do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.« less

Endoplasmic reticulum-plasma membrane junctions: structure, function and dynamics.

PubMed

Okeke, Emmanuel; Dingsdale, Hayley; Parker, Tony; Voronina, Svetlana; Tepikin, Alexei V

2016-06-01

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are contact sites between the ER and the PM; the distance between the two organelles in the junctions is below 40 nm and the membranes are connected by protein tethers. A number of molecular tools and technical approaches have been recently developed to visualise, modify and characterise properties of ER-PM junctions. The junctions serve as the platforms for lipid exchange between the organelles and for cell signalling, notably Ca(2+) and cAMP signalling. Vice versa, signalling events regulate the development and properties of the junctions. Two Ca(2+) -dependent mechanisms of de novo formation of ER-PM junctions have been recently described and characterised. The junction-forming proteins and lipids are currently the focus of vigorous investigation. Junctions can be relatively short-lived and simple structures, forming and dissolving on the time scale of a few minutes. However, complex, sophisticated and multifunctional ER-PM junctions, capable of attracting numerous protein residents and other cellular organelles, have been described in some cell types. The road from simplicity to complexity, i.e. the transformation from simple 'nascent' ER-PM junctions to advanced stable multiorganellar complexes, is likely to become an attractive research avenue for current and future junctologists. Another area of considerable research interest is the downstream cellular processes that can be activated by specific local signalling events in the ER-PM junctions. Studies of the cell physiology and indeed pathophysiology of ER-PM junctions have already produced some surprising discoveries, likely to expand with advances in our understanding of these remarkable organellar contact sites. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Earthquake Complex Network applied along the Chilean Subduction Zone.

NASA Astrophysics Data System (ADS)

Martin, F.; Pasten, D.; Comte, D.

2017-12-01

In recent years the earthquake complex networks have been used as a useful tool to describe and characterize the behavior of seismicity. The earthquake complex network is built in space, dividing the three dimensional space in cubic cells. If the cubic cell contains a hypocenter, we call this cell like a node. The connections between nodes follows the time sequence of the occurrence of the seismic events. In this sense, we have a spatio-temporal configuration of a specific region using the seismicity in that zone. In this work, we are applying complex networks to characterize the subduction zone along the coast of Chile using two networks: a directed and an undirected network. The directed network takes in consideration the time-direction of the connections, that is very important for the connectivity of the network: we are considering the connectivity, ki of the i-th node, like the number of connections going out from the node i and we add the self-connections (if two seismic events occurred successive in time in the same cubic cell, we have a self-connection). The undirected network is the result of remove the direction of the connections and the self-connections from the directed network. These two networks were building using seismic data events recorded by CSN (Chilean Seismological Center) in Chile. This analysis includes the last largest earthquakes occurred in Iquique (April 2014) and in Illapel (September 2015). The result for the directed network shows a change in the value of the critical exponent along the Chilean coast. The result for the undirected network shows a small-world behavior without important changes in the topology of the network. Therefore, the complex network analysis shows a new form to characterize the Chilean subduction zone with a simple method that could be compared with another methods to obtain more details about the behavior of the seismicity in this region.

Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding.

PubMed

Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

2015-01-01

Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

Structure, function and dynamics in adenovirus maturation

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

Mangel, Walter F.; San Martín, Carmen

2014-11-21

Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.

PubMed

Oliveira, Marco A S; Baura, Valter A; Aquino, Bruno; Huergo, Luciano F; Kadowaki, Marco A S; Chubatsu, Leda S; Souza, Emanuel M; Dixon, Ray; Pedrosa, Fábio O; Wassem, Roseli; Monteiro, Rose A

2009-01-01

Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.

Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy.

PubMed

Mancias, Joseph D; Wang, Xiaoxu; Gygi, Steven P; Harper, J Wade; Kimmelman, Alec C

2014-05-01

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.

THE LOCALIZATION OF HOMOLGOUS PLASMA PROTEINS IN THE TISSUES OF YOUNG HUMAN BEINGS AS DEMONSTRATED WITH FLUORESCENT ANTIBODIES

PubMed Central

Gitlin, David; Landing, Benjamin H.; Whipple, Ann

1953-01-01

Employing fluorescent antibodies for the detection of homologous plasma proteins in tissue sections, the distribution of plasma albumin, γ-globulin, β-lipoprotein, β1-metal-combining globulin, and fibrinogen has been studied in the tissues of infants and children. Plasma albumin, γ-globulin, and β1-metal-combining globulin were found in many cells and particularly cell nuclei, connective tissues and interstitial spaces, lymphatics, and blood vessels. β-Lipoprotein was found mostly in the nuclei of all cell types while fibrinogen was restricted largely to the lymphatic and vascular channels, connective tissues and the interstitial spaces. The widespread distribution of these plasma proteins in cells and connective tissues indicates the magnitude of the extravascular plasma protein pool which is in equilibrium with circulating plasma. Unfortunately, these results do not permit accurate localization of the sites of production of these plasma proteins, but do give some idea of their intimate relationship to the tissues. PMID:13022871

Toward Bridging the Mechanistic Gap Between Genes and Traits by Emphasizing the Role of Proteins in a Computational Environment

NASA Astrophysics Data System (ADS)

Haskel-Ittah, Michal; Yarden, Anat

2017-12-01

Previous studies have shown that students often ignore molecular mechanisms when describing genetic phenomena. Specifically, students tend to directly link genes to their encoded traits, ignoring the role of proteins as mediators in this process. We tested the ability of 10th grade students to connect genes to traits through proteins, using concept maps and reasoning questions. The context of this study was a computational learning environment developed specifically to foster this ability. This environment presents proteins as the mechanism-mediating genetic phenomena. We found that students' ability to connect genes, proteins, and traits, or to reason using this connection, was initially poor. However, significant improvement was obtained when using the learning environment. Our results suggest that visual representations of proteins' functions in the context of a specific trait contributed to this improvement. One significant aspect of these results is the indication that 10th graders are capable of accurately describing genetic phenomena and their underlying mechanisms, a task that has been shown to raise difficulties, even in higher grades of high school.

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

PubMed

Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

2011-12-09

Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

Effects of elasticity and geometry on the locomotion of a model bacterium

NASA Astrophysics Data System (ADS)

Nguyen, Frank; Graham, Michael

2017-11-01

The locomotion of flagellated bacteria in viscous fluid provides the blueprint for a number of micro-scale engineering applications. The elasticities of both the hook protein (connecting cell body and flagellum) and the flagella themselves play a key role in determining the stability of locomotion. We use a coarse-grained discretization of elastic flagella connected to a rigid cell body to examine trajectories and flow fields for free swimmers. We indeed find that hook and/or flagellar buckling occurs above a critical flexibility relative to the swimmer's torque input. This renders straight swimming ineffective, though not necessarily undesirable in practice. Simulations with multiple flagella show bundling may partially stabilize the buckling effect. For a single flagellum or single bundle, we define a parameter space of characteristic angles tracking the overall time-averaged shape of the swimmer while also delineating stability boundaries between different modes of buckling. Ultimately our results may provide insight on how swimmers move through complex environments and how to design microrobotic swimmers for specific applications. NHS, GERS.

Uniformity of nucleosome preservation pattern in Mammalian sperm and its connection to repetitive DNA elements.

PubMed

Samans, Birgit; Yang, Yang; Krebs, Stefan; Sarode, Gaurav Vilas; Blum, Helmut; Reichenbach, Myriam; Wolf, Eckhard; Steger, Klaus; Dansranjavin, Temuujin; Schagdarsurengin, Undraga

2014-07-14

Nucleosome-to-protamine exchange during mammalian spermiogenesis is essential for compaction and protection of paternal DNA. It is interesting that, depending on the species, 1% to 15% of nucleosomes are retained, but the generalizability and biological function of this retention are unknown. Here, we show concordantly in human and bovine that nucleosomes remained in sperm chromatin predominantly within distal intergenic regions and introns and associated with centromere repeats and retrotransposons (LINE1 and SINEs). In contrast, nucleosome depletion concerned particularly exons, 5'-UTR, 3'-UTR, TSS, and TTS and was associated with simple and low-complexity repeats. Overlap of human and bovine genes exhibiting nucleosome preservation in the promoter and gene body revealed a significant enrichment of signal transduction and RNA- and protein-processing factors. Our study demonstrates the genome-wide uniformity of the nucleosome preservation pattern in mammalian sperm and its connection to repetitive DNA elements and suggests a function in preimplantation processes for paternally derived nucleosomes. Copyright © 2014 Elsevier Inc. All rights reserved.

BinTree Seeking: A Novel Approach to Mine Both Bi-Sparse and Cohesive Modules in Protein Interaction Networks

PubMed Central

Shen, Hong-Bin

2011-01-01

Modern science of networks has brought significant advances to our understanding of complex systems biology. As a representative model of systems biology, Protein Interaction Networks (PINs) are characterized by a remarkable modular structures, reflecting functional associations between their components. Many methods were proposed to capture cohesive modules so that there is a higher density of edges within modules than those across them. Recent studies reveal that cohesively interacting modules of proteins is not a universal organizing principle in PINs, which has opened up new avenues for revisiting functional modules in PINs. In this paper, functional clusters in PINs are found to be able to form unorthodox structures defined as bi-sparse module. In contrast to the traditional cohesive module, the nodes in the bi-sparse module are sparsely connected internally and densely connected with other bi-sparse or cohesive modules. We present a novel protocol called the BinTree Seeking (BTS) for mining both bi-sparse and cohesive modules in PINs based on Edge Density of Module (EDM) and matrix theory. BTS detects modules by depicting links and nodes rather than nodes alone and its derivation procedure is totally performed on adjacency matrix of networks. The number of modules in a PIN can be automatically determined in the proposed BTS approach. BTS is tested on three real PINs and the results demonstrate that functional modules in PINs are not dominantly cohesive but can be sparse. BTS software and the supporting information are available at: www.csbio.sjtu.edu.cn/bioinf/BTS/. PMID:22140454

Propagation of Aß pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies.

PubMed

Eisele, Yvonne S; Duyckaerts, Charles

2016-01-01

In brains of patients with Alzheimer's disease (AD), Aβ peptides accumulate in parenchyma and, almost invariably, also in the vascular walls. Although Aβ aggregation is, by definition, present in AD, its impact is only incompletely understood. It occurs in a stereotypical spatiotemporal distribution within neuronal networks in the course of the disease. This suggests a role for synaptic connections in propagating Aβ pathology, and possibly of axonal transport in an antero- or retrograde way-although, there is also evidence for passive, extracellular diffusion. Striking, in AD, is the conjunction of tau and Aβ pathology. Tau pathology in the cell body of neurons precedes Aβ deposition in their synaptic endings in several circuits such as the entorhino-dentate, cortico-striatal or subiculo-mammillary connections. However, genetic evidence suggests that Aβ accumulation is the first step in AD pathogenesis. To model the complexity and consequences of Aβ aggregation in vivo, various transgenic (tg) rodents have been generated. In rodents tg for the human Aβ precursor protein, focal injections of preformed Aβ aggregates can induce Aβ deposits in the vicinity of the injection site, and over time in more distant regions of the brain. This suggests that Aβ shares with α-synuclein, tau and other proteins the property to misfold and aggregate homotypic molecules. We propose to group those proteins under the term "propagons". Propagons may lack the infectivity of prions. We review findings from neuropathological examinations of human brains in different stages of AD and from studies in rodent models of Aβ aggregation and discuss putative mechanisms underlying the initiation and spread of Aβ pathology.

QTL detection for forage quality and stem histology in four connected mapping populations of the model legume Medicago truncatula.

PubMed

Lagunes Espinoza, Luz Del Carmen; Julier, Bernadette

2013-02-01

Forage quality combines traits related to protein content and energy value. High-quality forages contribute to increase farm autonomy by reducing the use of energy or protein-rich supplements. Genetic analyses in forage legume species are complex because of their tetraploidy and allogamy. Indeed, no genetic studies of quality have been published at the molecular level on these species. Nonetheless, mapping populations of the model species M. truncatula can be used to detect QTL for forage quality. Here, we studied a crossing design involving four connected populations of M. truncatula. Each population was composed of ca. 200 recombinant inbred lines (RIL). We sought population-specific QTL and QTL explaining the whole design variation. We grew parents and RIL in a greenhouse for 2 or 3 seasons and analysed plants for chemical composition of vegetative organs (protein content, digestibility, leaf-to-stem ratio) and stem histology (stem cross-section area, tissue proportions). Over the four populations and all the traits, QTL were found on all chromosomes. Among these QTL, only four genomic regions, on chromosomes 1, 3, 7 and 8, contributed to explaining the variations in the whole crossing design. Surprisingly, we found that quality QTL were located in the same genomic regions as morphological QTL. We thus confirmed the quantitative inheritance of quality traits and tight relationships between quality and morphology. Our findings could be explained by a co-location of genes involved in quality and morphology. This study will help to detect candidate genes involved in quantitative variation for quality in forage legume species.

From pull-down data to protein interaction networks and complexes with biological relevance.

PubMed

Zhang, Bing; Park, Byung-Hoon; Karpinets, Tatiana; Samatova, Nagiza F

2008-04-01

Recent improvements in high-throughput Mass Spectrometry (MS) technology have expedited genome-wide discovery of protein-protein interactions by providing a capability of detecting protein complexes in a physiological setting. Computational inference of protein interaction networks and protein complexes from MS data are challenging. Advances are required in developing robust and seamlessly integrated procedures for assessment of protein-protein interaction affinities, mathematical representation of protein interaction networks, discovery of protein complexes and evaluation of their biological relevance. A multi-step but easy-to-follow framework for identifying protein complexes from MS pull-down data is introduced. It assesses interaction affinity between two proteins based on similarity of their co-purification patterns derived from MS data. It constructs a protein interaction network by adopting a knowledge-guided threshold selection method. Based on the network, it identifies protein complexes and infers their core components using a graph-theoretical approach. It deploys a statistical evaluation procedure to assess biological relevance of each found complex. On Saccharomyces cerevisiae pull-down data, the framework outperformed other more complicated schemes by at least 10% in F(1)-measure and identified 610 protein complexes with high-functional homogeneity based on the enrichment in Gene Ontology (GO) annotation. Manual examination of the complexes brought forward the hypotheses on cause of false identifications. Namely, co-purification of different protein complexes as mediated by a common non-protein molecule, such as DNA, might be a source of false positives. Protein identification bias in pull-down technology, such as the hydrophilic bias could result in false negatives.

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria.

PubMed

Asztalos, Emese; Sipka, Gábor; Kis, Mariann; Trotta, Massimo; Maróti, Péter

2012-06-01

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 μM) of mercury (Hg²⁺) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg²⁺ sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c₂ and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc₁ complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.

Chimera states in networks of logistic maps with hierarchical connectivities

NASA Astrophysics Data System (ADS)

zur Bonsen, Alexander; Omelchenko, Iryna; Zakharova, Anna; Schöll, Eckehard

2018-04-01

Chimera states are complex spatiotemporal patterns consisting of coexisting domains of coherence and incoherence. We study networks of nonlocally coupled logistic maps and analyze systematically how the dilution of the network links influences the appearance of chimera patterns. The network connectivities are constructed using an iterative Cantor algorithm to generate fractal (hierarchical) connectivities. Increasing the hierarchical level of iteration, we compare the resulting spatiotemporal patterns. We demonstrate that a high clustering coefficient and symmetry of the base pattern promotes chimera states, and asymmetric connectivities result in complex nested chimera patterns.

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

PubMed Central

Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

2016-01-01

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines.

PubMed

Hsu, Hwei-Jan; Drummond-Barbosa, Daniela

2017-01-01

The effect of diet on reproduction is well documented in a large number of organisms; however, much remains to be learned about the molecular mechanisms underlying this connection. The Drosophila ovary has a well described, fast and largely reversible response to diet. Ovarian stem cells and their progeny proliferate and grow faster on a yeast-rich diet than on a yeast-free (poor) diet, and death of early germline cysts, degeneration of early vitellogenic follicles and partial block in ovulation further contribute to the ∼60-fold decrease in egg laying observed on a poor diet. Multiple diet-dependent factors, including insulin-like peptides, the steroid ecdysone, the nutrient sensor Target of Rapamycin, AMP-dependent kinase, and adipocyte factors mediate this complex response. Here, we describe the results of a visual screen using a collection of green fluorescent protein (GFP) protein trap lines to identify additional factors potentially involved in this response. In each GFP protein trap line, an artificial GFP exon is fused in frame to an endogenous protein, such that the GFP fusion pattern parallels the levels and subcellular localization of the corresponding native protein. We identified 53 GFP-tagged proteins that exhibit changes in levels and/or subcellular localization in the ovary at 12-16 hours after switching females from rich to poor diets, suggesting them as potential candidates for future functional studies. Copyright © 2017 Elsevier B.V. All rights reserved.

From the Cover: Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

NASA Astrophysics Data System (ADS)

Ito, Takashi; Tashiro, Kosuke; Muta, Shigeru; Ozawa, Ritsuko; Chiba, Tomoko; Nishizawa, Mayumi; Yamamoto, Kiyoshi; Kuhara, Satoru; Sakaki, Yoshiyuki

2000-02-01

Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATα strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of ≈4 × 106 different combinations, constituting ≈10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

Prediction of protein tertiary structure from sequences using a very large back-propagation neural network

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

Liu, X.; Wilcox, G.L.

1993-12-31

We have implemented large scale back-propagation neural networks on a 544 node Connection Machine, CM-5, using the C language in MIMD mode. The program running on 512 processors performs backpropagation learning at 0.53 Gflops, which provides 76 million connection updates per second. We have applied the network to the prediction of protein tertiary structure from sequence information alone. A neural network with one hidden layer and 40 million connections is trained to learn the relationship between sequence and tertiary structure. The trained network yields predicted structures of some proteins on which it has not been trained given only their sequences.more » Presentation of the Fourier transform of the sequences accentuates periodicity in the sequence and yields good generalization with greatly increased training efficiency. Training simulations with a large, heterologous set of protein structures (111 proteins from CM-5 time) to solutions with under 2% RMS residual error within the training set (random responses give an RMS error of about 20%). Presentation of 15 sequences of related proteins in a testing set of 24 proteins yields predicted structures with less than 8% RMS residual error, indicating good apparent generalization.« less

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

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

Quantifying the deformation of the red blood cell skeleton in shear flow

NASA Astrophysics Data System (ADS)

Peng, Zhangli; Zhu, Qiang

2012-02-01

To quantitatively predict the response of red blood cell (RBC) membrane in shear flow, we carried out multiphysics simulations by coupling a three-level multiscale approach of RBC membranes with a Boundary Element Method (BEM) for surrounding flows. Our multiscale approach includes a model of spectrins with the domain unfolding feature, a molecular-based model of the junctional complex with detailed protein connectivity and a whole cell Finite Element Method (FEM) model with the bilayer-skeleton friction derived from measured transmembrane protein diffusivity based on the Einstein-Stokes relation. Applying this approach, we investigated the bilayer-skeleton slip and skeleton deformation of healthy RBCs and RBCs with hereditary spherocytosis anemia during tank-treading motion. Compared with healthy cells, cells with hereditary spherocytosis anemia sustain much larger skeleton-bilayer slip and area deformation of the skeleton due to deficiency of transmembrane proteins. This leads to extremely low skeleton density and large bilayer-skeleton interaction force, both of which may cause bilayer loss. This finding suggests a possible mechanism of the development of hereditary spherocytosis anemia.

Usher syndrome type 1–associated cadherins shape the photoreceptor outer segment

PubMed Central

Parain, Karine; Aghaie, Asadollah; Picaud, Serge

2017-01-01

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis, these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23, encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15–containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. PMID:28495838

Usher syndrome type 1-associated cadherins shape the photoreceptor outer segment.

PubMed

Schietroma, Cataldo; Parain, Karine; Estivalet, Amrit; Aghaie, Asadollah; Boutet de Monvel, Jacques; Picaud, Serge; Sahel, José-Alain; Perron, Muriel; El-Amraoui, Aziz; Petit, Christine

2017-06-05

Usher syndrome type 1 (USH1) causes combined hearing and sight defects, but how mutations in USH1 genes lead to retinal dystrophy in patients remains elusive. The USH1 protein complex is associated with calyceal processes, which are microvilli of unknown function surrounding the base of the photoreceptor outer segment. We show that in Xenopus tropicalis , these processes are connected to the outer-segment membrane by links composed of protocadherin-15 (USH1F protein). Protocadherin-15 deficiency, obtained by a knockdown approach, leads to impaired photoreceptor function and abnormally shaped photoreceptor outer segments. Rod basal outer disks displayed excessive outgrowth, and cone outer segments were curved, with lamellae of heterogeneous sizes, defects also observed upon knockdown of Cdh23 , encoding cadherin-23 (USH1D protein). The calyceal processes were virtually absent in cones and displayed markedly reduced F-actin content in rods, suggesting that protocadherin-15-containing links are essential for their development and/or maintenance. We propose that calyceal processes, together with their associated links, control the sizing of rod disks and cone lamellae throughout their daily renewal. © 2017 Schietroma et al.

Markov chain aggregation and its applications to combinatorial reaction networks.

PubMed

Ganguly, Arnab; Petrov, Tatjana; Koeppl, Heinz

2014-09-01

We consider a continuous-time Markov chain (CTMC) whose state space is partitioned into aggregates, and each aggregate is assigned a probability measure. A sufficient condition for defining a CTMC over the aggregates is presented as a variant of weak lumpability, which also characterizes that the measure over the original process can be recovered from that of the aggregated one. We show how the applicability of de-aggregation depends on the initial distribution. The application section is devoted to illustrate how the developed theory aids in reducing CTMC models of biochemical systems particularly in connection to protein-protein interactions. We assume that the model is written by a biologist in form of site-graph-rewrite rules. Site-graph-rewrite rules compactly express that, often, only a local context of a protein (instead of a full molecular species) needs to be in a certain configuration in order to trigger a reaction event. This observation leads to suitable aggregate Markov chains with smaller state spaces, thereby providing sufficient reduction in computational complexity. This is further exemplified in two case studies: simple unbounded polymerization and early EGFR/insulin crosstalk.

Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation

PubMed Central

Pratt, Metta B.; Johnson, Errin

2018-01-01

Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. Here, we examine Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane—although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. We show that PLP helps assemble “pericentriolar clouds” of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. We propose that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects we observe in Plp mutant cells. PMID:29425198

Using high-throughput barcode sequencing to efficiently map connectomes.

PubMed

Peikon, Ian D; Kebschull, Justus M; Vagin, Vasily V; Ravens, Diana I; Sun, Yu-Chi; Brouzes, Eric; Corrêa, Ivan R; Bressan, Dario; Zador, Anthony M

2017-07-07

The function of a neural circuit is determined by the details of its synaptic connections. At present, the only available method for determining a neural wiring diagram with single synapse precision-a 'connectome'-is based on imaging methods that are slow, labor-intensive and expensive. Here, we present SYNseq, a method for converting the connectome into a form that can exploit the speed and low cost of modern high-throughput DNA sequencing. In SYNseq, each neuron is labeled with a unique random nucleotide sequence-an RNA 'barcode'-which is targeted to the synapse using engineered proteins. Barcodes in pre- and postsynaptic neurons are then associated through protein-protein crosslinking across the synapse, extracted from the tissue, and joined into a form suitable for sequencing. Although our failure to develop an efficient barcode joining scheme precludes the widespread application of this approach, we expect that with further development SYNseq will enable tracing of complex circuits at high speed and low cost. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control.

PubMed

Yau, Richard G; Doerner, Kerstin; Castellanos, Erick R; Haakonsen, Diane L; Werner, Achim; Wang, Nan; Yang, X William; Martinez-Martin, Nadia; Matsumoto, Marissa L; Dixit, Vishva M; Rape, Michael

2017-11-02

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information. Copyright © 2017 Elsevier Inc. All rights reserved.

Network-based prediction and knowledge mining of disease genes

PubMed Central

2015-01-01

Background In recent years, high-throughput protein interaction identification methods have generated a large amount of data. When combined with the results from other in vivo and in vitro experiments, a complex set of relationships between biological molecules emerges. The growing popularity of network analysis and data mining has allowed researchers to recognize indirect connections between these molecules. Due to the interdependent nature of network entities, evaluating proteins in this context can reveal relationships that may not otherwise be evident. Methods We examined the human protein interaction network as it relates to human illness using the Disease Ontology. After calculating several topological metrics, we trained an alternating decision tree (ADTree) classifier to identify disease-associated proteins. Using a bootstrapping method, we created a tree to highlight conserved characteristics shared by many of these proteins. Subsequently, we reviewed a set of non-disease-associated proteins that were misclassified by the algorithm with high confidence and searched for evidence of a disease relationship. Results Our classifier was able to predict disease-related genes with 79% area under the receiver operating characteristic (ROC) curve (AUC), which indicates the tradeoff between sensitivity and specificity and is a good predictor of how a classifier will perform on future data sets. We found that a combination of several network characteristics including degree centrality, disease neighbor ratio, eccentricity, and neighborhood connectivity help to distinguish between disease- and non-disease-related proteins. Furthermore, the ADTree allowed us to understand which combinations of strongly predictive attributes contributed most to protein-disease classification. In our post-processing evaluation, we found several examples of potential novel disease-related proteins and corresponding literature evidence. In addition, we showed that first- and second-order neighbors in the PPI network could be used to identify likely disease associations. Conclusions We analyzed the human protein interaction network and its relationship to disease and found that both the number of interactions with other proteins and the disease relationship of neighboring proteins helped to determine whether a protein had a relationship to disease. Our classifier predicted many proteins with no annotated disease association to be disease-related, which indicated that these proteins have network characteristics that are similar to disease-related proteins and may therefore have disease associations not previously identified. By performing a post-processing step after the prediction, we were able to identify evidence in literature supporting this possibility. This method could provide a useful filter for experimentalists searching for new candidate protein targets for drug repositioning and could also be extended to include other network and data types in order to refine these predictions. PMID:26043920

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

Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor

PubMed Central

Pandini, Alessandro; Kleinjung, Jens; Rasool, Shafqat; Khan, Shahid

2015-01-01

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) “torque” helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity. PMID:26561852

On the connection between inherent DNA flexure and preferred binding of hydroxymethyluracil-containing DNA by the type II DNA-binding protein TF1.

PubMed

Grove, A; Galeone, A; Mayol, L; Geiduschek, E P

1996-07-12

TF1 is a member of the family of type II DNA-binding proteins, which also includes the bacterial HU proteins and the Escherichia coli integration host factor (IHF). Distinctive to TF1, which is encoded by the Bacillus subtilis bacteriophage SPO1, is its preferential binding to DNA in which thymine is replaced by 5-hydroxymethyluracil (hmU), as it is in the phage genome. TF1 binds to preferred sites within the phage genome and generates pronounced DNA bending. The extent to which DNA flexibility contributes to the sequence-specific binding of TF1, and the connection between hmU preference and DNA flexibility has been examined. Model flexible sites, consisting of consecutive mismatches, increase the affinity of thymine-containing DNA for TF1. In particular, tandem mismatches separated by nine base-pairs generate an increase, by orders of magnitude, in the affinity of TF1 for T-containing DNA with the sequence of a preferred TF1 binding site, and fully match the affinity of TF1 for this cognate site in hmU-containing DNA (Kd approximately 3 nM). Other placements of loops generate suboptimal binding. This is consistent with a significant contribution of site-specific DNA flexibility to complex formation. Analysis of complexes with hmU-DNA of decreasing length shows that a major part of the binding affinity is generated within a central 19 bp segment (delta G0 = 41.7 kJ mol-1) with more-distal DNA contributing modestly to the affinity (delta delta G = -0.42 kJ mol-1 bp-1 on increasing duplex length to 37 bp). However, a previously characterised thermostable and more tightly binding mutant TF1, TF1(E15G/T32I), derives most of its extra affinity from interaction with flanking DNA. We propose that inherent but sequence-dependent deformability of hmU-containing DNA underlies the preferential binding of TF1 and that TF1-induced DNA bendings is a result of distortions at two distinct sites separated by 9 bp of duplex DNA.

Multiple intermediates on the energy landscape of a 15-HEAT-repeat protein

PubMed Central

Tsytlonok, Maksym; Craig, Patricio O.; Sivertsson, Elin; Serquera, David; Perrett, Sarah; Best, Robert B.; Wolynes, Peter G.; Itzhaki, Laura S.

2014-01-01

Repeat proteins are a special class of modular, non-globular proteins composed of small structural motifs arrayed to form elongated architectures and stabilised solely by short-range contacts. We find a remarkable complexity in the unfolding of the large HEAT repeat protein PR65/A. In contrast to what has been seen for small repeat proteins in which unfolding propagates from one end, the HEAT array of PR65/A ruptures at multiple distant sites, leading to intermediate states with non-contiguous folded subdomains. Kinetic analysis allows us to define a network of intermediates and to delineate the pathways that connect them. There is a dominant sequence of unfolding, reflecting a non-uniform distribution of stability across the repeat array; however the unfolding of certain intermediates is competitive, leading to parallel pathways. Theoretical models accounting for the heterogeneous contact density in the folded structure are able to rationalize the variation in stability across the array. This variation in stability also suggests how folding may direct function in a large repeat protein: The stability distribution enables certain regions to present rigid motifs for molecular recognition while affording others flexibility to broaden the search area as in a fly-casting mechanism. Thus PR65/A uses the two ends of the repeat array to bind diverse partners and thereby coordinate the dephosphorylation of many different substrates and of multiple sites within hyperphosphorylated substrates. PMID:24120762

Complexity, Connections, and Soul-Work

ERIC Educational Resources Information Center

Bloch, Deborah P.

2008-01-01

Organizational theory and personal behaviors are both shaped by contemporary thinking and theories regarding spirituality, history, and the order, shape, and direction of modern culture. Complexity theory, discussed in this article, offers some helpful insights into appreciating the relationships and connections often overlooked in today's…

Functional connectivity supporting the selective maintenance of feature-location binding in visual working memory

PubMed Central

Takahama, Sachiko; Saiki, Jun

2014-01-01

Information on an object's features bound to its location is very important for maintaining object representations in visual working memory. Interactions with dynamic multi-dimensional objects in an external environment require complex cognitive control, including the selective maintenance of feature-location binding. Here, we used event-related functional magnetic resonance imaging to investigate brain activity and functional connectivity related to the maintenance of complex feature-location binding. Participants were required to detect task-relevant changes in feature-location binding between objects defined by color, orientation, and location. We compared a complex binding task requiring complex feature-location binding (color-orientation-location) with a simple binding task in which simple feature-location binding, such as color-location, was task-relevant and the other feature was task-irrelevant. Univariate analyses showed that the dorsolateral prefrontal cortex (DLPFC), hippocampus, and frontoparietal network were activated during the maintenance of complex feature-location binding. Functional connectivity analyses indicated cooperation between the inferior precentral sulcus (infPreCS), DLPFC, and hippocampus during the maintenance of complex feature-location binding. In contrast, the connectivity for the spatial updating of simple feature-location binding determined by reanalyzing the data from Takahama et al. (2010) demonstrated that the superior parietal lobule (SPL) cooperated with the DLPFC and hippocampus. These results suggest that the connectivity for complex feature-location binding does not simply reflect general memory load and that the DLPFC and hippocampus flexibly modulate the dorsal frontoparietal network, depending on the task requirements, with the infPreCS involved in the maintenance of complex feature-location binding and the SPL involved in the spatial updating of simple feature-location binding. PMID:24917833