Application of linker technique to trap transiently interacting protein complexes for structural studies

PubMed Central

Reddy Chichili, Vishnu Priyanka; Kumar, Veerendra; Sivaraman, J.

2016-01-01

Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes. PMID:26985443

Computation of pH-Dependent Binding Free Energies

PubMed Central

Kim, M. Olivia; McCammon, J. Andrew

2015-01-01

Protein-ligand binding accompanies changes in the surrounding electrostatic environments of the two binding partners and may lead to changes in protonation upon binding. In cases where the complex formation results in a net transfer of protons, the binding process is pH-dependent. However, conventional free energy computations or molecular docking protocols typically employ fixed protonation states for the titratable groups in both binding partners set a priori, which are identical for the free and bound states. In this review, we draw attention to these important yet largely ignored binding-induced protonation changes in protein-ligand association by outlining physical origins and prevalence of the protonation changes upon binding. Following a summary of various theoretical methods for pKa prediction, we discuss the theoretical framework to examine the pH dependence of protein-ligand binding processes. PMID:26202905

Effect of redox partner binding on CYP101D1 conformational dynamics.

PubMed

Batabyal, Dipanwita; Poulos, Thomas L

2018-06-01

We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDX D38 which forms an important ion pair with P450cam R112 at the center of the interface is Arx L39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with Arx L39 . Copyright © 2018. Published by Elsevier Inc.

Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions.

PubMed

Gruet, Antoine; Dosnon, Marion; Blocquel, David; Brunel, Joanna; Gerlier, Denis; Das, Rahul K; Bonetti, Daniela; Gianni, Stefano; Fuxreiter, Monika; Longhi, Sonia; Bignon, Christophe

2016-02-01

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (NTAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of NTAIL remains conspicuously disordered, with α-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on NTAIL /XD binding, we generated N-terminal truncation variants of NTAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV NTAIL binding partner), and for the homologous NTAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV NTAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region. © 2015 Federation of European Biochemical Societies.

Speeding up biomolecular interactions by molecular sledding

DOE PAGES

Turkin, Alexander; Zhang, Lei; Marcozzi, Alessio; ...

2015-10-07

In numerous biological processes associations involve a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Moreover, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We also demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptidemore » and demonstrate that the ability of the peptide to one-dimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions.« less

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

PubMed

Rudolph, Markus G; Klostermeier, Dagmar

2015-08-01

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

Molecular tweezers modulate 14-3-3 protein-protein interactions

NASA Astrophysics Data System (ADS)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

A data-mining approach to rank candidate protein-binding partners-The case of biogenesis of lysosome-related organelles complex-1 (BLOC-1).

PubMed

Rodriguez-Fernandez, I A; Dell'Angelica, E C

2009-04-01

The study of protein-protein interactions is a powerful approach to uncovering the molecular function of gene products associated with human disease. Protein-protein interaction data are accumulating at an unprecedented pace owing to interactomics projects, although it has been recognized that a significant fraction of these data likely represents false positives. During our studies of biogenesis of lysosome-related organelles complex-1 (BLOC-1), a protein complex involved in protein trafficking and containing the products of genes mutated in Hermansky-Pudlak syndrome, we faced the problem of having too many candidate binding partners to pursue experimentally. In this work, we have explored ways of efficiently gathering high-quality information about candidate binding partners and presenting the information in a visually friendly manner. We applied the approach to rank 70 candidate binding partners of human BLOC-1 and 102 candidates of its counterpart from Drosophila melanogaster. The top candidate for human BLOC-1 was the small GTPase encoded by the RAB11A gene, which is a paralogue of the Rab38 and Rab32 proteins in mammals and the lightoid gene product in flies. Interestingly, genetic analyses in D. melanogaster uncovered a synthetic sick/lethal interaction between Rab11 and lightoid. The data-mining approach described herein can be customized to study candidate binding partners for other proteins or possibly candidates derived from other types of 'omics' data.

26 CFR 301.6224(c)-2 - Pass-thru partner binds indirect partners.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the following example: Example. Partnership J is a partner in partnership P. C is a partner in J but... C holds in P is through J. The tax matters partner of J enters into a settlement agreement with the... (CONTINUED) PROCEDURE AND ADMINISTRATION PROCEDURE AND ADMINISTRATION Assessment In General § 301.6224(c)-2...

Partner-Mediated Polymorphism of an Intrinsically Disordered Protein.

PubMed

Bignon, Christophe; Troilo, Francesca; Gianni, Stefano; Longhi, Sonia

2017-11-29

Intrinsically disordered proteins (IDPs) recognize their partners through molecular recognition elements (MoREs). The MoRE of the C-terminal intrinsically disordered domain of the measles virus nucleoprotein (N TAIL ) is partly pre-configured as an α-helix in the free form and undergoes α-helical folding upon binding to the X domain (XD) of the viral phosphoprotein. Beyond XD, N TAIL also binds the major inducible heat shock protein 70 (hsp70). So far, no structural information is available for the N TAIL /hsp70 complex. Using mutational studies combined with a protein complementation assay based on green fluorescent protein reconstitution, we have investigated both N TAIL /XD and N TAIL /hsp70 interactions. Although the same N TAIL region binds the two partners, the binding mechanisms are different. Hsp70 binding is much more tolerant of MoRE substitutions than XD, and the majority of substitutions lead to an increased N TAIL /hsp70 interaction strength. Furthermore, while an increased and a decreased α-helicity of the MoRE lead to enhanced and reduced interaction strength with XD, respectively, the impact on hsp70 binding is negligible, suggesting that the MoRE does not adopt an α-helical conformation once bound to hsp70. Here, by showing that the α-helical conformation sampled by the free form of the MoRE does not systematically commit it to adopt an α-helical conformation in the bound form, we provide an example of partner-mediated polymorphism of an IDP and of the relative insensitiveness of the bound structure to the pre-recognition state. The present results therefore contribute to shed light on the molecular mechanisms by which IDPs recognize different partners. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural biology of intrinsically disordered proteins: Revisiting unsolved mysteries.

PubMed

Sigalov, Alexander B

2016-06-01

The emergence of intrinsically disordered proteins (IDPs) has challenged the classical protein structure-function paradigm by introducing a new paradigm of "coupled binding and folding". This paradigm suggests that IDPs fold upon binding to their partners. Further studies, however, revealed a novel and previously unrecognized phenomenon of "uncoupled binding and folding" suggesting that IDPs do not necessarily fold upon interaction with their lipid and protein partners. The complex and often unusual biophysics of IDPs makes structural characterization of these proteins and their complexes not only challenging but often resulting in opposite conclusions. For this reason, some crucial questions in this field remain unsolved for well over a decade. Considering an important role of IDPs in cellular regulation, signaling and control in health and disease, more efforts are needed to solve these mysteries. Here, I focus on two long-standing contradictions in the literature concerning dimerization and membrane-binding activities of IDPs. Molecular explanation of these discrepancies is provided. I also demonstrate how resolution of these critical issues in the field of IDPs results in our expanded understanding of cell function and has multiple applications in biology and medicine. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

PubMed

Dahal, Liza; Shammas, Sarah L; Clarke, Jane

2017-12-19

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo. Here we show that, contrary to expectation, phosphorylation has no effect on association rates-unphosphorylated KID binds just as rapidly as pKID, the phosphorylated form-but rather, acts by increasing the lifetime of the complex. We propose that by controlling the lifetime of the bound complex of pKID:KIX via altering the dissociation rate, phosphorylation can facilitate effective control of transcription regulation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.

PubMed

Weidmann, Chase A; Qiu, Chen; Arvola, René M; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

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

Weidmann, Chase A.; Qiu, Chen; Arvola, René M.

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that aremore » not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.« less

Protein-Protein Interface Predictions by Data-Driven Methods: A Review

PubMed Central

Xue, Li C; Dobbs, Drena; Bonvin, Alexandre M.J.J.; Honavar, Vasant

2015-01-01

Reliably pinpointing which specific amino acid residues form the interface(s) between a protein and its binding partner(s) is critical for understanding the structural and physicochemical determinants of protein recognition and binding affinity, and has wide applications in modeling and validating protein interactions predicted by high-throughput methods, in engineering proteins, and in prioritizing drug targets. Here, we review the basic concepts, principles and recent advances in computational approaches to the analysis and prediction of protein-protein interfaces. We point out caveats for objectively evaluating interface predictors, and discuss various applications of data-driven interface predictors for improving energy model-driven protein-protein docking. Finally, we stress the importance of exploiting binding partner information in reliably predicting interfaces and highlight recent advances in this emerging direction. PMID:26460190

Targeting binding partners of the CBFβ-SMMHC fusion protein for the treatment of inversion 16 acute myeloid leukemia

PubMed Central

Hyde, R. Katherine

2016-01-01

Inversion of chromosome 16 (inv(16)) generates the CBFβ-SMMHC fusion protein and is found in nearly all patients with acute myeloid leukemia subtype M4 with Eosinophilia (M4Eo). Expression of CBFβ-SMMHC is causative for leukemia development, but the molecular mechanisms underlying its activity are unclear. Recently, there have been important advances in defining the role of CBFβ-SMMHC and its binding partners, the transcription factor RUNX1 and the histone deacetylase HDAC8. Importantly, initial trials demonstrate that small molecules targeting these binding partners are effective against CBFβ-SMMHC induced leukemia. This review will discuss recent advances in defining the mechanism of CBFβ-SMMHC activity, as well as efforts to develop new therapies for inv(16) AML. PMID:27542261

Comprehensive identification of proteins binding to RNA G-quadruplex motifs in the 5' UTR of tumor-associated mRNAs.

PubMed

Serikawa, Tatsuo; Spanos, Christos; von Hacht, Annekathrin; Budisa, Nediljko; Rappsilber, Juri; Kurreck, Jens

2018-01-01

G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. The use of a control composed of samples from all G-quadruplex-forming sequences and their mutated controls ensured that the identified proteins are specific for RNA G-quadruplex structures and are not general RNA-binding proteins. Independent validation experiments based on pull-down assays and Western blotting confirmed the MS data. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). Several of the candidate proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. Interestingly, additional proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions are not only important for the fine-tuning of translation but are also relevant to the regulation of mRNA maturation and may play an important role in tumor biology. Proteomic data are available via ProteomeXchange with identifier PXD005761. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson's disease.

PubMed

Merzetti, Eric M; Dolomount, Lindsay A; Staveley, Brian E

2017-01-01

Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson's disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

Phosphorylated c-MPL tyrosine 591 regulates thrombopoietin-induced signaling

PubMed Central

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2018-01-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. PMID:24607955

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

PubMed Central

Huang, Bill X.; Kim, Hee-Yong

2013-01-01

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

Sequence-Based Prediction of RNA-Binding Residues in Proteins.

PubMed

Walia, Rasna R; El-Manzalawy, Yasser; Honavar, Vasant G; Dobbs, Drena

2017-01-01

Identifying individual residues in the interfaces of protein-RNA complexes is important for understanding the molecular determinants of protein-RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein-RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein-RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner.

Sequence-Based Prediction of RNA-Binding Residues in Proteins

PubMed Central

Walia, Rasna R.; EL-Manzalawy, Yasser; Honavar, Vasant G.; Dobbs, Drena

2017-01-01

Identifying individual residues in the interfaces of protein–RNA complexes is important for understanding the molecular determinants of protein–RNA recognition and has many potential applications. Recent technical advances have led to several high-throughput experimental methods for identifying partners in protein–RNA complexes, but determining RNA-binding residues in proteins is still expensive and time-consuming. This chapter focuses on available computational methods for identifying which amino acids in an RNA-binding protein participate directly in contacting RNA. Step-by-step protocols for using three different web-based servers to predict RNA-binding residues are described. In addition, currently available web servers and software tools for predicting RNA-binding sites, as well as databases that contain valuable information about known protein–RNA complexes, RNA-binding motifs in proteins, and protein-binding recognition sites in RNA are provided. We emphasize sequence-based methods that can reliably identify interfacial residues without the requirement for structural information regarding either the RNA-binding protein or its RNA partner. PMID:27787829

Understanding the impact of Fc glycosylation on its conformational changes by molecular dynamics simulations and bioinformatics.

PubMed

Zhang, Yubo

2015-12-01

N-linked glycosylation of Fc at N297 plays an important role in its effector function, aberrance of which would cause disease pathogenesis. Here, we performed all-atom molecular dynamics simulations to explore the effects of Fc glycosylation on its dynamics behaviors. Firstly, equilibrium simulations suggested that Fc deglycosylation was able to induce residual flexibility in its CH2 domain. Besides, the free energy landscape revealed three minimum energy wells in deglycosylated Fc, representing its "open", "semi-closed" and "closed" states. However, we could only observe the "open" state of glycosylated Fc. Supportively, principal component analysis emphasized the prominent motion of delyclosylated Fc and dynamically depicted how it changed from the "open" state to its "closed" state. Secondly, we studied the recognition mechanism of the Fc binding to its partners. Energy decomposition analysis identified key residues of Fc to recognize its two partners P13 and P34. Evidently, electrostatic potential surfaces showed that electrostatic attraction helped to stabilize the interaction between Fc and its partners. Also, relative binding free energies explained different binding affinities in Fc-P13 and Fc-P34. Collectively, these results together provided the structural basis for understanding conformational changes of deglycosylated Fc and the recognition mechanism of the Fc binding to its partners.

From DCPD to NTCP: The long journey towards identifying a functional hepatitis B virus receptor

PubMed Central

2015-01-01

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection. PMID:26523264

From DCPD to NTCP: the long journey towards identifying a functional hepatitis B virus receptor.

PubMed

Li, Jisu; Tong, Shuping

2015-09-01

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.

NMR Characterization of Self-Association Domains Promoted by Interactions with LC8 Hub Protein

PubMed Central

Barbar, Elisar; Nyarko, Afua

2014-01-01

Most proteins in interaction networks have a small number of partners, while a few, called hubs, participate in a large number of interactions and play a central role in cell homeostasis. One highly conserved hub is a protein called LC8 that was originally identified as an essential component of the multi-subunit complex dynein but later shown to be also critical in multiple protein complexes in diverse systems. What is intriguing about this hub protein is that it does not passively bind its various partners but emerging evidence suggests that LC8 acts as a dimerization engine that promotes self-association and/or higher order organization of its primarily disordered monomeric partners. This structural organization process does not require ATP but is triggered by long-range allosteric regulation initiated by LC8 binding a pair of disordered chains forming a bivalent or polybivalent scaffold. This review focuses on the role of LC8 in promoting self-association of two of its binding partners, a dynein intermediate chain and a non dynein protein called Swallow. PMID:24757501

PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

PubMed Central

Hong, Nan Hyung; Qi, Aidong

2015-01-01

Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

PubMed Central

Weidmann, Chase A; Qiu, Chen; Arvola, René M; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2016-01-01

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics. DOI: http://dx.doi.org/10.7554/eLife.17096.001 PMID:27482653

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

DOE PAGES

Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; ...

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAsmore » that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.« less

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

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

Weidmann, Chase A.; Qiu, Chen; Arvola, René M.

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAsmore » that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.« less

Monte Carlo replica-exchange based ensemble docking of protein conformations.

PubMed

Zhang, Zhe; Ehmann, Uwe; Zacharias, Martin

2017-05-01

A replica-exchange Monte Carlo (REMC) ensemble docking approach has been developed that allows efficient exploration of protein-protein docking geometries. In addition to Monte Carlo steps in translation and orientation of binding partners, possible conformational changes upon binding are included based on Monte Carlo selection of protein conformations stored as ordered pregenerated conformational ensembles. The conformational ensembles of each binding partner protein were generated by three different approaches starting from the unbound partner protein structure with a range spanning a root mean square deviation of 1-2.5 Å with respect to the unbound structure. Because MC sampling is performed to select appropriate partner conformations on the fly the approach is not limited by the number of conformations in the ensemble compared to ensemble docking of each conformer pair in ensemble cross docking. Although only a fraction of generated conformers was in closer agreement with the bound structure the REMC ensemble docking approach achieved improved docking results compared to REMC docking with only the unbound partner structures or using docking energy minimization methods. The approach has significant potential for further improvement in combination with more realistic structural ensembles and better docking scoring functions. Proteins 2017; 85:924-937. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Role of tyrosine hot-spot residues at the interface of colicin E9 and immunity protein 9: a comparative free energy simulation study.

PubMed

Luitz, Manuel P; Zacharias, Martin

2013-03-01

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high-affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot-spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein-protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr-Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein-protein interactions dominating the free energy change due to the Tyr54Ala mutation. Copyright © 2012 Wiley Periodicals, Inc.

Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation.

PubMed

Tang, Xiaoling; Jang, Sung-Wuk; Wang, Xuerong; Liu, Zhixue; Bahr, Scott M; Sun, Shi-Yong; Brat, Daniel; Gutmann, David H; Ye, Keqiang

2007-10-01

The neurofibromatosis-2 (NF2) tumour-suppressor gene encodes an intracellular membrane-associated protein, called merlin, whose growth-suppressive function is dependent on its ability to form interactions through its intramolecular amino-terminal domain (NTD) and carboxy-terminal domain (CTD). Merlin phosphorylation plays a critical part in dictating merlin NTD/CTD interactions as well as in controlling binding to its effector proteins. Merlin is partially regulated by phosphorylation of Ser 518, such that hyperphosphorylated merlin is inactive and fails to form productive intramolecular and intermolecular interactions. Here, we show that the protein kinase Akt directly binds to and phosphorylates merlin on residues Thr 230 and Ser 315, which abolishes merlin NTD/CTD interactions and binding to merlin's effector protein PIKE-L and other binding partners. Furthermore, Akt-mediated phosphorylation leads to merlin degradation by ubiquitination. These studies demonstrate that Akt-mediated merlin phosphorylation regulates the function of merlin in the absence of an inactivating mutation.

Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners

NASA Astrophysics Data System (ADS)

Herling, Therese; Linse, Sara; Knowles, Tuomas

2015-03-01

Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution.

Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

PubMed

Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

2014-06-01

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.

PubMed

Choudhury, Nila Roy; Heikel, Gregory; Trubitsyna, Maryia; Kubik, Peter; Nowak, Jakub Stanislaw; Webb, Shaun; Granneman, Sander; Spanos, Christos; Rappsilber, Juri; Castello, Alfredo; Michlewski, Gracjan

2017-11-08

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25's endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity.

Controllable activation of nanoscale dynamics in a disordered protein alters binding kinetics

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

Callaway, David J. E.; Matsui, Tsutomu; Weiss, Thomas

The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the tip of the C-terminal tail in the phosphomimic S339D/S340D mutant. The “tip of the whip” that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the bindingmore » of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. Furthermore NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.« less

Controllable activation of nanoscale dynamics in a disordered protein alters binding kinetics

DOE PAGES

Callaway, David J. E.; Matsui, Tsutomu; Weiss, Thomas; ...

2017-03-08

The phosphorylation of specific residues in a flexible disordered activation loop yields precise control of signal transduction. One paradigm is the phosphorylation of S339/S340 in the intrinsically disordered tail of the multi-domain scaffolding protein NHERF1, which affects the intracellular localization and trafficking of NHERF1 assembled signaling complexes. Using neutron spin echo spectroscopy (NSE), we show salt-concentration-dependent excitation of nanoscale motion at the tip of the C-terminal tail in the phosphomimic S339D/S340D mutant. The “tip of the whip” that is unleashed is near the S339/S340 phosphorylation site and flanks the hydrophobic Ezrin-binding motif. The kinetic association rate constant of the bindingmore » of the S339D/S340D mutant to the FERM domain of Ezrin is sensitive to buffer salt concentration, correlating with the excited nanoscale dynamics. The results suggest that electrostatics modulates the activation of nanoscale dynamics of an intrinsically disordered protein, controlling the binding kinetics of signaling partners. Furthermore NSE can pinpoint the nanoscale dynamics changes in a highly specific manner.« less

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

PubMed Central

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

2013-01-01

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

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

PubMed

Rowland, Meng M; Bostic, Heidi E; Gong, Denghuang; Speers, Anna E; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F; Best, Michael D

2011-12-27

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.

Phosphatidylinositol (3,4,5)-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

PubMed Central

Rowland, Meng M.; Bostic, Heidi E.; Gong, Denghuang; Speers, Anna E.; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F.; Best, Michael D.

2013-01-01

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins. PMID:22074223

Identification of discrete sites in Yip1A necessary for regulation of endoplasmic reticulum structure.

PubMed

Dykstra, Kaitlyn M; Ulengin, Idil; Delrose, Nicholas; Lee, Tina H

2013-01-01

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.

Designing a Binding Interface for Control of Cancer Cell Adhesion via 3D Topography and Metabolic Oligosaccharide Engineering

PubMed Central

Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J.

2011-01-01

This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac5ManNTGc, a thiol-bearing analogue of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bioorthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

Cellular Localization and Characterization of Cytosolic Binding Partners for Gla Domain-containing Proteins PRRG4 and PRRG2*

PubMed Central

Yazicioglu, Mustafa N.; Monaldini, Luca; Chu, Kirk; Khazi, Fayaz R.; Murphy, Samuel L.; Huang, Heshu; Margaritis, Paris; High, Katherine A.

2013-01-01

The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins. PMID:23873930

Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates.

PubMed

Longobardi, E; Penkov, D; Mateos, D; De Florian, G; Torres, M; Blasi, Francesco

2014-01-01

TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions, and are involved in important pathologies. This review focuses on the PREP, MEIS, and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e., structure, interactors, and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1, and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEIS proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions. Copyright © 2013 Wiley Periodicals, Inc.

Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway*

PubMed Central

Madsen, Kenneth L.; Thorsen, Thor S.; Rahbek-Clemmensen, Troels; Eriksen, Jacob; Gether, Ulrik

2012-01-01

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β2-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway. PMID:22303009

Functional advantages of dynamic protein disorder.

PubMed

Berlow, Rebecca B; Dyson, H Jane; Wright, Peter E

2015-09-14

Intrinsically disordered proteins participate in many important cellular regulatory processes. The absence of a well-defined structure in the free state of a disordered domain, and even on occasion when it is bound to physiological partners, is fundamental to its function. Disordered domains are frequently the location of multiple sites for post-translational modification, the key element of metabolic control in the cell. When a disordered domain folds upon binding to a partner, the resulting complex buries a far greater surface area than in an interaction of comparably-sized folded proteins, thus maximizing specificity at modest protein size. Disorder also maintains accessibility of sites for post-translational modification. Because of their inherent plasticity, disordered domains frequently adopt entirely different structures when bound to different partners, increasing the repertoire of available interactions without the necessity for expression of many different proteins. This feature also adds to the faithfulness of cellular regulation, as the availability of a given disordered domain depends on competition between various partners relevant to different cellular processes. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin

NASA Astrophysics Data System (ADS)

Picas, Laura; Viaud, Julien; Schauer, Kristine; Vanni, Stefano; Hnia, Karim; Fraisier, Vincent; Roux, Aurélien; Bassereau, Patricia; Gaits-Iacovoni, Frédérique; Payrastre, Bernard; Laporte, Jocelyn; Manneville, Jean-Baptiste; Goud, Bruno

2014-12-01

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Ligand-promoted protein folding by biased kinetic partitioning.

PubMed

Hingorani, Karan S; Metcalf, Matthew C; Deming, Derrick T; Garman, Scott C; Powers, Evan T; Gierasch, Lila M

2017-04-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems.

Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

PubMed Central

Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

2017-01-01

Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control.

PubMed

Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi

2016-11-01

The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.

Imaging dynamic and selective low-complexity domain interactions that control gene transcription.

PubMed

Chong, Shasha; Dugast-Darzacq, Claire; Liu, Zhe; Dong, Peng; Dailey, Gina M; Cattoglio, Claudia; Heckert, Alec; Banala, Sambashiva; Lavis, Luke; Darzacq, Xavier; Tjian, Robert

2018-06-21

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity domains (LCDs), but how they drive transactivation remains unclear. Here, live-cell single-molecule imaging reveals that TF-LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF-LCD hubs stabilize DNA binding, recruit RNA polymerase II (Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for novel drugs targeting gene regulatory interactions implicated in disease. Copyright © 2018, American Association for the Advancement of Science.

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant

PubMed Central

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A.; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K.; Altenbach, Christian; Hong, Yuan; Hanson, Susan M.; Palazzo, Maria C.; Chen, Jeannie; Hubbell, Wayne L.; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2013-01-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. PMID:24012956

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

PubMed

Song, Xiufeng; Seo, Jungwon; Baameur, Faiza; Vishnivetskiy, Sergey A; Chen, Qiuyan; Kook, Seunghyi; Kim, Miyeon; Brooks, Evan K; Altenbach, Christian; Hong, Yuan; Hanson, Susan M; Palazzo, Maria C; Chen, Jeannie; Hubbell, Wayne L; Gurevich, Eugenia V; Gurevich, Vsevolod V

2013-12-01

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization. © 2013.

Intrinsically Disordered Energy Landscapes

PubMed Central

Chebaro, Yassmine; Ballard, Andrew J.; Chakraborty, Debayan; Wales, David J.

2015-01-01

Analysis of an intrinsically disordered protein (IDP) reveals an underlying multifunnel structure for the energy landscape. We suggest that such ‘intrinsically disordered’ landscapes, with a number of very different competing low-energy structures, are likely to characterise IDPs, and provide a useful way to address their properties. In particular, IDPs are present in many cellular protein interaction networks, and several questions arise regarding how they bind to partners. Are conformations resembling the bound structure selected for binding, or does further folding occur on binding the partner in a induced-fit fashion? We focus on the p53 upregulated modulator of apoptosis (PUMA) protein, which adopts an -helical conformation when bound to its partner, and is involved in the activation of apoptosis. Recent experimental evidence shows that folding is not necessary for binding, and supports an induced-fit mechanism. Using a variety of computational approaches we deduce the molecular mechanism behind the instability of the PUMA peptide as a helix in isolation. We find significant barriers between partially folded states and the helix. Our results show that the favoured conformations are molten-globule like, stabilised by charged and hydrophobic contacts, with structures resembling the bound state relatively unpopulated in equilibrium. PMID:25999294

Free-energy landscape of intrinsically disordered proteins investigated by all-atom multicanonical molecular dynamics.

PubMed

Higo, Junichi; Umezawa, Koji

2014-01-01

We introduce computational studies on intrinsically disordered proteins (IDPs). Especially, we present our multicanonical molecular dynamics (McMD) simulations of two IDP-partner systems: NRSF-mSin3 and pKID-KIX. McMD is one of enhanced conformational sampling methods useful for conformational sampling of biomolecular systems. IDP adopts a specific tertiary structure upon binding to its partner molecule, although it is unstructured in the unbound state (i.e. the free state). This IDP-specific property is called "coupled folding and binding". The McMD simulation treats the biomolecules with an all-atom model immersed in an explicit solvent. In the initial configuration of simulation, IDP and its partner molecules are set to be distant from each other, and the IDP conformation is disordered. The computationally obtained free-energy landscape for coupled folding and binding has shown that native- and non-native-complex clusters distribute complicatedly in the conformational space. The all-atom simulation suggests that both of induced-folding and population-selection are coupled complicatedly in the coupled folding and binding. Further analyses have exemplified that the conformational fluctuations (dynamical flexibility) in the bound and unbound states are essentially important to characterize IDP functioning.

Structural features and ligand binding properties of tandem WW domains from YAP and TAZ, nuclear effectors of the Hippo pathway.

PubMed

Webb, Claire; Upadhyay, Abhishek; Giuntini, Francesca; Eggleston, Ian; Furutani-Seiki, Makoto; Ishima, Rieko; Bagby, Stefan

2011-04-26

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

PubMed Central

Aragón, Eric; Goerner, Nina; Zaromytidou, Alexia-Ileana; Xi, Qiaoran; Escobedo, Albert; Massagué, Joan; Macias, Maria J.

2011-01-01

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events—Smad activation first and destruction later—and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers. PMID:21685363

Gab1 Is Required for Cell Cycle Transition, Cell Proliferation, and Transformation Induced by an Oncogenic Met Receptor

PubMed Central

Mood, Kathleen; Saucier, Caroline; Bong, Yong-Sik; Lee, Hyun-Shik; Park, Morag

2006-01-01

We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cγ binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met–mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor. PMID:16775003

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.

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane[OPEN

PubMed Central

Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R.

2015-01-01

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners. PMID:25747882

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth*

PubMed Central

Mertz, Joseph; Tan, Haiyan; Pagala, Vishwajeeth; Bai, Bing; Chen, Ping-Chung; Li, Yuxin; Cho, Ji-Hoon; Shaw, Timothy; Wang, Xusheng; Peng, Junmin

2015-01-01

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development. PMID:25931508

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

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

Rudenko, Gabby

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, formingtrans-complexes spanning the synaptic cleft orcis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affectmore » their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics.« less

Dynamic Control of Synaptic Adhesion and Organizing Molecules in Synaptic Plasticity

PubMed Central

2017-01-01

Synapses play a critical role in establishing and maintaining neural circuits, permitting targeted information transfer throughout the brain. A large portfolio of synaptic adhesion/organizing molecules (SAMs) exists in the mammalian brain involved in synapse development and maintenance. SAMs bind protein partners, forming trans-complexes spanning the synaptic cleft or cis-complexes attached to the same synaptic membrane. SAMs play key roles in cell adhesion and in organizing protein interaction networks; they can also provide mechanisms of recognition, generate scaffolds onto which partners can dock, and likely take part in signaling processes as well. SAMs are regulated through a portfolio of different mechanisms that affect their protein levels, precise localization, stability, and the availability of their partners at synapses. Interaction of SAMs with their partners can further be strengthened or weakened through alternative splicing, competing protein partners, ectodomain shedding, or astrocytically secreted factors. Given that numerous SAMs appear altered by synaptic activity, in vivo, these molecules may be used to dynamically scale up or scale down synaptic communication. Many SAMs, including neurexins, neuroligins, cadherins, and contactins, are now implicated in neuropsychiatric and neurodevelopmental diseases, such as autism spectrum disorder, schizophrenia, and bipolar disorder and studying their molecular mechanisms holds promise for developing novel therapeutics. PMID:28255461

Regulation of BDNF chromatin status and promoter accessibility in a neural correlate of associative learning

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce

2015-01-01

Brain-derived neurotrophic factor (BDNF) gene expression critically controls learning and its aberrant regulation is implicated in Alzheimer's disease and a host of neurodevelopmental disorders. The BDNF gene is target of known DNA regulatory mechanisms but details of its activity-dependent regulation are not fully characterized. We performed a comprehensive analysis of the epigenetic regulation of the turtle BDNF gene (tBDNF) during a neural correlate of associative learning using an in vitro model of eye blink classical conditioning. Shortly after conditioning onset, the results from ChIP-qPCR show conditioning-dependent increases in methyl-CpG-binding protein 2 (MeCP2) and repressor basic helix-loop-helix binding protein 2 (BHLHB2) binding to tBDNF promoter II that corresponds with transcriptional repression. In contrast, enhanced binding of ten-eleven translocation protein 1 (Tet1), extracellular signal-regulated kinase 1/2 (ERK1/2), and cAMP response element-binding protein (CREB) to promoter III corresponds with transcriptional activation. These actions are accompanied by rapid modifications in histone methylation and phosphorylation status of RNA polymerase II (RNAP II). Significantly, these remarkably coordinated changes in epigenetic factors for two alternatively regulated tBDNF promoters during conditioning are controlled by Tet1 and ERK1/2. Our findings indicate that Tet1 and ERK1/2 are critical partners that, through complementary functions, control learning-dependent tBDNF promoter accessibility required for rapid transcription and acquisition of classical conditioning. PMID:26336984

NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

PubMed

Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

2016-09-01

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

Tes, a specific Mena interacting partner, breaks the rules for EVH1 binding.

PubMed

Boëda, Batiste; Briggs, David C; Higgins, Theresa; Garvalov, Boyan K; Fadden, Andrew J; McDonald, Neil Q; Way, Michael

2007-12-28

The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.

Phosphorylation of an intrinsically disordered segment in Ets1 shifts conformational sampling toward binding-competent substates.

PubMed

Bui, Jennifer M; Gsponer, Jörg

2014-08-05

Functions of many proteins are affected by posttranslational modifications of intrinsically disordered (ID) regions, yet little is known about the underlying molecular mechanisms. By combining molecular dynamics simulations and protein docking, we demonstrate that the addition of phosphates to an ID segment adjacent to the PNT domain of Ets1 directs conformational sampling toward substates that are most compatible with high-affinity binding of the TAZ1 domain of its coactivator CBP. The phosphate charges disrupt salt bridges and thereby open a hydrophobic cleft and expose hydrophobic residues at the ID N terminus. The structure of the PNT-TAZ1 complex that we determined shows that PNT binds to TAZ1 via these hydrophobic regions in a similar manner to how it interacts with other partners. Our calculations reveal a dual effect of phosphorylation in that it changes the dynamics of PNT so that it becomes more compatible for TAZ1 binding and increases complementarity with this binding partner. Copyright © 2014 Elsevier Ltd. All rights reserved.

Probing protein flexibility reveals a mechanism for selective promiscuity

PubMed Central

Pabon, Nicolas A; Camacho, Carlos J

2017-01-01

Many eukaryotic regulatory proteins adopt distinct bound and unbound conformations, and use this structural flexibility to bind specifically to multiple partners. However, we lack an understanding of how an interface can select some ligands, but not others. Here, we present a molecular dynamics approach to identify and quantitatively evaluate the interactions responsible for this selective promiscuity. We apply this approach to the anticancer target PD-1 and its ligands PD-L1 and PD-L2. We discover that while unbound PD-1 exhibits a hard-to-drug hydrophilic interface, conserved specific triggers encoded in the cognate ligands activate a promiscuous binding pathway that reveals a flexible hydrophobic binding cavity. Specificity is then established by additional contacts that stabilize the PD-1 cavity into distinct bound-like modes. Collectively, our studies provide insight into the structural basis and evolution of multiple binding partners, and also suggest a biophysical approach to exploit innate binding pathways to drug seemingly undruggable targets. DOI: http://dx.doi.org/10.7554/eLife.22889.001 PMID:28432789

Concerted application of LC-MS and ligand binding assays to better understand exposure of a large molecule drug.

PubMed

Xu, Weifeng; Jiang, Hao; Titsch, Craig; Gadkari, Snaehal; Batog, Alicja; Wang, Bonnie; Hippeli, Lauren; Yamamoto, Brent; Chadwick, Kristina; Wheeler, Jennifer; Thompson, Chris; Stahl, James; Willett, Scott; DeSilva, Binodh S; Myler, Heather; Dodge, Robert W; Pillutla, Renuka C

2018-06-20

A ligand-binding assay (LBA) was used to measure exposure of PRM-151, the recombinant form of human pentraxin-2 (PTX-2), a complex pentamer with multiple binding partners. However, the assay showed a lack of dose-dependent exposure in select preclinical species and it could not differentiate the infused PRM-151 from the endogenous PTX-2 in nonhuman primates. Instead of assessing interference from its multiple binding partners, which could be time consuming and laborious, a LC-MS assay avoid of these interference was implemented to measure 'total' drug without the use of immunoaffinity capture reagents. The resultant LC-MS data confirmed the original data and the lack of dose-dependent exposure is now understood to be due to the multiple and diverse targets and functions and resultant complex biodistribution rather than an assay artifact.

Specialized rules of gene transcription in male germ cells: the CREM paradigm.

PubMed

Monaco, Lucia; Kotaja, Noora; Fienga, Giulia; Hogeveen, Kevin; Kolthur, Ullas S; Kimmins, Sarah; Brancorsini, Stefano; Macho, Betina; Sassone-Corsi, Paolo

2004-12-01

Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.

Designing photoswitchable peptides using the AsLOV2 domain.

PubMed

Lungu, Oana I; Hallett, Ryan A; Choi, Eun Jung; Aiken, Mary J; Hahn, Klaus M; Kuhlman, Brian

2012-04-20

Photocontrol of functional peptides is a powerful tool for spatial and temporal control of cell signaling events. We show that the genetically encoded light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2) can be used to reversibly photomodulate the affinity of peptides for their binding partners. Sequence analysis and molecular modeling were used to embed two peptides into the Jα helix of the AsLOV2 domain while maintaining AsLOV2 structure in the dark but allowing for binding to effector proteins when the Jα helix unfolds in the light. Caged versions of the ipaA and SsrA peptides, LOV-ipaA and LOV-SsrA, bind their targets with 49- and 8-fold enhanced affinity in the light, respectively. These switches can be used as general tools for light-dependent colocalization, which we demonstrate with photo-activable gene transcription in yeast. Copyright © 2012 Elsevier Ltd. All rights reserved.

High-stringency screening of target-binding partners using a microfluidic device

DOEpatents

Soh, Hyongsok; Lou, Xinhui; Lagally, Eric

2015-12-01

The invention provides a method of screening a library of candidate agents by contacting the library with a target in a reaction mixture under a condition of high stringency, wherein the target includes a tag that responds to a controllable force applied to the tag, and passing the members of the library through a microfluidic device in a manner that exposes the library members to the controllable force, thereby displacing members of the library that are bound to the target relative to their unbound counterparts. Kits and systems for use with the methods of the invention are also provided.

An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)

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

Song, Mia; He, Qianjing; Berk, Benjamin-Andreas

2016-01-15

Filamin A (FLNA) is an actin filament crosslinking protein with multiple intracellular binding partners. Mechanical force exposes cryptic FLNA binding sites for some of these ligands. To identify new force-dependent binding interactions, we used a fusion construct composed of two FLNA domains, one of which was previously identified as containing a force-dependent binding site as a bait in a yeast two-hybrid system and identified the Rho dissociation inhibitor 2 (RhoGDI2) as a potential interacting partner. A RhoGDI2 truncate with 81 N-terminal amino acid residues and a phosphomimetic mutant, RhoGDI(Tyr153Glu) interacted with the FLNA construct. However, neither wild-type or full-length RhoGDI2 phosphorylatedmore » at Y153 interacted with FLNA. Our interpretation of these contradictions is that truncation and/or mutation of RhoGDI2 perturbs its conformation to expose a site that adventitiously binds FLNA and is not a bona–fide interaction. Therefore, previous studies reporting that a RhoGDI(Y153E) mutant suppresses the metastasis of human bladder cancer cells must be reinvestigated in light of artificial interaction of this point mutant with FLNA. - Highlights: • RhoGDI2 is identified as a potential filamin A (FLNA)-binding partner. • Phosphomimetic mutant, RhoGDI2(Tyr153Glu) interacts with FLNA. • RhoGDI2 phosphorylated (Tyr153) by src kinase does not interact with FLNA. • Mutation of Tyr-153 to Glu of RhoGDI2 does not mimic phosphorylation. • RhoGDI2(Tyr153Glu) provokes an adventitious interaction with FLNA.« less

Repression of transcriptional activity of C/EBPalpha by E2F-dimerization partner complexes.

PubMed

Zaragoza, Katrin; Bégay, Valérie; Schuetz, Anja; Heinemann, Udo; Leutz, Achim

2010-05-01

The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPalpha transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPalpha BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPalpha mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPalpha interacts with the dimerization partner (DP) of E2F and that C/EBPalpha-E2F/DP interaction prevents both binding of C/EBPalpha to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPalpha, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis.

Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions.

PubMed

Glover, Karen; Mei, Yang; Sinha, Sangita C

2016-10-01

Many proteins contain intrinsically disordered regions (IDRs) lacking stable secondary and ordered tertiary structure. IDRs are often implicated in macromolecular interactions, and may undergo structural transitions upon binding to interaction partners. However, as binding partners of many protein IDRs are unknown, these structural transitions are difficult to verify and often are poorly understood. In this study we describe a method to identify IDRs that are likely to undergo helical transitions upon binding. This method combines bioinformatics analyses followed by circular dichroism spectroscopy to monitor 2,2,2-trifluoroethanol (TFE)-induced changes in secondary structure content of these IDRs. Our results demonstrate that there is no significant change in the helicity of IDRs that are not predicted to fold upon binding. IDRs that are predicted to fold fall into two groups: one group does not become helical in the presence of TFE and includes examples of IDRs that form β-strands upon binding, while the other group becomes more helical and includes examples that are known to fold into helices upon binding. Therefore, we propose that bioinformatics analyses combined with experimental evaluation using TFE may provide a general method to identify IDRs that undergo binding-induced disorder-to-helix transitions. Copyright © 2016 Elsevier B.V. All rights reserved.

Structures of the APC–ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners

PubMed Central

Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC–Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC–ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC–ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC–ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC–ARM binding partners. PMID:27462415

Structures of the APC-ARM domain in complexes with discrete Amer1/WTX fragments reveal that it uses a consensus mode to recognize its binding partners.

PubMed

Zhang, Zhenyi; Akyildiz, Senem; Xiao, Yafei; Gai, Zhongchao; An, Ying; Behrens, Jürgen; Wu, Geng

2015-01-01

The tumor suppressor APC employs its conserved armadillo repeat (ARM) domain to recognize many of its binding partners, including Amer1/WTX, which is mutated in Wilms' tumor and bone overgrowth syndrome. The APC-Amer1 complex has important roles in regulating Wnt signaling and cell adhesion. Three sites A1, A2, and A3 of Amer1 have been reported to mediate its interaction with APC-ARM. In this study, crystal structures of APC-ARM in complexes with Amer1-A1, -A2, and -A4, which is newly identified in this work, were determined. Combined with our GST pull-down, yeast two-hybrid, and isothermal titration calorimetry (ITC) assay results using mutants of APC and Amer1 interface residues, our structures demonstrate that Amer1-A1, -A2, and -A4, as well as other APC-binding proteins such as Asef and Sam68, all employ a common recognition pattern to associate with APC-ARM. In contrast, Amer1-A3 binds to the C-terminal side of APC-ARM through a bipartite interaction mode. Composite mutations on either APC or Amer1 disrupting all four interfaces abrogated their association in cultured cells and impaired the membrane recruitment of APC by Amer1. Our study thus comprehensively elucidated the recognition mechanism between APC and Amer1, and revealed a consensus recognition sequence employed by various APC-ARM binding partners.

Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

DOE PAGES

Guntas, Gurkan; Hallett, Ryan A.; Zimmerman, Seth P.; ...

2014-12-22

The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. In this study, to create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2more » (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. Finally, we demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.« less

Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

PubMed Central

Capitanio, Juliana S; Montpetit, Ben; Wozniak, Richard W

2017-01-01

Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases. DOI: http://dx.doi.org/10.7554/eLife.18825.001 PMID:28221134

Intramolecular interactions regulate SAP97 binding to GKAP

PubMed Central

Wu, Hongju; Reissner, Carsten; Kuhlendahl, Sven; Coblentz, Blake; Reuver, Susanne; Kindler, Stefan; Gundelfinger, Eckart D.; Garner, Craig C.

2000-01-01

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP–GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners. PMID:11060025

Molecular Mechanism Underlying the Entomotoxic Effect of Colocasia esculenta Tuber Agglutinin against Dysdercus cingulatus

PubMed Central

Roy, Amit; Das, Sampa

2015-01-01

Colocasia esculenta tuber agglutinin (CEA), a mannose binding lectin, exhibits insecticidal efficacy against different hemipteran pests. Dysdercus cingulatus, red cotton bug (RCB), has also shown significant susceptibility to CEA intoxication. However, the molecular basis behind such entomotoxicity of CEA has not been addressed adequately. The present study elucidates the mechanism of insecticidal efficacy of CEA against RCB. Confocal and scanning electron microscopic analyses documented CEA binding to insect midgut tissue, resulting in an alteration of perimicrovillar membrane (PMM) morphology. Internalization of CEA into insect haemolymph and ovary was documented by western blotting analyses. Ligand blot followed by mass spectrometric identification revealed the cognate binding partners of CEA as actin, ATPase and cytochrome P450. Deglycosylation and mannose inhibition assays indicated the interaction to probably be mannose mediated. Bioinformatic identification of putative glycosylation or mannosylation sites in the binding partners further supports the sugar mediated interaction. Correlating entomotoxicity of CEA with immune histological and binding assays to the insect gut contributes to a better understanding of the insecticidal potential of CEA and endorses its future biotechnological application.

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

Mays, Suzanne G.; Okafor, C. Denise; Tuntland, Micheal L.

Peroxisome proliferator-activated gamma coactivator 1-α (PGC1α) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1α is known to bind and activate LRH-1, mechanisms through which PGC1α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1–PGC1α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), inmore » coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1–PGC1α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1α but promoted allosteric signaling from the helix 6/β-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1–PGC1α interaction and may illuminate strategies for selective therapeutic targeting of PGC1α-dependent LRH-1 signaling pathways.« less

Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

PubMed

Enz, Ralf

2012-01-01

Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

Great interactions: How binding incorrect partners can teach us about protein recognition and function.

PubMed

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra; Sacquin-Mora, Sophie

2016-10-01

Protein-protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross-docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross-docking predictions using the area under the specificity-sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding-site predictions resulting from the cross-docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408-1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

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

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

A tool for calculating binding-site residues on proteins from PDB structures.

PubMed

Hu, Jing; Yan, Changhui

2009-08-03

In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB) that consists of the protein of interest and its interacting partner(s) and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. The developed tool is very useful for the research on protein binding site analysis and prediction.

Protein social behavior makes a stronger signal for partner identification than surface geometry

PubMed Central

Laine, Elodie

2016-01-01

ABSTRACT Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico‐chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross‐docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S‐index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface‐based (ranking) score to discriminate partners from non‐interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137–154. © 2016 Wiley Periodicals, Inc. PMID:27802579

Protein social behavior makes a stronger signal for partner identification than surface geometry.

PubMed

Laine, Elodie; Carbone, Alessandra

2017-01-01

Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico-chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross-docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S-index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface-based (ranking) score to discriminate partners from non-interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137-154. © 2016 Wiley Periodicals, Inc. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Effects of the Insulin-like Growth Factor Pathway on the Regulation of Mammary Gland Development.

PubMed

Ha, Woo Tae; Jeong, Ha Yeon; Lee, Seung Yoon; Song, Hyuk

2016-09-01

The insulin-like growth factor (IGF) pathway is a key signal transduction pathway involved in cell proliferation, migration, and apoptosis. In dairy cows, IGF family proteins and binding receptors, including their intracellular binding partners, regulate mammary gland development. IGFs and IGF receptor interactions in mammary glands influence the early stages of mammogenesis, i.e., mammary ductal genesis until puberty. The IGF pathway includes three major components, IGFs (such as IGF-I, IGF-II, and insulin), their specific receptors, and their high-affinity binding partners (IGF binding proteins [IGFBPs]; i.e., IGFBP1-6), including specific proteases for each IGFBP. Additionally, IGFs and IGFBP interactions are critical for the bioactivities of various intracellular mechanisms, including cell proliferation, migration, and apoptosis. Notably, the interactions between IGFs and IGFBPs in the IGF pathway have been difficult to characterize during specific stages of bovine mammary gland development. In this review, we aim to describe the role of the interaction between IGFs and IGFBPs in overall mammary gland development in dairy cows.

TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.

PubMed

Lin, Jiangguo; Countryman, Preston; Buncher, Noah; Kaur, Parminder; E, Longjiang; Zhang, Yiyun; Gibson, Greg; You, Changjiang; Watkins, Simon C; Piehler, Jacob; Opresko, Patricia L; Kad, Neil M; Wang, Hong

2014-02-01

Human telomeres are maintained by the shelterin protein complex in which TRF1 and TRF2 bind directly to duplex telomeric DNA. How these proteins find telomeric sequences among a genome of billions of base pairs and how they find protein partners to form the shelterin complex remains uncertain. Using single-molecule fluorescence imaging of quantum dot-labeled TRF1 and TRF2, we study how these proteins locate TTAGGG repeats on DNA tightropes. By virtue of its basic domain TRF2 performs an extensive 1D search on nontelomeric DNA, whereas TRF1's 1D search is limited. Unlike the stable and static associations observed for other proteins at specific binding sites, TRF proteins possess reduced binding stability marked by transient binding (∼ 9-17 s) and slow 1D diffusion on specific telomeric regions. These slow diffusion constants yield activation energy barriers to sliding ∼ 2.8-3.6 κ(B)T greater than those for nontelomeric DNA. We propose that the TRF proteins use 1D sliding to find protein partners and assemble the shelterin complex, which in turn stabilizes the interaction with specific telomeric DNA. This 'tag-team proofreading' represents a more general mechanism to ensure a specific set of proteins interact with each other on long repetitive specific DNA sequences without requiring external energy sources.

Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

PubMed

Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

2017-03-01

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

PubMed Central

Kleino, Iivari; Järviluoma, Annika; Hepojoki, Jussi; Huovila, Ari Pekka; Saksela, Kalle

2015-01-01

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior. PMID:25825872

EHD proteins: Key conductors of endocytic transport

PubMed Central

Naslavsky, Naava; Caplan, Steve

2010-01-01

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

Collybistin binds and inhibits mTORC1 signaling: a potential novel mechanism contributing to intellectual disability and autism.

PubMed

Machado, Camila Oliveira Freitas; Griesi-Oliveira, Karina; Rosenberg, Carla; Kok, Fernando; Martins, Stephanie; Passos-Bueno, Maria Rita; Sertie, Andrea Laurato

2016-01-01

Protein synthesis regulation via mammalian target of rapamycin complex 1 (mTORC1) signaling pathway has key roles in neural development and function, and its dysregulation is involved in neurodevelopmental disorders associated with autism and intellectual disability. mTOR regulates assembly of the translation initiation machinery by interacting with the eukaryotic initiation factor eIF3 complex and by controlling phosphorylation of key translational regulators. Collybistin (CB), a neuron-specific Rho-GEF responsible for X-linked intellectual disability with epilepsy, also interacts with eIF3, and its binding partner gephyrin associates with mTOR. Therefore, we hypothesized that CB also binds mTOR and affects mTORC1 signaling activity in neuronal cells. Here, by using induced pluripotent stem cell-derived neural progenitor cells from a male patient with a deletion of entire CB gene and from control individuals, as well as a heterologous expression system, we describe that CB physically interacts with mTOR and inhibits mTORC1 signaling pathway and protein synthesis. These findings suggest that disinhibited mTORC1 signaling may also contribute to the pathological process in patients with loss-of-function variants in CB.

Collybistin binds and inhibits mTORC1 signaling: a potential novel mechanism contributing to intellectual disability and autism

PubMed Central

Machado, Camila Oliveira Freitas; Griesi-Oliveira, Karina; Rosenberg, Carla; Kok, Fernando; Martins, Stephanie; Rita Passos-Bueno, Maria; Sertie, Andrea Laurato

2016-01-01

Protein synthesis regulation via mammalian target of rapamycin complex 1 (mTORC1) signaling pathway has key roles in neural development and function, and its dysregulation is involved in neurodevelopmental disorders associated with autism and intellectual disability. mTOR regulates assembly of the translation initiation machinery by interacting with the eukaryotic initiation factor eIF3 complex and by controlling phosphorylation of key translational regulators. Collybistin (CB), a neuron-specific Rho-GEF responsible for X-linked intellectual disability with epilepsy, also interacts with eIF3, and its binding partner gephyrin associates with mTOR. Therefore, we hypothesized that CB also binds mTOR and affects mTORC1 signaling activity in neuronal cells. Here, by using induced pluripotent stem cell-derived neural progenitor cells from a male patient with a deletion of entire CB gene and from control individuals, as well as a heterologous expression system, we describe that CB physically interacts with mTOR and inhibits mTORC1 signaling pathway and protein synthesis. These findings suggest that disinhibited mTORC1 signaling may also contribute to the pathological process in patients with loss-of-function variants in CB. PMID:25898924

Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control

PubMed Central

Grant, Gavin D.; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K.; Mahoney, J. Matthew; Loros, Jennifer J.; Dunlap, Jay C.; Whitfield, Michael L.

2012-01-01

We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant. PMID:22740631

NM23 proteins: innocent bystanders or local energy boosters for CFTR?

PubMed

Muimo, Richmond; Alothaid, Hani Mm; Mehta, Anil

2018-03-01

NM23 proteins NDPK-A and -B bind to the cystic fibrosis (CF) protein CFTR in different ways from kinases such as PKA, CK2 and AMPK or linkers to cell calcium such as calmodulin and annexins. NDPK-A (not -B) interacts with CFTR through reciprocal AMPK binding/control, whereas NDPK-B (not -A) binds directly to CFTR. NDPK-B can activate G proteins without ligand-receptor coupling, so perhaps NDPK-B's binding influences energy supply local to a nucleotide-binding site (NBD1) needed for CFTR to function. Curiously, CFTR (ABC-C7) is a member of the ATP-binding cassette (ABC) protein family that does not obey 'clan rules'; CFTR channels anions and is not a pump, regulates disparate processes, is itself regulated by multiple means and is so pleiotropic that it acts as a hub that orchestrates calcium signaling through its consorts such as calmodulin/annexins. Furthermore, its multiple partners make CFTR dance to different tunes in different cellular and subcellular locations as it recycles from the plasma membrane to endosomes. CFTR function in airway apical membranes is inhibited by smoking which has been dubbed 'acquired CF'. CFTR alone among family members possesses a trap for other proteins that it unfurls as a 'fish-net' and which bears consensus phosphorylation sites for many protein kinases, with PKA being the most canonical. Recently, the site of CFTR's commonest mutation has been proposed as a knock-in mutant that alters allosteric control of kinase CK2 by log orders of activity towards calmodulin and other substrates after CFTR fragmentation. This link from CK2 to calmodulin that binds the R region invokes molecular paths that control lumen formation, which is incomplete in the tracheas of some CF-affected babies. Thus, we are poised to understand the many roles of NDPK-A and -B in CFTR function and, especially lumen formation, which is defective in the gut and lungs of many CF babies.

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

PubMed

Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Complex Interdependence Regulates Heterotypic Transcription Factor Distribution and Coordinates Cardiogenesis.

PubMed

Luna-Zurita, Luis; Stirnimann, Christian U; Glatt, Sebastian; Kaynak, Bogac L; Thomas, Sean; Baudin, Florence; Samee, Md Abul Hassan; He, Daniel; Small, Eric M; Mileikovsky, Maria; Nagy, Andras; Holloway, Alisha K; Pollard, Katherine S; Müller, Christoph W; Bruneau, Benoit G

2016-02-25

Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

Allosteric conformational barcodes direct signaling in the cell.

PubMed

Nussinov, Ruth; Ma, Buyong; Tsai, Chung-Jung; Csermely, Peter

2013-09-03

The cellular network is highly interconnected. Pathways merge and diverge. They proceed through shared proteins and may change directions. How are cellular pathways controlled and their directions decided, coded, and read? These questions become particularly acute when we consider that a small number of pathways, such as signaling pathways that regulate cell fates, cell proliferation, and cell death in development, are extensively exploited. This review focuses on these signaling questions from the structural standpoint and discusses the literature in this light. All co-occurring allosteric events (including posttranslational modifications, pathogen binding, and gain-of-function mutations) collectively tag the protein functional site with a unique barcode. The barcode shape is read by an interacting molecule, which transmits the signal. A conformational barcode provides an intracellular address label, which selectively favors binding to one partner and quenches binding to others, and, in this way, determines the pathway direction, and, eventually, the cell's response and fate. Copyright © 2013 Elsevier Ltd. All rights reserved.

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

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

Lim, So-Hee; Moon, Jeonghee; Lee, Myungkyu

2013-09-13

Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified asmore » a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.« less

Interacting protein partners of Arabidopsis RNA binding protein AtRBP45b

USDA-ARS?s Scientific Manuscript database

RNA binding proteins (RBPs) are important players in post-transcriptional gene regulation and shown to play an important role in normal development and in response to environmental perturbations. Arabidopsis RBP, AtRBP45b with triple RNA recognition motifs (RRMs) have are closely related to the yeas...

MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

PubMed

Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

2016-10-20

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms*

PubMed Central

Radnai, László; Rapali, Péter; Hódi, Zsuzsa; Süveges, Dániel; Molnár, Tamás; Kiss, Bence; Bécsi, Bálint; Erdödi, Ferenc; Buday, László; Kardos, József; Kovács, Mihály; Nyitray, László

2010-01-01

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with Kd values of 9 and 40 μm, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: Kd values of 37 and 3.5 nm for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent Kd value (3 μm). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network. PMID:20889982

Replication protein A 32 interacts through a similar binding interface with TIPIN, XPA, and UNG2.

PubMed

Ali, Seikh Imtiaz; Shin, Jae-Sun; Bae, Sung-Hun; Kim, Byoungkook; Choi, Byong-Seok

2010-07-01

The 32kDa subunit of replication protein A (RPA32) is involved in various DNA repair systems such as nucleotide excision repair, base excision repair, and homologous recombination. In these processes, RPA32 interacts with different binding partners via its C-terminal domain (RPA32C; residues 172-270). It has been reported recently that RPA32C also interacts with TIPIN during the intra-S checkpoint. To determine the significance of the interaction of RPA32C with TIPIN, we have examined the interaction mode using NMR spectroscopy and an in silico modeling approach. Here, we show that TIPIN(185-218), which shares high sequence similarity with XPA(10-43) and UNG2(56-89), is less ordered in the free state and then forms a longer alpha-helix upon binding to RPA32C. The binding interface between TIPIN(185-218) and RPA32C is similar to those of XPA and UNG2, but its mode of interaction is different. The results suggest that RPA32 is an exchange point for multiple proteins involved in DNA repair, homologous recombination, and checkpoint processes and that it binds to different partners with comparable binding affinity using a single site. Copyright 2010 Elsevier Ltd. All rights reserved.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

PubMed

Lim, Raelene; Winteringham, Louise N; Williams, James H; McCulloch, Ross K; Ingley, Evan; Tiao, Jim Y-H; Lalonde, Jean-Philippe; Tsai, Schickwann; Tilbrook, Peta A; Sun, Yi; Wu, Xiaohua; Morris, Stephan W; Klinken, S Peter

2002-10-25

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

RanBPM: a potential therapeutic target for modulating diverse physiological disorders.

PubMed

Das, Soumyadip; Suresh, Bharathi; Kim, Hyongbum Henry; Ramakrishna, Suresh

2017-12-01

The Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein involved in a variety of intracellular signaling pathways that control diverse cellular functions. RanBPM interacts with proteins that are linked to various diseases, including Alzheimer's disease (AD), schizophrenia (SCZ), and cancer. In this article, we define the characteristics of the scaffolding protein RanBPM and focus on its interaction partners in diverse physiological disorders, such as neurological diseases, fertility disorders, and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence

PubMed Central

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-01-01

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction. PMID:26578773

PML IV/ARF interaction enhances p53 SUMO-1 conjugation, activation, and senescence.

PubMed

Ivanschitz, Lisa; Takahashi, Yuki; Jollivet, Florence; Ayrault, Olivier; Le Bras, Morgane; de Thé, Hugues

2015-11-17

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) recruit multiple partners, including p53 and many of its regulators. NBs are believed to facilitate several posttranslational modifications and are key regulators of senescence. PML, the organizer of NBs, is expressed as a number of splice variants that all efficiently recruit p53 partners. However, overexpression of only one of them, PML IV, triggers p53-driven senescence. Here, we show that PML IV specifically binds ARF, a key p53 regulator. Similar to ARF, PML IV enhances global SUMO-1 conjugation, particularly that of p53, resulting in p53 stabilization and activation. ARF interacts with and stabilizes the NB-associated UBC9 SUMO-conjugating enzyme, possibly explaining PML IV-enhanced SUMOylation. These results unexpectedly link two key tumor suppressors, highlighting their convergence for global control of SUMO conjugation, p53 activation, and senescence induction.

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions

PubMed Central

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-01-01

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. PMID:27604871

Structure-based multiscale approach for identification of interaction partners of PDZ domains.

PubMed

Tiwari, Garima; Mohanty, Debasisa

2014-04-28

PDZ domains are peptide recognition modules which mediate specific protein-protein interactions and are known to have a complex specificity landscape. We have developed a novel structure-based multiscale approach which identifies crucial specificity determining residues (SDRs) of PDZ domains from explicit solvent molecular dynamics (MD) simulations on PDZ-peptide complexes and uses these SDRs in combination with knowledge-based scoring functions for proteomewide identification of their interaction partners. Multiple explicit solvent simulations ranging from 5 to 50 ns duration have been carried out on 28 PDZ-peptide complexes with known binding affinities. MM/PBSA binding energy values calculated from these simulations show a correlation coefficient of 0.755 with the experimental binding affinities. On the basis of the SDRs of PDZ domains identified by MD simulations, we have developed a simple scoring scheme for evaluating binding energies for PDZ-peptide complexes using residue based statistical pair potentials. This multiscale approach has been benchmarked on a mouse PDZ proteome array data set by calculating the binding energies for 217 different substrate peptides in binding pockets of 64 different mouse PDZ domains. Receiver operating characteristic (ROC) curve analysis indicates that, the area under curve (AUC) values for binder vs nonbinder classification by our structure based method is 0.780. Our structure based method does not require experimental PDZ-peptide binding data for training.

Understanding the mechanisms of protein-DNA interactions

NASA Astrophysics Data System (ADS)

Lavery, Richard

2004-03-01

Structural, biochemical and thermodynamic data on protein-DNA interactions show that specific recognition cannot be reduced to a simple set of binary interactions between the partners (such as hydrogen bonds, ion pairs or steric contacts). The mechanical properties of the partners also play a role and, in the case of DNA, variations in both conformation and flexibility as a function of base sequence can be a significant factor in guiding a protein to the correct binding site. All-atom molecular modeling offers a means of analyzing the role of different binding mechanisms within protein-DNA complexes of known structure. This however requires estimating the binding strengths for the full range of sequences with which a given protein can interact. Since this number grows exponentially with the length of the binding site it is necessary to find a method to accelerate the calculations. We have achieved this by using a multi-copy approach (ADAPT) which allows us to build a DNA fragment with a variable base sequence. The results obtained with this method correlate well with experimental consensus binding sequences. They enable us to show that indirect recognition mechanisms involving the sequence dependent properties of DNA play a significant role in many complexes. This approach also offers a means of predicting protein binding sites on the basis of binding energies, which is complementary to conventional lexical techniques.

Abundance of intrinsic structural disorder in the histone H1 subtypes.

PubMed

Kowalski, Andrzej

2015-12-01

The intrinsically disordered proteins consist of partially structured regions linked to the unstructured stretches, which consequently form the transient and dynamic conformational ensembles. They undergo disorder to order transition upon binding their partners. Intrinsic disorder is attributed to histones H1, perceived as assemblers of chromatin structure and the regulators of DNA and proteins activity. In this work, the comparison of intrinsic disorder abundance in the histone H1 subtypes was performed both by the analysis of their amino acid composition and by the prediction of disordered stretches, as well as by identifying molecular recognition features (MoRFs) and ANCHOR protein binding regions (APBR) that are responsible for recognition and binding. Both human and model organisms-animals, plants, fungi and protists-have H1 histone subtypes with the properties typical of disordered state. They possess a significantly higher content of hydrophilic and charged amino acid residues, arranged in the long regions, covering over half of the whole amino acid residues in chain. Almost complete disorder corresponds to histone H1 terminal domains, including MoRFs and ANCHOR. Those motifs were also identified in a more ordered histone H1 globular domain. Compared to the control (globular and fibrous) proteins, H1 histones demonstrate the increased folding rate and a higher proportion of low-complexity segments. The results of this work indicate that intrinsic disorder is an inherent structural property of histone H1 subtypes and it is essential for establishing a protein conformation which defines functional outcomes affecting on DNA- and/or partner protein-dependent cell processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structure and Dynamics of the Liver Receptor Homolog 1-PGC1α Complex.

PubMed

Mays, Suzanne G; Okafor, C Denise; Tuntland, Micheal L; Whitby, Richard J; Dharmarajan, Venkatasubramanian; Stec, Józef; Griffin, Patrick R; Ortlund, Eric A

2017-07-01

Peroxisome proliferator-activated gamma coactivator 1- α (PGC1 α ) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1 α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1 α is known to bind and activate LRH-1, mechanisms through which PGC1 α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1-PGC1 α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1 α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1-PGC1 α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1 α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1 α but promoted allosteric signaling from the helix 6/ β -sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1-PGC1 α interaction and may illuminate strategies for selective therapeutic targeting of PGC1 α -dependent LRH-1 signaling pathways. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Rapid, Optimized Interactomic Screening

PubMed Central

Hakhverdyan, Zhanna; Domanski, Michal; Hough, Loren; Oroskar, Asha A.; Oroskar, Anil R.; Keegan, Sarah; Dilworth, David J.; Molloy, Kelly R.; Sherman, Vadim; Aitchison, John D.; Fenyö, David; Chait, Brian T.; Jensen, Torben Heick; Rout, Michael P.; LaCava, John

2015-01-01

We must reliably map the interactomes of cellular macromolecular complexes in order to fully explore and understand biological systems. However, there are no methods to accurately predict how to capture a given macromolecular complex with its physiological binding partners. Here, we present a screen that comprehensively explores the parameters affecting the stability of interactions in affinity-captured complexes, enabling the discovery of physiological binding partners and the elucidation of their functional interactions in unparalleled detail. We have implemented this screen on several macromolecular complexes from a variety of organisms, revealing novel profiles even for well-studied proteins. Our approach is robust, economical and automatable, providing an inroad to the rigorous, systematic dissection of cellular interactomes. PMID:25938370

Great interactions: How binding incorrect partners can teach us about protein recognition and function

PubMed Central

Vamparys, Lydie; Laurent, Benoist; Carbone, Alessandra

2016-01-01

ABSTRACT Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:27287388

How the extra methylene group affects the ligation properties of Glu vs. Asp and Gln vs. Asn amino acids: a DFT/PCM study.

PubMed

Dudev, Todor; Doudeva, Lyudmila

2017-02-01

The effect of the extra methylene group on the ligation properties of glutamic (Glu) vs. aspartic (Asp) acid, and glutamine (Gln) vs. asparagine (Asn) amino acids-two pairs of protein building blocks differing by the length of their side chains-has been studied by employing DFT calculations combined with polarizable continuum model (PCM) computations. Complexes of the nominal species with partner ligands of various structures, charge states, and degree of solvent exposure have been examined. The results obtained reveal that the difference in the alkyl chain length of these amino acid residues does not affect the mode of their binding. This, however, influences the thermodynamics of the ligand-ligand and ligand-metal recognition thus bestowing unique ligation characteristics on the competing entities. The calculations reveal that the competition between the longer-chain and shorter-chain analogs is entropy driven and that the differential electronic effects are of minor importance for the process. Thus, the outcome of the rivalry between Asp and Glu, and Asn and Gln is almost unaffected by the nature of the partner ligand, its charge state and, in most cases, the dielectric properties of the binding site. The longer-chain Glu, as opposed to its shorter-chain Asp counterpart, is the preferred partner ligand in various protein binding sites. Contrariwise, the shorter-chain Asn binds more favorably to the respective binding sites than its longer-chain Gln analog. The results obtained shed additional light on the intimate mechanism of the ligand-ligand and ligand-metal recognition in proteins and could be employed as guidelines in protein engineering and design.

Integrated In Silico-In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma.

PubMed

Liu, Jiangang; Wang, Dapeng; Li, Yanyan; Yao, Hui; Zhang, Nan; Zhang, Xuewen; Zhong, Fangping; Huang, Yulun

2018-06-01

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: 161 LGPPSPVK 168 and m2p: 168 KMPSPPWE 175 of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in most performance measures. To the best of our knowledge, this is the first sequence-based prediction of protein-binding nucleotides in RNA which considers the binding partner of RNA. The new model will provide valuable information for designing biochemical experiments to find putative protein-binding sites in RNA with unknown structure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins.

PubMed

Castello, Alfredo; Hentze, Matthias W; Preiss, Thomas

2015-12-01

In the past century, few areas of biology advanced as much as our understanding of the pathways of intermediary metabolism. Initially considered unimportant in terms of gene regulation, crucial cellular fate changes, cell differentiation, or malignant transformation are now known to involve 'metabolic remodeling' with profound changes in the expression of many metabolic enzyme genes. This review focuses on the recent identification of RNA-binding activity of numerous metabolic enzymes. We discuss possible roles of this unexpected second activity in feedback gene regulation ('moonlighting') and/or in the control of enzymatic function. We also consider how metabolism-driven post-translational modifications could regulate enzyme-RNA interactions. Thus, RNA emerges as a new partner of metabolic enzymes with far-reaching possible consequences to be unraveled in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom

PubMed Central

Xu, Yanjie; Liu, Suxuan; Xia, Jixiang; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

2016-01-01

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair. PMID:28199211

Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

PubMed

Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

2015-12-15

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

A Study of the Interaction of Bovine Hemoglobin with Synthetic Dyes Using Spectroscopic Techniques and Molecular Docking.

PubMed

Kamaljeet; Bansal, Saurabh; SenGupta, Uttara

2016-01-01

Synthetic dyes are a very efficient class of dyes that are ingested or come into contact with the skin from numerous sources (cosmetics, textiles, leather, paper, and drugs). An important component of their safety profile is the interactions that they form after they enter the body. Hemoglobin is a functionally important protein that can form multiple interactions with soluble compounds present in the blood, and hence forms an important aspect of the toxicological or safety profile of the dyes. Here we study the interaction between bovine hemoglobin and organic dyes using UV-Vis absorbance and fluorescence spectroscopy. Molecular modeling was used to visualize the binding site and partners of the dye molecules, within the hemoglobin molecule. We find that all four dyes studied form sufficiently strong interactions with hemoglobin to allow for the formation of potentially toxic interactions. Molecular modeling showed that all four dyes bind within the central cavity of the hemoglobin molecule. However, binding partners could not be identified as multiple binding conformations with very similar energies were possible for each dye.

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning.

PubMed

Waskiewicz, A J; Rikhof, H A; Hernandez, R E; Moens, C B

2001-11-01

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr(-) phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Effects of organophosphates on the regulation of mesenchymal stem cell proliferation and differentiation.

PubMed

Prugh, Amber M; Cole, Stephanie D; Glaros, Trevor; Angelini, Daniel J

2017-03-25

Mesenchymal stem cells (MSCs) are multipotent cells located within various adult tissues. Recent literature has reported that human bone marrow-derived MSCs express active acetylcholinesterase (AChE) and that disruption of AChE activity by organophosphate (OP) chemicals decreases the ability of MSCs to differentiate into osteoblasts. The potential role of AChE in regulating MSC proliferation and differentiation is currently unknown. In the present study, we demonstrate that MSCs exposed to OPs have both decreased AChE activity and abundance. In addition, exposure to these OPs induced cellular death while decreasing cellular proliferation. Exposures to these compounds also reduced the adipogenic/osteogenic differentiation potentials of the MSCs. To elucidate the possible role of AChE in MSCs signaling following OP exposure, we captured potential AChE binding partners by performing polyhistidine (His 8 )-tagged AChE pulldowns, followed by protein identification using liquid chromatography mass spectrometry (LC-MS). Using this method, we determined that the focal adhesion protein, vinculin, is a potential binding partner with AChE in MSCs and these initial findings were confirmed with follow-up co-immunoprecipitation experiments. Identifying AChE binding partners helps to determine potential pathways associated with MSC proliferation and differentiation, and this understanding could lead to the development of future MSC-based tissue repair therapies. Published by Elsevier B.V.

The Protein Kingdom Extended: Ordered and Intrinsically Disordered Proteins, Their Folding, Supramolecular Complex Formation, and Aggregation

PubMed Central

Turoverov, Konstantin K.; Kuznetsova, Irina M.; Uversky, Vladimir N.

2010-01-01

The native state of a protein is usually associated with a compact globular conformation possessing a rigid and highly ordered structure. At the turn of the last century certain studies arose which concluded that many proteins cannot, in principle, form a rigid globular structure in an aqueous environment, but they are still able to fulfill their specific functions — i.e., they are native. The existence of the disordered regions allows these proteins to interact with their numerous binding partners. Such interactions are often accompanied by the formation of complexes that possess a more ordered structure than the original components. The functional diversity of these proteins, combined with the variability of signals related to the various intra-and intercellular processes handled by these proteins and their capability to produce multi-variant and multi-directional responses allow them to form a unique regulatory net in a cell. The abundance of disordered proteins inside the cell is precisely controlled at the synthesis and clearance levels as well as via interaction with specific binding partners and posttranslational modifications. Another recently recognized biologically active state of proteins is the functional amyloid. The formation of such functional amyloids is tightly controlled and therefore differs from the uncontrolled formation of pathogenic amyloids which are associated with the pathogenesis of several conformational diseases, the development of which is likely to be determined by the failures of the cellular regulatory systems rather than by the formation of the proteinaceous deposits and/or by the protofibril toxicity. PMID:20097220

The PDZ Ligand Domain of the Human Papillomavirus Type 16 E6 Protein Is Required for E6's Induction of Epithelial Hyperplasia In Vivo

PubMed Central

Nguyen, Marie L.; Nguyen, Minh M.; Lee, Denis; Griep, Anne E.; Lambert, Paul F.

2003-01-01

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6WT) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6Δ146-151, from the human keratin 14 (K14) promoter. The K14E6Δ146-151 mice exhibit a radiation response similar to that of the K14E6WT mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6Δ146-151 mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia. PMID:12768014

Mechanistic insights into phosphoprotein-binding FHA domains.

PubMed

Liang, Xiangyang; Van Doren, Steven R

2008-08-01

[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

PubMed Central

Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-01-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

PubMed

Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-03-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

PubMed Central

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5’-end including the 5’-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer. PMID:26221730

In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

PubMed

Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

2015-01-01

A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA.

PubMed

Jayachandran, Uma; Grey, Heather; Cook, Atlanta G

2016-02-29

Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA-Directed Assembly of Capture Tools for Constitutional Studies of Large Protein Complexes.

PubMed

Meyer, Rebecca; Faesen, Alex; Vogel, Katrin; Jeganathan, Sadasivam; Musacchio, Andrea; Niemeyer, Christof M

2015-06-10

Large supramolecular protein complexes, such as the molecular machinery involved in gene regulation, cell signaling, or cell division, are key in all fundamental processes of life. Detailed elucidation of structure and dynamics of such complexes can be achieved by reverse-engineering parts of the complexes in order to probe their interactions with distinctive binding partners in vitro. The exploitation of DNA nanostructures to mimic partially assembled supramolecular protein complexes in which the presence and state of two or more proteins are decisive for binding of additional building blocks is reported here. To this end, four-way DNA Holliday junction motifs bearing a fluorescein and a biotin tag, for tracking and affinity capture, respectively, are site-specifically functionalized with centromeric protein (CENP) C and CENP-T. The latter serves as baits for binding of the so-called KMN component, thereby mimicking early stages of the assembly of kinetochores, structures that mediate and control the attachment of microtubules to chromosomes in the spindle apparatus. Results from pull-down experiments are consistent with the hypothesis that CENP-C and CENP-T may bind cooperatively to the KMN network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamics, Conformational Entropy, and Frustration in Protein-Protein Interactions Involving an Intrinsically Disordered Protein Domain.

PubMed

Lindström, Ida; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are abundant in the eukaryotic proteome. However, little is known about the role of subnanosecond dynamics and the conformational entropy that it represents in protein-protein interactions involving IDPs. Using nuclear magnetic resonance side chain and backbone relaxation, stopped-flow kinetics, isothermal titration calorimetry, and computational studies, we have characterized the interaction between the globular TAZ1 domain of the CREB binding protein and the intrinsically disordered transactivation domain of STAT2 (TAD-STAT2). We show that the TAZ1/TAD-STAT2 complex retains considerable subnanosecond motions, with TAD-STAT2 undergoing only a partial disorder-to-order transition. We report here the first experimental determination of the conformational entropy change for both binding partners in an IDP binding interaction and find that the total change even exceeds in magnitude the binding enthalpy and is comparable to the contribution from the hydrophobic effect, demonstrating its importance in the binding energetics. Furthermore, we show that the conformational entropy change for TAZ1 is also instrumental in maintaining a biologically meaningful binding affinity. Strikingly, a spatial clustering of very high amplitude motions and a cluster of more rigid sites in the complex exist, which through computational studies we found to overlap with regions that experience energetic frustration and are less frustrated, respectively. Thus, the residual dynamics in the bound state could be necessary for faster dissociation, which is important for proteins that interact with multiple binding partners.

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

PubMed

Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

2016-11-02

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of chlorophyll binding to LIL3.

PubMed

Mork-Jansson, Astrid Elisabeth; Eichacker, Lutz Andreas

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

PubMed

Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

2016-04-01

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

Pbx3 Deficiency Results in Central Hypoventilation

PubMed Central

Rhee, Joon Whan; Arata, Akiko; Selleri, Licia; Jacobs, Yakop; Arata, Satoru; Onimaru, Hiroshi; Cleary, Michael L.

2004-01-01

Pbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder. PMID:15466398

The Role of Control in Intimate Partner Violence: A Study in Dutch Forensic Outpatients.

PubMed

Verschuere, Bruno; van Horn, Joan; Buitelaar, Nannet

2018-05-01

Johnson argued that coercive control is crucial in explaining heterogeneity in intimate partner violence, with such violence being more frequent, less reciprocal, and more often male-to-female aggression when it serves to exercise control over the partner. We assessed 280 Dutch forensic outpatients who had recently engaged in intimate partner violence on nonaggressive coercive control. Control showed significant, small to moderate, associations with more frequent past year acts of psychological aggression, physical assault, and sexual coercion and more frequently resulted in partner injury. Control was unrelated to reciprocity of partner violence. High controlling violence was enacted mostly, but not exclusively by men. Overall, while perhaps not having a uniquely strong association, our findings provide partial support for the role of coercive control in intimate partner violence and suggest it may benefit intimate partner violence risk assessment.

Relationship between Hot Spot Residues and Ligand Binding Hot Spots in Protein-Protein Interfaces

PubMed Central

Zerbe, Brandon S.; Hall, David R.

2013-01-01

In the context of protein-protein interactions, the term “hot spot” refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening. PMID:22770357

Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces.

PubMed

Zerbe, Brandon S; Hall, David R; Vajda, Sandor; Whitty, Adrian; Kozakov, Dima

2012-08-27

In the context of protein-protein interactions, the term "hot spot" refers to a residue or cluster of residues that makes a major contribution to the binding free energy, as determined by alanine scanning mutagenesis. In contrast, in pharmaceutical research, a hot spot is a site on a target protein that has high propensity for ligand binding and hence is potentially important for drug discovery. Here we examine the relationship between these two hot spot concepts by comparing alanine scanning data for a set of 15 proteins with results from mapping the protein surfaces for sites that can bind fragment-sized small molecules. We find the two types of hot spots are largely complementary; the residues protruding into hot spot regions identified by computational mapping or experimental fragment screening are almost always themselves hot spot residues as defined by alanine scanning experiments. Conversely, a residue that is found by alanine scanning to contribute little to binding rarely interacts with hot spot regions on the partner protein identified by fragment mapping. In spite of the strong correlation between the two hot spot concepts, they fundamentally differ, however. In particular, while identification of a hot spot by alanine scanning establishes the potential to generate substantial interaction energy with a binding partner, there are additional topological requirements to be a hot spot for small molecule binding. Hence, only a minority of hot spots identified by alanine scanning represent sites that are potentially useful for small inhibitor binding, and it is this subset that is identified by experimental or computational fragment screening.

Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

PubMed

Kucejová, B; Foury, F

2003-01-01

RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

Structure-Function Based Molecular Relationships in Ewing's Sarcoma

PubMed Central

2015-01-01

Ewing's Sarcoma Oncogene (ews) on chromosome 22q12 is encoding a ubiquitously expressed RNA-binding protein (EWS) with unknown function that is target of tumor-specific chromosomal translocations in Ewing's sarcoma family of tumors. A model of transcription complex was proposed in which the heterodimer Rpb4/7 binds to EAD, connecting it to Core RNA Pol II. The DNA-binding domain, provided by EFP, is bound to the promoter. Rpb4/7 binds RNA, stabilizing the transcription complex. The complex Rpb4/7 can stabilize the preinitiation complexes by converting the conformation of RNA Pol II. EWS may change its conformation, so that NTD becomes accessible. Two different mechanisms of interaction between EWS and RNA Pol II are proposed: (I) an intermolecular EWS-EWS interaction between two molecules, pushing conformation from “closed” to “open” state, or (II) an intramolecular interaction inside the molecule of EWS, pushing conformation of the molecule from “closed” to “open” state. The modified forms of EWS may interact with Pol II subunits hsRpb5 and hsRpb7. The EWS and EFPs binding partners are described schematically in a model, an attempt to link the transcription with the splicing. The proposed model helps to understand the functional molecular interactions in cancer, to find new partners and ways to treat cancer. PMID:25688366

The genetic and functional basis of isolated 17,20-lyase deficiency.

PubMed

Geller, D H; Auchus, R J; Mendonça, B B; Miller, W L

1997-10-01

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.

To Punish or to Leave: Distinct Cognitive Processes Underlie Partner Control and Partner Choice Behaviors

PubMed Central

Martin, Justin W.; Cushman, Fiery

2015-01-01

When a cooperative partner defects, at least two types of response are available: Punishment, aimed at modifying behavior, and ostracism, aimed at avoiding further social interaction with the partner. These options, termed partner control and partner choice, have been distinguished at behavioral and evolutionary levels. However, little work has compared their cognitive bases. Do these disparate behaviors depend on common processes of moral evaluation? Specifically, we assess whether they show identical patterns of dependence on two key dimensions of moral evaluation: A person’s intentions, and the outcomes that they cause. We address this issue in a “trembling hand” economic game. In this game, an allocator divides a monetary stake between themselves and a responder based on a stochastic mechanism. This allows for dissociations between the allocator’s intent and the actual outcome. Responders were either given the opportunity to punish or reward the allocator (partner control) or to switch to a different partner for a subsequent round of play (partner choice). Our results suggest that partner control and partner choice behaviors are supported by distinct underlying cognitive processes: Partner control exhibits greater sensitivity to the outcomes a partner causes, while partner choice is influenced almost exclusively by a partner’s intentions. This cognitive dissociation can be understood in light of the unique adaptive functions of partner control and partner choice. PMID:25915550

The centrosomal component CEP161 of Dictyostelium discoideum interacts with the Hippo signaling pathway

PubMed Central

Sukumaran, Salil K.; Blau-Wasser, Rosemarie; Rohlfs, Meino; Gallinger, Christoph; Schleicher, Michael; Noegel, Angelika A

2015-01-01

CEP161 is a novel component of the Dictyostelium discoideum centrosome which was identified as binding partner of the pericentriolar component CP250. Here we show that the amino acids 1-763 of the 1381 amino acids CEP161 are sufficient for CP250 binding, centrosomal targeting and centrosome association. Analysis of AX2 cells over-expressing truncated and full length CEP161 proteins revealed defects in growth and development. By immunoprecipitation experiments we identified the Hippo related kinase SvkA (Hrk-svk) as binding partner for CEP161. Both proteins colocalize at the centrosome. In in vitro kinase assays the N-terminal domain of CEP161 (residues 1-763) inhibited the kinase activity of Hrk-svk. A comparison of D. discoideum Hippo kinase mutants with mutants overexpressing CEP161 polypeptides revealed similar defects. We propose that the centrosomal component CEP161 is a novel player in the Hippo signaling pathway and affects various cellular properties through this interaction. PMID:25607232

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin.

PubMed

Miclet, Emeric; Bourgoin-Voillard, Sandrine; Byrne, Cillian; Jacquot, Yves

2016-01-01

The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17β-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca(2+)-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca(2+)-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (K d) of this interaction.

Analysis of Glycosaminoglycans Using Mass Spectrometry

PubMed Central

Staples, Gregory O.; Zaia, Joseph

2015-01-01

The glycosaminoglycans (GAGs) are linear polysaccharides expressed on animal cell surfaces and in extracellular matrices. Their biosynthesis is under complex control and confers a domain structure that is essential to their ability to bind to protein partners. Key to understanding the functions of GAGs are methods to determine accurately and rapidly patterns of sulfation, acetylation and uronic acid epimerization that correlate with protein binding or other biological activities. Mass spectrometry (MS) is particularly suitable for the analysis of GAGs for biomedical purposes. Using modern ionization techniques it is possible to accurately determine molecular weights of GAG oligosaccharides and their distributions within a mixture. Methods for direct interfacing with liquid chromatography have been developed to permit online mass spectrometric analysis of GAGs. New tandem mass spectrometric methods for fine structure determination of GAGs are emerging. This review summarizes MS-based approaches for analysis of GAGs, including tissue extraction and chromatographic methods compatible with LC/MS and tandem MS. PMID:25705143

Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

PubMed Central

Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh G.; Beld, Joris; La Clair, James J.; Burkart, Michael D.

2016-01-01

An ideal target for metabolic engineering, fatty acid biosynthesis remains poorly understood on a molecular level. These carrier protein-dependent pathways require fundamental protein–protein interactions to guide reactivity and processivity, and their control has become one of the major hurdles in successfully adapting these biological machines. Our laboratory has developed methods to prepare acyl carrier proteins (ACPs) loaded with substrate mimetics and cross-linkers to visualize and trap interactions with partner enzymes, and we continue to expand the tools for studying these pathways. We now describe application of the slow-onset, tight-binding inhibitor triclosan to explore the interactions between the type II fatty acid ACP from Escherichia coli, AcpP, and its corresponding enoyl-ACP reductase, FabI. We show that the AcpP–triclosan complex demonstrates nM binding, inhibits in vitro activity, and can be used to isolate FabI in complex proteomes. PMID:26938266

Allostery: Absence of a change in shape does not imply that allostery is not at play

PubMed Central

Tsai, Chung-Jung; Sol, Antonio del; Nussinov, Ruth

2009-01-01

Allostery is essential for controlled catalysis, signal transmission, receptor trafficking, turning genes on and off, and apoptosis. It governs the organism’s response to environmental and metabolic cues, dictating transient partner interactions in the cellular network. Textbooks taught us that allostery is a change of shape at one site on the protein surface brought about by ligand binding to another. For already several years it has been broadly accepted that the change of shape is not induced; rather, it is observed simply because a larger protein population presents it. Current data indicate that while side-chains can reorient and rewire, allostery may not even involve a change of (backbone) shape. Assuming that the enthalpy change does not reverse the free energy change due to the change in entropy, entropy is mainly responsible for binding. PMID:18353365

Four Amino Acids within a Tandem QxVx Repeat in a Predicted Extended α-Helix of the Smad-Binding Domain of Sip1 Are Necessary for Binding to Activated Smad Proteins

PubMed Central

Conidi, Andrea; van den Berghe, Veronique; Leslie, Kris; Stryjewska, Agata; Xue, Hua; Chen, Ye-Guang; Seuntjens, Eve; Huylebroeck, Danny

2013-01-01

The zinc finger transcription factor Smad-interacting protein-1 (Sip1; Zeb2, Zfhx1b) plays an important role during vertebrate embryogenesis in various tissues and differentiating cell types, and during tumorigenesis. Previous biochemical analysis suggests that interactions with several partner proteins, including TGFβ family receptor-activated Smads, regulate the activities of Sip1 in the nucleus both as a DNA-binding transcriptional repressor and activator. Using a peptide aptamer approach we mapped in Sip1 its Smad-binding domain (SBD), initially defined as a segment of 51 amino acids, to a shorter stretch of 14 amino acids within this SBD. Modelling suggests that this short SBD stretch is part of an extended α-helix that may fit the binding to a hydrophobic corridor within the MH2 domain of activated Smads. Four amino acids (two polar Q residues and two non-polar V residues) that form the tandem repeat (QxVx)2 in this 14-residue stretch were found to be crucial for binding to both TGFβ/Nodal/Activin-Smads and BMP-Smads. A full-length Sip1 with collective mutation of these Q and V residues (to A) no longer binds to Smads, while it retains its binding activity to its cognate bipartite target DNA sequence. This missense mutant Sip1(AxAx)2 provides a new molecular tool to identify SBD (in)dependent target genes in Sip1-controlled TGFβ and/or BMP (de)regulated cellular, developmental and pathological processes. PMID:24146916

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

PubMed Central

Rosenfeld, Robin J.; Bonaventura, Joseph; Szymczyna, Blair R.; MacCoss, Michael J.; Arvai, Andrew S.; Yates, John R.; Tainer, John A.; Getzoff, Elizabeth D.

2010-01-01

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS4 site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions. PMID:20659888

Analysis of the interaction between membrane proteins and soluble binding partners by surface plasmon resonance.

PubMed

Wu, Zht Cheng; de Keyzer, Jeanine; Kusters, Ilja; Driessen, Arnold J M

2013-01-01

The interaction between membrane proteins and their (protein) ligands is conventionally investigated by nonequilibrium methods such as co-sedimentation or pull-down assays. Surface Plasmon Resonance can be used to monitor such binding events in real-time using isolated membranes immobilized to a surface providing insights in the kinetics of binding under equilibrium conditions. This application provides a fast, automated way to detect interacting species and to determine the kinetics and affinity (Kd) of the interaction.

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

PubMed

Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

2018-02-01

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver

PubMed Central

2018-01-01

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. PMID:29757144

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

PubMed

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein

PubMed Central

Ferrar, Tony; Chamousset, Delphine; De Wever, Veerle; Nimick, Mhairi; Andersen, Jens; Trinkle-Mulcahy, Laura; Moorhead, Greg B. G.

2012-01-01

Summary The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway. PMID:23213405

Characterization of chlorophyll binding to LIL3

PubMed Central

Mork-Jansson, Astrid Elisabeth

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3. PMID:29390011

Functional relationship between CABIT, SAM and 14-3-3 binding domains of GAREM1 that play a role in its subcellular localization

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

Nishino, Tasuku; Matsunaga, Ryota; Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp

2015-08-21

GAREM1 (Grb2-associated regulator of Erk/MAPK1) is an adaptor protein that is involved in the epidermal growth factor (EGF) pathway. The nuclear localization of GAREM1 depends on the nuclear localization sequence (NLS), which is located at the N-terminal CABIT (cysteine-containing, all in Themis) domain. Here, we identified 14-3-3ε as a GAREM-binding protein, and its binding site is closely located to the NLS. This 14-3-3 binding site was of the atypical type and independent of GAREM phosphorylation. Moreover, the binding of 14-3-3 had an effect on the nuclear localization of GAREM1. Unexpectedly, we observed that the CABIT domain had intramolecular association withmore » the C-terminal SAM (sterile alpha motif) domain. This association might be inhibited by binding of 14-3-3 at the CABIT domain. Our results demonstrate that the mechanism underlying the nuclear localization of GAREM1 depends on its NLS in the CABIT domain, which is controlled by the binding of 14-3-3 and the C-terminal SAM domain. We suggest that the interplay between 14-3-3, SAM domain and CABIT domain might be responsible for the distribution of GAREM1 in mammalian cells. - Highlights: • 14-3-3ε regulated the nuclear localization of GAREM1 as its binding partner. • The atypical 14-3-3 binding site of GAREM1 is located near the NLS in CABIT domain. • The CABIT domain had intramolecular association with the SAM domain in GAREM1. • Subcellular localization of GAREM1 is affected with its CABIT-SAM interaction.« less

The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

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

Pertinhez, Thelma A.; Ferrari, Elena; Casali, Emanuela

2009-12-25

{sup 15}N and {sup 1}HN chemical shift data and {sup 15}N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the {beta}-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the {beta}-barrel cavity. These finding justify why MUPmore » is able to adapt to allow for many successful binding partners.« less

Electrostatics, structure prediction, and the energy landscapes for protein folding and binding.

PubMed

Tsai, Min-Yeh; Zheng, Weihua; Balamurugan, D; Schafer, Nicholas P; Kim, Bobby L; Cheung, Margaret S; Wolynes, Peter G

2016-01-01

While being long in range and therefore weakly specific, electrostatic interactions are able to modulate the stability and folding landscapes of some proteins. The relevance of electrostatic forces for steering the docking of proteins to each other is widely acknowledged, however, the role of electrostatics in establishing specifically funneled landscapes and their relevance for protein structure prediction are still not clear. By introducing Debye-Hückel potentials that mimic long-range electrostatic forces into the Associative memory, Water mediated, Structure, and Energy Model (AWSEM), a transferable protein model capable of predicting tertiary structures, we assess the effects of electrostatics on the landscapes of thirteen monomeric proteins and four dimers. For the monomers, we find that adding electrostatic interactions does not improve structure prediction. Simulations of ribosomal protein S6 show, however, that folding stability depends monotonically on electrostatic strength. The trend in predicted melting temperatures of the S6 variants agrees with experimental observations. Electrostatic effects can play a range of roles in binding. The binding of the protein complex KIX-pKID is largely assisted by electrostatic interactions, which provide direct charge-charge stabilization of the native state and contribute to the funneling of the binding landscape. In contrast, for several other proteins, including the DNA-binding protein FIS, electrostatics causes frustration in the DNA-binding region, which favors its binding with DNA but not with its protein partner. This study highlights the importance of long-range electrostatics in functional responses to problems where proteins interact with their charged partners, such as DNA, RNA, as well as membranes. © 2015 The Protein Society.

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane.

PubMed

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J; Galan, Jacob A; Tao, W Andy; Low, Philip S

2013-01-11

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

How Robust Is the Mechanism of Folding-Upon-Binding for an Intrinsically Disordered Protein?

PubMed

Bonetti, Daniela; Troilo, Francesca; Brunori, Maurizio; Longhi, Sonia; Gianni, Stefano

2018-04-24

The mechanism of interaction of an intrinsically disordered protein (IDP) with its physiological partner is characterized by a disorder-to-order transition in which a recognition and a binding step take place. Even if the mechanism is quite complex, IDPs tend to bind their partner in a cooperative manner such that it is generally possible to detect experimentally only the disordered unbound state and the structured complex. The interaction between the disordered C-terminal domain of the measles virus nucleoprotein (N TAIL ) and the X domain (XD) of the viral phosphoprotein allows us to detect and quantify the two distinct steps of the overall reaction. Here, we analyze the robustness of the folding of N TAIL upon binding to XD by measuring the effect on both the folding and binding steps of N TAIL when the structure of XD is modified. Because it has been shown that wild-type XD is structurally heterogeneous, populating an on-pathway intermediate under native conditions, we investigated the binding to 11 different site-directed variants of N TAIL of one particular variant of XD (I504A XD) that populates only the native state. Data reveal that the recognition and the folding steps are both affected by the structure of XD, indicating a highly malleable pathway. The experimental results are briefly discussed in the light of previous experiments on other IDPs. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane*

PubMed Central

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J.; Galan, Jacob A.; Tao, W. Andy; Low, Philip S.

2013-01-01

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction. PMID:23150667

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

PubMed Central

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

2012-01-01

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

Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

PubMed

He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

2000-08-01

Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach.

PubMed

Opalińska, Magdalena; Parys, Katarzyna; Jańska, Hanna

2017-11-18

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i -AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4's in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4's physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome.

Identification of Physiological Substrates and Binding Partners of the Plant Mitochondrial Protease FTSH4 by the Trapping Approach

PubMed Central

Parys, Katarzyna; Jańska, Hanna

2017-01-01

Maintenance of functional mitochondria is vital for optimal cell performance and survival. This is accomplished by distinct mechanisms, of which preservation of mitochondrial protein homeostasis fulfills a pivotal role. In plants, inner membrane-embedded i-AAA protease, FTSH4, contributes to the mitochondrial proteome surveillance. Owing to the limited knowledge of FTSH4’s in vivo substrates, very little is known about the pathways and mechanisms directly controlled by this protease. Here, we applied substrate trapping coupled with mass spectrometry-based peptide identification in order to extend the list of FTSH4’s physiological substrates and interaction partners. Our analyses revealed, among several putative targets of FTSH4, novel (mitochondrial pyruvate carrier 4 (MPC4) and Pam18-2) and known (Tim17-2) substrates of this protease. Furthermore, we demonstrate that FTSH4 degrades oxidatively damaged proteins in mitochondria. Our report provides new insights into the function of FTSH4 in the maintenance of plant mitochondrial proteome. PMID:29156584

Enhanced sampling simulations to construct free-energy landscape of protein-partner substrate interaction.

PubMed

Ikebe, Jinzen; Umezawa, Koji; Higo, Junichi

2016-03-01

Molecular dynamics (MD) simulations using all-atom and explicit solvent models provide valuable information on the detailed behavior of protein-partner substrate binding at the atomic level. As the power of computational resources increase, MD simulations are being used more widely and easily. However, it is still difficult to investigate the thermodynamic properties of protein-partner substrate binding and protein folding with conventional MD simulations. Enhanced sampling methods have been developed to sample conformations that reflect equilibrium conditions in a more efficient manner than conventional MD simulations, thereby allowing the construction of accurate free-energy landscapes. In this review, we discuss these enhanced sampling methods using a series of case-by-case examples. In particular, we review enhanced sampling methods conforming to trivial trajectory parallelization, virtual-system coupled multicanonical MD, and adaptive lambda square dynamics. These methods have been recently developed based on the existing method of multicanonical MD simulation. Their applications are reviewed with an emphasis on describing their practical implementation. In our concluding remarks we explore extensions of the enhanced sampling methods that may allow for even more efficient sampling.

Charge effects in the selection of NPF motifs by the EH domain of EHD1.

PubMed

Henry, Gillian D; Corrigan, Daniel J; Dineen, Joseph V; Baleja, James D

2010-04-27

The Eps15 homology (EH) domain is found in proteins associated with endocytosis and vesicle trafficking. EH domains bind to their target proteins through an asparagine-proline-phenylalanine (NPF) motif. We have measured the interaction energetics of the EH domain from EHD1 with peptides derived from two of its binding partners: Rabenosyn-5 (Ac-GPSLNPFDEED-NH(2)) and Rab11-Fip2 (Ac-YESTNPFTAK-NH(2)). Heteronuclear single quantum coherence (HSQC) spectroscopy shows that both peptides bind in the canonical binding pocket of EHD1 EH and induce identical structural changes, yet the affinity of the negatively charged Ac-GPSLNPFDEED-NH(2) (K(a) = 8 x 10(5) M(-1)) is tighter by 2 orders of magnitude. The thermodynamic profiles (DeltaG, DeltaH, DeltaS) were measured for both peptides as a function of temperature. The enthalpies of binding are essentially identical, and the difference in affinity is a consequence of the difference in entropic cost. Ac-GPSLNPFDEED-NH(2) binding is salt-dependent, demonstrating an electrostatic component to the interaction, whereas Ac-YESTNPFTAK-NH(2) binding is independent of salt. Successive replacement of acidic residues in Ac-GPSLNPFDEED-NH(2) with neutral residues showed that all are important. Lysine side chains in EHD1 EH create a region of strong positive surface potential near the NPF binding pocket. Contributions by lysine epsilon-amino groups to complex formation with Ac-GPSLNPFDEED-NH(2) was shown using direct-observe (15)N NMR spectroscopy. These experiments have enabled us to define a new extended interaction motif for EHD proteins, N-P-F-[DE]-[DE]-[DE], which we have used to predict new interaction partners and hence broaden the range of cellular activities involving the EHD proteins.

The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading.

PubMed

Tarallo, Roberta; Giurato, Giorgio; Bruno, Giuseppina; Ravo, Maria; Rizzo, Francesca; Salvati, Annamaria; Ricciardi, Luca; Marchese, Giovanna; Cordella, Angela; Rocco, Teresa; Gigantino, Valerio; Pierri, Biancamaria; Cimmino, Giovanni; Milanesi, Luciano; Ambrosino, Concetta; Nyman, Tuula A; Nassa, Giovanni; Weisz, Alessandro

2017-10-06

The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

PubMed

Nicholson, Judith; Scherl, Alex; Way, Luke; Blackburn, Elizabeth A; Walkinshaw, Malcolm D; Ball, Kathryn L; Hupp, Ted R

2014-06-01

Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors. Copyright © 2014 Elsevier Inc. All rights reserved.

Reciprocity in group-living animals: partner control versus partner choice.

PubMed

Schino, Gabriele; Aureli, Filippo

2017-05-01

Reciprocity is probably the most debated of the evolutionary explanations for cooperation. Part of the confusion surrounding this debate stems from a failure to note that two different processes can result in reciprocity: partner control and partner choice. We suggest that the common observation that group-living animals direct their cooperative behaviours preferentially to those individuals from which they receive most cooperation is to be interpreted as the result of the sum of the two separate processes of partner control and partner choice. We review evidence that partner choice is the prevalent process in primates and propose explanations for this pattern. We make predictions that highlight the need for studies that separate the effects of partner control and partner choice in a broader variety of group-living taxa. © 2016 Cambridge Philosophical Society.

Optogenetic Control of Cells and Circuits

PubMed Central

Miesenböck, Gero

2013-01-01

The absorption of light by bound or diffusible chromophores causes conformational rearrangements in natural and artificial photoreceptor proteins. These rearrangements are coupled to the opening or closing of ion transport pathways, the association or dissociation of binding partners, the enhancement or suppression of catalytic activity, or the transcription or repression of genetic information. Illumination of cells, tissues, or organisms engineered genetically to express photoreceptor proteins can thus be used to perturb biochemical and electrical signaling with exquisite cellular and molecular specificity. First demonstrated in 2002, this principle of optogenetic control has had a profound impact on neuroscience, where it provides a direct and stringent means of probing the organization of neural circuits and of identifying the neural substrates of behavior. The impact of optogenetic control is also beginning to be felt in other areas of cell and organismal biology. PMID:21819234

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

PubMed

Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

2017-07-01

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma.

PubMed

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function.

Toll like receptors TLR1/2, TLR6 and MUC5B as binding interaction partners with cytostatic proline rich polypeptide 1 in human chondrosarcoma

PubMed Central

Galoian, Karina; Abrahamyan, Silva; Chailyan, Gor; Qureshi, Amir; Patel, Parthik; Metser, Gil; Moran, Alexandra; Sahakyan, Inesa; Tumasyan, Narine; Lee, Albert; Davtyan, Tigran; Chailyan, Samvel; Galoyan, Armen

2018-01-01

Metastatic chondrosarcoma is a bone malignancy not responsive to conventional therapies; new approaches and therapies are urgently needed. We have previously reported that mTORC1 inhibitor, antitumorigenic cytostatic proline rich polypeptide 1 (PRP-1), galarmin caused a significant upregulation of tumor suppressors including TET1/2 and SOCS3 (known to be involved in inflammatory processes), downregulation of oncoproteins and embryonic stem cell marker miR-302C and its targets Nanog, c-Myc and Bmi-1 in human chondrosarcoma. To understand better the mechanism of PRP-1 action it was very important to identify the receptor it binds to. Nuclear pathway receptor and GPCR assays indicated that PRP-1 receptors are not G protein coupled, neither do they belong to family of nuclear or orphan receptors. In the present study, we have demonstrated that PRP-1 binding interacting partners belong to innate immunity pattern recognition toll like receptors TLR1/2 and TLR6 and gel forming secreted mucin MUC5B. MUC5B was identified as PRP-1 receptor in human chondrosarcoma JJ012 cell line using Ligand-receptor capture technology. Toll like receptors TLR1/2 and TLR6 were identified as binding interaction partners with PRP-1 by western blot analysis in human chondrosarcoma JJ012 cell line lysates. Immunocytochemistry experiments confirmed the finding and indicated the localization of PRP-1 receptors in the tumor nucleus predominantly. TLR1/2, TLR6 and MUC5B were downregulated in human chondrosarcoma and upregulated in dose-response manner upon PRP-1 treatment. Experimental data indicated that in this cellular context the mentioned receptors had tumor suppressive function. PMID:29138803

SH2 Ligand Prediction-Guidance for In-Silico Screening.

PubMed

Li, Shawn S C; Li, Lei

2017-01-01

Systematic identification of binding partners for SH2 domains is important for understanding the biological function of the corresponding SH2 domain-containing proteins. Here, we describe two different web-accessible computer programs, SMALI and DomPep, for predicting binding ligands for SH2 domains. The former was developed using a Scoring Matrix method and the latter based on the Support Vector Machine model.

Interaction of CSFV E2 Protein with Swine Host Factors as Detected by Yeast Two-Hybrid System

PubMed Central

Gladue, Douglas P.; Baker-Bransetter, Ryan; Holinka, Lauren G.; Fernandez-Sainz, Ignacio J.; O’Donnell, Vivian; Fletcher, Paige; Lu, Zhiqiang; Borca, Manuel V.

2014-01-01

E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle. PMID:24416391

Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

PubMed

Disney, Matthew D; Winkelsas, Audrey M; Velagapudi, Sai Pradeep; Southern, Mark; Fallahi, Mohammad; Childs-Disney, Jessica L

2016-06-17

The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

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

Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.

2008-09-03

LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}).more » Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.« less

A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

PubMed

Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

2017-07-11

Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson's disease.

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription

PubMed Central

Aydin, Özge Z.; Marteijn, Jurgen A.; Ribeiro-Silva, Cristina; Rodríguez López, Aida; Wijgers, Nils; Smeenk, Godelieve; van Attikum, Haico; Poot, Raymond A.; Vermeulen, Wim; Lans, Hannes

2014-01-01

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA. PMID:24990377

Unrelated solubility-enhancing fusion partners MBP and NusA utilize a similar mode of action

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2014-01-01

The tendency of recombinant proteins to accumulate in the form of insoluble aggregates in Escherichia coli is a major hindrance to their overproduction. One of the more effective approaches to circumvent this problem is to use translation fusion partners (solubility-enhancers, SEs). E. coli maltose binding protein (MBP) and N-utilization substance A (NusA) are arguably the most effective solubilizing agents that have been discovered so far. Here, we show that although these two proteins are structurally, functionally, and physiochemically distinct, they influence the solubility and folding of their fusion partners in a very similar manner. These SEs act as “holdases” that prevent the aggregation of their fusion partners. Subsequent folding of the passenger proteins, when it occurs, is either spontaneous or chaperone-mediated. PMID:24942647

Structural deformation upon protein-protein interaction: A structural alphabet approach

PubMed Central

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-01-01

Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking. PMID:18307769

Structural deformation upon protein-protein interaction: a structural alphabet approach.

PubMed

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-02-28

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Protein Arginine Methylation in Mammals: Who, What, and Why

PubMed Central

Bedford, Mark T.; Clarke, Steven G.

2012-01-01

The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. PMID:19150423

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

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

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 inducedmore » polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.« less

The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

PubMed

An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

2008-09-05

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

A study of the Interaction of bovine Hemoglobin with Synthetic dyes using Spectroscopic techniques and Molecular docking

NASA Astrophysics Data System (ADS)

Kamaljeet; Bansal, Saurabh; SenGupta, Uttara

2016-12-01

Synthetic dyes are a very efficient class of dyes that are ingested or come into contact with the skin from numerous sources (cosmetics, textiles, leather, paper, drugs). An important component of their safety profile is the interactions that they form after they enter the body. Hemoglobin is a functionally important protein that can form multiple interactions with soluble compounds present in the blood, and hence forms an important aspect of the toxicological or safety profile of the dyes. Here we study the interaction between bovine haemoglobin and organic dyes using UV-Vis absorbance and fluorescence spectroscopy. Molecular modelling was used to visualise the binding site and partners of the dye molecules, within the hemoglobin molecule. We find that all four dyes studied form sufficiently strong interactions with haemoglobin to allow for the formation of potentially toxic interactions. Molecular modelling showed that all 4 dyes bound within the central cavity of the haemoglobin molecule. However, binding partners could not be identified as multiple binding conformations with very similar energies were possible for each dye.

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

Structural perturbations on huntingtin N17 domain during its folding on 2D-nanomaterials

NASA Astrophysics Data System (ADS)

Zhang, Leili; Feng, Mei; Zhou, Ruhong; Luan, Binquan

2017-09-01

A globular protein’s folded structure in its physiological environment is largely determined by its amino acid sequence. Recently, newly discovered transformer proteins as well as intrinsically disordered proteins may adopt the folding-upon-binding mechanism where their secondary structures are highly dependent on their binding partners. Due to the various applications of nanomaterials in biological sensors and potential wearable devices, it is important to discover possible conformational changes of proteins on nanomaterials. Here, through molecular dynamics simulations, we show that the first 17 residues of the huntingtin protein (HTT-N17) exhibit appreciable differences during its folding on 2D-nanomaterials, such as graphene and MoS2 nanosheets. Namely, the protein is disordered on the graphene surface but is helical on the MoS2 surface. Despite that the amphiphilic environment at the nanosheet-water interface promotes the folding of the amphipathic proteins (such as HTT-N17), competitions between protein-nanosheet and intra-protein interactions yield very different protein conformations. Therefore, as engineered binding partners, nanomaterials might significantly affect the structures of adsorbed proteins.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

PubMed Central

Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

2011-01-01

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

PubMed Central

Maertens, Goedele N.

2016-01-01

To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

Tomato pistil factor STIG1 promotes in vivo pollen tube growth by binding to phosphatidylinositol 3-phosphate and the extracellular domain of the pollen receptor kinase LePRK2

USDA-ARS?s Scientific Manuscript database

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown ...

SH2 Binding Site Protection Assay: A Method for Identification of SH2 Domain Interaction Partners by Exploiting SH2 Mediated Phosphosite Protection.

PubMed

Jadwin, Joshua A

2017-01-01

Over the last two decades there has been a significant effort in the field to characterize the phosphosite binding specificities of SH2 domains with the goal of deciphering the pY signaling code. Although high throughput studies in various formats using most SH2 domains have collectively provided a rich resource of in vitro SH2-pTyr site specificity maps, this data can only be used approximate what is happening in the cell where protein concentrations and localization are not homogenous, as they are for in vitro experiments. Here we describe an in vivo approach, SH2 site protection assay, which can capture the pTyr binding specificity of SH2 domains in the cell. The basis of this approach is SH2-pY site protection, the ability of SH2 domains to prevent the PTP-dependent dephosphorylation of their pY site binding partners. We overexpress a tracer SH2 domain in cells and quantify the change in abundance of tyrosine phosphorylated sites using MS. Since the method is performed in vivo, it has the advantage of identifying SH2-pY interactions as they occur within in the cell.

Gas6 - Axl receptor signaling is regulated by glucose in vascular smooth muscle cells

PubMed Central

Cavet, Megan E.; Smolock, Elaine M.; Ozturk, Oktay H.; World, Cameron; Pang, Jinjiang; Konishi, Atsushi; Berk, Bradford C.

2009-01-01

Objective The receptor tyrosine kinase Axl and its ligand Gas6 are involved in the development of renal diabetic disease. In vascular smooth muscle cells (VSMC) Axl is activated by reactive oxygen species and stimulates migration and cell survival, suggesting a role for Axl in the vascular complications of diabetes. Methods and Results We investigated the effect of varying glucose concentration on Axl signaling in VSMC. Glucose exerted powerful effects on Gas6-Axl signaling with greater activation of Akt and mTOR in low glucose, and greater activation of ERK1/2 in high glucose. Plasma membrane distribution and tyrosine phosphorylation of Axl were not affected by glucose. However, co-immunoprecipitation studies demonstrated that glucose changed the interaction of Axl with its binding partners. Specifically, binding of Axl to the p85 subunit of PI3-kinase was increased in low glucose, whereas binding to SHP-2 was increased in high glucose. Furthermore, Gas6-Axl induced migration was increased in high glucose, while Gas6-Axl mediated inhibition of apoptosis was greater in low glucose. Conclusion This study demonstrates a role for glucose in altering Axl signaling through coupling to binding partners, and suggests a mechanism by which Axl contributes to VSMC dysfunction in diabetes. PMID:18292389

Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage.

PubMed

Reesink, Heidi L; Bonnevie, Edward D; Liu, Sherry; Shurer, Carolyn R; Hollander, Michael J; Bonassar, Lawrence J; Nixon, Alan J

2016-05-09

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin's mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.

Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage

PubMed Central

Reesink, Heidi L.; Bonnevie, Edward D.; Liu, Sherry; Shurer, Carolyn R.; Hollander, Michael J.; Bonassar, Lawrence J.; Nixon, Alan J.

2016-01-01

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin’s mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis. PMID:27157803

Structural properties of the promiscuous VP16 activation domain.

PubMed

Jonker, Hendrik R A; Wechselberger, Rainer W; Boelens, Rolf; Folkers, Gert E; Kaptein, Rob

2005-01-25

Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to alpha-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an alpha-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.

Peroxisomal ATP-binding cassette transporters form mainly tetramers

PubMed Central

Geillon, Flore; Gondcaille, Catherine; Raas, Quentin; Dias, Alexandre M. M.; Pecqueur, Delphine; Truntzer, Caroline; Lucchi, Géraldine; Ducoroy, Patrick; Falson, Pierre; Savary, Stéphane; Trompier, Doriane

2017-01-01

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane. PMID:28258215

Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage

NASA Astrophysics Data System (ADS)

Reesink, Heidi L.; Bonnevie, Edward D.; Liu, Sherry; Shurer, Carolyn R.; Hollander, Michael J.; Bonassar, Lawrence J.; Nixon, Alan J.

2016-05-01

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin’s mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.

RACK1 binds to Smad3 to modulate transforming growth factor-beta1-stimulated alpha2(I) collagen transcription in renal tubular epithelial cells.

PubMed

Okano, Kazuhiro; Schnaper, H William; Bomsztyk, Karol; Hayashida, Tomoko

2006-09-08

Although it is clear that transforming growth factor-beta1 (TGF-beta1) is critical for renal fibrogenesis, the complexity of the involved mechanisms is increasingly apparent. TGF-beta1 stimulates phosphorylation of Smad2/3 and activates other signaling molecules as well. The molecular link between these other kinases and Smads is not known. We sought new binding partners for Smad3 in renal cells and identified receptor for activated protein kinase C 1 (RACK1) as a novel binding partner of Smad3. The linker region of Smad3 and the tryptophan-aspartic acid repeat 6 and 7 of RACK1 are sufficient for the association. RACK1 also interacts with Smad3 in the human kidney epithelial cell line, HKC. Silencing RACK1 increases transcriptional activity of TGF-beta1-responsive promoter sequences of the Smad binding element (SBE), p3TP-Lux, and alpha2(I) collagen. Conversely, overexpressed RACK1 negatively modulates alpha2(I) collagen transcriptional activity in TGF-beta1-stimulated cells. RACK1 did not affect phosphorylation of Smad3 at the C terminus or in the linker region. However, RACK1 reduced direct binding of Smad3 to the SBE motif. Mutating a RACK1 tyrosine at residue 246, but not at 228, decreased the inhibitory effect of RACK1 on both alpha2(I) collagen promoter activity and Smad binding to SBE induced by TGF-beta1. These results suggest that RACK1 modulates transcription of alpha2(I) collagen by TGF-beta1 through interference with Smad3 binding to the gene promoter.

Daily Associations Among Self-control, Heavy Episodic Drinking, and Relationship Functioning: An Examination of Actor and Partner Effects

PubMed Central

Crane, Cory A.; Testa, Maria; Derrick, Jaye L.; Leonard, Kenneth E.

2014-01-01

An emerging literature suggests that temporary deficits in the ability to inhibit impulsive urges may be proximally associated with intimate partner aggression. The current study examined the experience of alcohol use and the depletion of self-control in the prediction of relationship functioning. Daily diary data collected from 118 heterosexual couples were analyzed using parallel multi-level Actor Partner Interdependence Models to assess the effects of heavy episodic drinking and depletion of self-control across partners on outcomes of participant-reported daily arguing with and anger toward an intimate partner. Heavy episodic drinking among actors predicted greater arguing but failed to interact with either actor or partner depletion. We also found that greater arguing was reported on days of high congruent actor and partner depletion. Both actor and partner depletion, as well as their interaction, predicted greater partner-specific anger. Greater partner-specific anger was generally reported on days of congruent actor and partner depletion, particularly on days of high partner depletion. The current results highlight the importance of independently assessing partner effects (i.e., depletion of self-control), which interact dynamically with disinhibiting actor effects, in the prediction of daily adverse relationship functioning. Results offer further support for the development of prospective individualized and couples-based interventions for partner conflict. PMID:24700558

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

PubMed

Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

2015-11-01

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence*

PubMed Central

Li, Shijun; Tan, Min; Juillard, Franceline; Ponnusamy, Rajesh; Correia, Bruno; Simas, J. Pedro; Carrondo, Maria A.; McVey, Colin E.; Kaye, Kenneth M.

2015-01-01

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes. PMID:26420481

Novel Interactions of the TRTK12 Peptide with S100 Protein Family Members: Specificity and Thermodynamic Characterization

PubMed Central

Wafer, Lucas N.; Tzul, Franco O.; Pandharipande, Pranav P.; Makhatadze, George I.

2013-01-01

The S100 protein family consists of small, dimeric proteins that exert their biological functions in response to changing calcium concentrations. S100B is the best studied member and has been shown to interact with over 20 binding partners in a calcium-dependent manner. The TRTK12 peptide, derived from the consensus binding sequence for S100B, has previously been found to interact with S100A1 and has been proposed to be a general binding partner of the S100 family. To test this hypothesis and gain a better understanding of the specificity of binding for the S100 proteins sixteen members of the human S100 family were screened against this peptide and its alanine variants. Novel interactions were only found with two family members: S100P and S100A2, indicating that TRTK12 selectively interacts with a small subset of the S100 proteins. Substantial promiscuity was observed in the binding site of S100B to accommodate variations in the peptide sequence, while S100A1, S100A2, and S100P exhibited larger differences in the binding constants for the TRTK12 alanine variants. This suggests that single-point substitutions can be used to selectively modulate the affinity of TRTK12 peptides for individual S100 proteins. This study has important implications for the rational drug design of inhibitors for the S100 proteins, which are involved in a variety of cancers and neurodegenerative diseases. PMID:23899389

The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

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

Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidencemore » towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.« less

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks

PubMed Central

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions. PMID:27857564

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

PubMed

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long - range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

PubMed

Wszelaka-Rylik, Małgorzata; Witkiewicz-Kucharczyk, Aleksandra; Wójcik, Jacek; Bal, Wojciech

2007-05-01

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

Capillarity theory for the fly-casting mechanism

PubMed Central

Trizac, Emmanuel; Levy, Yaakov; Wolynes, Peter G.

2010-01-01

Biomolecular folding and function are often coupled. During molecular recognition events, one of the binding partners may transiently or partially unfold, allowing more rapid access to a binding site. We describe a simple model for this fly-casting mechanism based on the capillarity approximation and polymer chain statistics. The model shows that fly casting is most effective when the protein unfolding barrier is small and the part of the chain which extends toward the target is relatively rigid. These features are often seen in known examples of fly casting in protein–DNA binding. Simulations of protein–DNA binding based on well-funneled native-topology models with electrostatic forces confirm the trends of the analytical theory. PMID:20133683

Sexual communication, dyadic adjustment, and psychosexual well-being in premenopausal women with self-reported dyspareunia and their partners: a controlled study.

PubMed

Pazmany, Els; Bergeron, Sophie; Verhaeghe, Johan; Van Oudenhove, Lukas; Enzlin, Paul

2014-07-01

Although research that takes into account partner and relationship factors in dyspareunia is slowly emerging, little is known about how these couples communicate about their sexuality. Additionally, partner psychosexual adjustment has not been examined in a controlled fashion. This study aimed to compare dyadic sexual communication, dyadic adjustment, psychological adjustment, and sexual well-being of women with self-reported dyspareunia and their partners with those of pain-free control women and their partners. Premenopausal women (n = 38; mean [M] age = 24.92) with self-reported dyspareunia, their partners (n = 38; M age = 26.71), as well as pain-free control women (n = 44; M age = 25.86) and their partners (n = 44; M age = 27.95) completed an online survey measuring dyadic sexual communication, dyadic adjustment, anxiety, depression, sexual functioning, and sexual distress. Assessments of women and men's (i) dyadic sexual communication; (ii) dyadic adjustment; (iii) anxiety; (iv) depression; (v) sexual functioning; and (vi) women's sexual distress were the main outcome measures. Compared with pain-free controls, women with dyspareunia reported significantly poorer dyadic sexual communication, a difference not found between partners of women with dyspareunia and control partners. Compared with partners of control women, those of women with dyspareunia reported significantly more impaired sexual functioning. No differences in dyadic adjustment were found between women with dyspareunia and pain-free control women, or between their respective partners. Finally, compared with control women, those with dyspareunia reported significantly more impaired psychological and sexual well-being. Findings suggest that dyspareunia impacts not only the psychosexual adjustment of affected women but also that of their partners. It seems relevant to include both members of the couple in future research and treatment for dyspareunia. © 2014 International Society for Sexual Medicine.

Metallomics investigations on potential binding partners of methylmercury in tuna fish muscle tissue using complementary mass spectrometric techniques.

PubMed

Kutscher, Daniel J; Sanz-Medel, Alfredo; Bettmer, Jörg

2012-08-01

In this study, the binding behaviour of methylmercury (MeHg(+)) towards proteins is investigated. Free sulfhydryl groups in cysteine residues are known to be the most likely binding partners, due to the high affinity of mercury to sulphur. However, detailed knowledge about discrete binding sites in living organisms has been so far scarce. A metallomics approach using different methods like size-exclusion chromatography (SEC) and liquid chromatography (LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) as well as complementary mass spectrometric techniques (electrospray ionisation-tandem mass spectrometry, ESI-MS/MS) are combined to sequence and identify possible target proteins or peptides after enzymatic digestion. Potential targets for MeHg(+) in tuna fish muscle tissue are investigated using the certified reference material CRM464 as a model tissue. Different extraction procedures appropriate for the extraction of proteins are evaluated for their efficiency using isotope dilution analysis for the determination of total Hg in the extracts. Due to the high chemical stability of the mercury-sulphur bond, the bioconjugate can be quantitatively extracted with a combination of tris(hydroxymethyl)aminomethane (TRIS) and sodium dodecyl sulphate (SDS). Using different separation techniques such as SEC and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) it can be shown that major binding occurs to a high-molecular weight protein (M(w) > 200 kDa). A potential target protein, skeletal muscle myosin heavy chain, could be identified after tryptic digestion and capillary LC-ESI-MS/MS.

Tissue Specificity of Human Angiotensin I-Converting Enzyme

PubMed Central

Kryukova, Olga V.; Tikhomirova, Victoria E.; Golukhova, Elena Z.; Evdokimov, Valery V.; Kalantarov, Gavreel F.; Trakht, Ilya N.; Schwartz, David E.; Dull, Randal O.; Gusakov, Alexander V.; Uporov, Igor V.; Kost, Olga A.; Danilov, Sergei M.

2015-01-01

Background Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distingushing tissue-specific ACEs. PMID:26600189

modPDZpep: a web resource for structure based analysis of human PDZ-mediated interaction networks.

PubMed

Sain, Neetu; Mohanty, Debasisa

2016-09-21

PDZ domains recognize short sequence stretches usually present in C-terminal of their interaction partners. Because of the involvement of PDZ domains in many important biological processes, several attempts have been made for developing bioinformatics tools for genome-wide identification of PDZ interaction networks. Currently available tools for prediction of interaction partners of PDZ domains utilize machine learning approach. Since, they have been trained using experimental substrate specificity data for specific PDZ families, their applicability is limited to PDZ families closely related to the training set. These tools also do not allow analysis of PDZ-peptide interaction interfaces. We have used a structure based approach to develop modPDZpep, a program to predict the interaction partners of human PDZ domains and analyze structural details of PDZ interaction interfaces. modPDZpep predicts interaction partners by using structural models of PDZ-peptide complexes and evaluating binding energy scores using residue based statistical pair potentials. Since, it does not require training using experimental data on peptide binding affinity, it can predict substrates for diverse PDZ families. Because of the use of simple scoring function for binding energy, it is also fast enough for genome scale structure based analysis of PDZ interaction networks. Benchmarking using artificial as well as real negative datasets indicates good predictive power with ROC-AUC values in the range of 0.7 to 0.9 for a large number of human PDZ domains. Another novel feature of modPDZpep is its ability to map novel PDZ mediated interactions in human protein-protein interaction networks, either by utilizing available experimental phage display data or by structure based predictions. In summary, we have developed modPDZpep, a web-server for structure based analysis of human PDZ domains. It is freely available at http://www.nii.ac.in/modPDZpep.html or http://202.54.226.235/modPDZpep.html . This article was reviewed by Michael Gromiha and Zoltán Gáspári.

Challenges and opportunities in the high-resolution cryo-EM visualization of microtubules and their binding partners.

PubMed

Nogales, Eva; Kellogg, Elizabeth H

2017-10-01

As non-crystallizable polymers, microtubules have been the target of cryo-electron microscopy (cryo-EM) studies since the technique was first established. Over the years, image processing strategies have been developed that take care of the unique, pseudo-helical symmetry of the microtubule. With recent progress in data quality and data processing, cryo-EM reconstructions are now reaching resolutions that allow the generation of atomic models of microtubules and the factors that bind them. These include cellular partners that contribute to microtubule cellular functions, or small ligands that interfere with those functions in the treatment of cancer. The stage is set to generate a family portrait for all identified microtubule interacting proteins and to use cryo-EM as a drug development tool in the targeting of tubulin. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cooperative macromolecular device revealed by meta-analysis of static and time-resolved structures

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

Ren, Zhong; Šrajer, Vukica; Knapp, James E.

2013-04-08

Here we present a meta-analysis of a large collection of static structures of a protein in the Protein Data Bank in order to extract the progression of structural events during protein function. We apply this strategy to the homodimeric hemoglobin HbI from Scapharca inaequivalvis. We derive a simple dynamic model describing how binding of the first ligand in one of the two chemically identical subunits facilitates a second binding event in the other partner subunit. The results of our ultrafast time-resolved crystallographic studies support this model. We demonstrate that HbI functions like a homodimeric mechanical device, such as pliers ormore » scissors. Ligand-induced motion originating in one subunit is transmitted to the other via conserved pivot points, where the E and F' helices from two partner subunits are 'bolted' together to form a stable dimer interface permitting slight relative rotation but preventing sliding.« less

Role of BAG3 in cancer progression: A therapeutic opportunity.

PubMed

De Marco, Margot; Basile, Anna; Iorio, Vittoria; Festa, Michelina; Falco, Antonia; Ranieri, Bianca; Pascale, Maria; Sala, Gianluca; Remondelli, Paolo; Capunzo, Mario; Firpo, Matthew A; Pezzilli, Raffaele; Marzullo, Liberato; Cavallo, Pierpaolo; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra

2018-06-01

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rigid-Docking Approaches to Explore Protein-Protein Interaction Space.

PubMed

Matsuzaki, Yuri; Uchikoga, Nobuyuki; Ohue, Masahito; Akiyama, Yutaka

Protein-protein interactions play core roles in living cells, especially in the regulatory systems. As information on proteins has rapidly accumulated on publicly available databases, much effort has been made to obtain a better picture of protein-protein interaction networks using protein tertiary structure data. Predicting relevant interacting partners from their tertiary structure is a challenging task and computer science methods have the potential to assist with this. Protein-protein rigid docking has been utilized by several projects, docking-based approaches having the advantages that they can suggest binding poses of predicted binding partners which would help in understanding the interaction mechanisms and that comparing docking results of both non-binders and binders can lead to understanding the specificity of protein-protein interactions from structural viewpoints. In this review we focus on explaining current computational prediction methods to predict pairwise direct protein-protein interactions that form protein complexes.

Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation.

PubMed

Wong, Lilly; Lieser, Scot A; Miyashita, Osamu; Miller, Meghan; Tasken, Kjetil; Onuchic, Josè N; Adams, Joseph A; Woods, Virgil L; Jennings, Patricia A

2005-08-05

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Investigation of binding phenomenon of NSP3 and p130Cas mutants and their effect on cell signalling.

PubMed

Balu K; Rajendran, Vidya; Sethumadhavan, Rao; Purohit, Rituraj

2013-11-01

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3(L469R), NSP3(L623E), NSP3(R627E)) and p130Cas (p130Cas(F794R), p130Cas(L787E), p130Cas(D797R)) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3(L469R) and p130Cas(D797R) mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3(L469R) and p130Cas(D797R) showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.

SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

PubMed Central

Zhang, Lei; Jablonski, Angela Marie; Neve, Rachael; Zhai, JinBin

2017-01-01

Abstract The dendritic tree is a key determinant of neuronal information processing. In the motor system, the dendritic tree of spinal cord neurons undergoes dramatic remodeling in an activity-dependent manner during early postnatal life. This leads to the proper segmental spinal cord connectivity that subserves normal locomotor behavior. One molecular system driving the establishment of dendrite architecture of mammalian motor neurons relies on AMPA receptors (AMPA-Rs) assembled with the GluA1 subunit, and this occurs in an NMDA receptor (NMDA-R)-independent manner. The dendrite growth promoting activity of GluA1-containing AMPA-Rs depends on its intracellular binding partner, SAP97, and SAP97’s PDZ3 domain. We show here that cysteine-rich interactor of PDZ3 (CRIPT) is a bona fide SAP97 PDZ3-domain binding partner, localizes to synapses with GluA1 and SAP97 along the dendritic tree, and is a determinant of the dendritic growth of mammalian spinal cord neurons. We further show that CRIPT has a well-conserved ortholog in the nematode, Caenorhabditis elegans, and animals lacking CRIPT display decreased dendrite branching of the well-studied PVD neuron in vivo. The lack of CRIPT leads to a selective defect in touch perception, and this is rescued by expression of wild-type (WT) human CRIPT (hCRIPT) in the nervous system. This work brings new light into the molecular machinery that drives dendritic growth during development and may prove relevant to the promotion of nervous system plasticity following insult. PMID:29218323

A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

PubMed

DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

2014-07-15

Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. © 2014 DeMille et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Competitive tuning: Competition's role in setting the frequency-dependence of Ca2+-dependent proteins

PubMed Central

Patel, Neal M.; Kinzer-Ursem, Tamara L.

2017-01-01

A number of neurological disorders arise from perturbations in biochemical signaling and protein complex formation within neurons. Normally, proteins form networks that when activated produce persistent changes in a synapse’s molecular composition. In hippocampal neurons, calcium ion (Ca2+) flux through N-methyl-D-aspartate (NMDA) receptors activates Ca2+/calmodulin signal transduction networks that either increase or decrease the strength of the neuronal synapse, phenomena known as long-term potentiation (LTP) or long-term depression (LTD), respectively. The calcium-sensor calmodulin (CaM) acts as a common activator of the networks responsible for both LTP and LTD. This is possible, in part, because CaM binding proteins are “tuned” to different Ca2+ flux signals by their unique binding and activation dynamics. Computational modeling is used to describe the binding and activation dynamics of Ca2+/CaM signal transduction and can be used to guide focused experimental studies. Although CaM binds over 100 proteins, practical limitations cause many models to include only one or two CaM-activated proteins. In this work, we view Ca2+/CaM as a limiting resource in the signal transduction pathway owing to its low abundance relative to its binding partners. With this view, we investigate the effect of competitive binding on the dynamics of CaM binding partner activation. Using an explicit model of Ca2+, CaM, and seven highly-expressed hippocampal CaM binding proteins, we find that competition for CaM binding serves as a tuning mechanism: the presence of competitors shifts and sharpens the Ca2+ frequency-dependence of CaM binding proteins. Notably, we find that simulated competition may be sufficient to recreate the in vivo frequency dependence of the CaM-dependent phosphatase calcineurin. Additionally, competition alone (without feedback mechanisms or spatial parameters) could replicate counter-intuitive experimental observations of decreased activation of Ca2+/CaM-dependent protein kinase II in knockout models of neurogranin. We conclude that competitive tuning could be an important dynamic process underlying synaptic plasticity. PMID:29107982

Increasing Partner Attendance in Antenatal Care and HIV Testing Services: Comparable Outcomes Using Written versus Verbal Invitations in an Urban Facility-Based Controlled Intervention Trial in Mbeya, Tanzania.

PubMed

Theuring, Stefanie; Jefferys, Laura F; Nchimbi, Philo; Mbezi, Paulina; Sewangi, Julius

2016-01-01

In many Sub-Saharan African settings male partner involvement in antenatal care (ANC) remains low, although great benefits for maternal and infant health outcomes have been long recognised, in particular regarding the prevention of HIV transmission. Yet there is paucity on evidence regarding the effectiveness of strategies to increase male partner involvement. This controlled intervention trial in Ruanda Health Centre in Mbeya, Tanzania, assessed the effectiveness of invitation letters for male involvement in ANC. Pregnant women approaching ANC without partners received official letters inviting the partner to attend ANC. A control group was instructed to verbally invite partners. Partner attendance was recorded at two subsequent ANC visits. Rates for male partner return, couple voluntary counselling and testing (CVCT), and influencing factors were analysed. From 199 ANC clients in total, 97 were assigned to the invitation letter group; 30 of these (30.9%) returned with their male partners for ANC. In the control group of 102 women, 28 (27.5%) returned with their partner. In both groups CVCT rates among jointly returning couples were 100%. Partner return/CVCT rate was not statistically different in intervention and control group (OR 1.2, p = 0.59). Former partner attendance at ANC during a previous pregnancy was the only factor found to be significantly linked with partner return (p = 0.03). Our study demonstrates that rather simple measures to increase male partner attendance in ANC and CVCT can be effective, with written and verbal invitations having comparable outcomes. In terms of practicability in Sub-Saharan African settings, we recommend systematic coaching of ANC clients on how to verbally invite male partners in the first instance, followed by written invitation letters for partners in case of their non-attendance. Further studies covering both urban and rural settings will be more informative for effective translation into policy.

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

PubMed

Eble, Johannes A

2018-02-15

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles

PubMed Central

2013-01-01

The ability to interact with different partners is one of the most important features in proteins. Proteins that bind a large number of partners (hubs) have been often associated with intrinsic disorder. However, many examples exist of hubs with an ordered structure, and evidence of a general mechanism promoting promiscuity in ordered proteins is still elusive. An intriguing hypothesis is that promiscuous binding sites have specific dynamical properties, distinct from the rest of the interface and pre-existing in the protein isolated state. Here, we present the first comprehensive study of the intrinsic dynamics of promiscuous residues in a large protein data set. Different computational methods, from coarse-grained elastic models to geometry-based sampling methods and to full-atom Molecular Dynamics simulations, were used to generate conformational ensembles for the isolated proteins. The flexibility and dynamic correlations of interface residues with a different degree of binding promiscuity were calculated and compared considering side chain and backbone motions, the latter both on a local and on a global scale. The study revealed that (a) promiscuous residues tend to be more flexible than nonpromiscuous ones, (b) this additional flexibility has a higher degree of organization, and (c) evolutionary conservation and binding promiscuity have opposite effects on intrinsic dynamics. Findings on simulated ensembles were also validated on ensembles of experimental structures extracted from the Protein Data Bank (PDB). Additionally, the low occurrence of single nucleotide polymorphisms observed for promiscuous residues indicated a tendency to preserve binding diversity at these positions. A case study on two ubiquitin-like proteins exemplifies how binding promiscuity in evolutionary related proteins can be modulated by the fine-tuning of the interface dynamics. The interplay between promiscuity and flexibility highlighted here can inspire new directions in protein–protein interaction prediction and design methods. PMID:24250278

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.

PubMed

Li, Shijun; Tan, Min; Juillard, Franceline; Ponnusamy, Rajesh; Correia, Bruno; Simas, J Pedro; Carrondo, Maria A; McVey, Colin E; Kaye, Kenneth M

2015-11-20

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

PubMed

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

2007-07-03

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat.

PubMed

Urayama, Satoshi; Harada, Yoshito; Nakagawa, Yoko; Ban, Susumu; Akasaka, Mari; Kawasaki, Nana; Sawada, Hitoshi

2008-08-01

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.

Functional Advantages of Conserved Intrinsic Disorder in RNA-Binding Proteins.

PubMed

Varadi, Mihaly; Zsolyomi, Fruzsina; Guharoy, Mainak; Tompa, Peter

2015-01-01

Proteins form large macromolecular assemblies with RNA that govern essential molecular processes. RNA-binding proteins have often been associated with conformational flexibility, yet the extent and functional implications of their intrinsic disorder have never been fully assessed. Here, through large-scale analysis of comprehensive protein sequence and structure datasets we demonstrate the prevalence of intrinsic structural disorder in RNA-binding proteins and domains. We addressed their functionality through a quantitative description of the evolutionary conservation of disordered segments involved in binding, and investigated the structural implications of flexibility in terms of conformational stability and interface formation. We conclude that the functional role of intrinsically disordered protein segments in RNA-binding is two-fold: first, these regions establish extended, conserved electrostatic interfaces with RNAs via induced fit. Second, conformational flexibility enables them to target different RNA partners, providing multi-functionality, while also ensuring specificity. These findings emphasize the functional importance of intrinsically disordered regions in RNA-binding proteins.

Perceived verbal conflict behaviors associated with physical aggression and sexual coercion in dating relationships: a gender-sensitive analysis.

PubMed

Katz, Jennifer; Carino, Andrew; Hilton, Angela

2002-02-01

We studied perceived partner verbal behaviors associated with participants' use of dating aggression. Men's reports of their partners' demanding, controlling, and psychologically abusive behaviors during conflicts were expected to predict men's perpetration against partners. In contrast, women's reports of their partners' withdrawal were expected to predict women's perpetration. Data were collected from heterosexual undergraduates (N = 223) in exclusive dating relationships. Participants' reports of partner demands and partner psychological abuse were associated with participants' use of physical aggression and sexual coercion. Reports of partner withdrawal and partner controlling behaviors were associated with participants' sexual coercion only. Significant moderating effects of gender emerged. As expected, partner demands, controlling behaviors, and psychological abuse were associated with physical aggression and sexual coercion in men, but not women. Partner withdrawal was associated with sexual coercion in both women and men. We conclude that gender-sensitive approaches are necessary to understand and prevent verbal conflict patterns associated with physical aggression and sexual coercion in intimate relationships.

Binding of Disordered Peptides to Kelch: Insights from Enhanced Sampling Simulations.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2016-01-12

Keap1 protein plays an essential role in regulating cellular oxidative stress response and is a crucial binding hub for multiple proteins, several of which are intrinsically disordered proteins (IDP). Among Kelch's IDP binding partners, NRF2 and PTMA are the two most interesting cases. They share a highly similar binding motif; however, NRF2 binds to Kelch with a binding affinity of approximately 100-fold higher than that of PTMA. In this study, we perform an exhaustive sampling composed of 6 μs well-tempered metadynamics and 2 μs unbiased molecular dynamics (MD) simulations aiming at characterizing the binding mechanisms and structural properties of these two peptides. Our results agree with previous experimental observations that PTMA is remarkably more disordered than NRF2 in both the free and bound states. This explains PTMA's lower binding affinity. Our extensive sampling also provides valuable insights into the vast conformational ensembles of both NRF2 and PTMA, supports the hypothesis of coupled folding-binding, and confirms the essential role of linear motifs in IDP binding.

Identification of novel binding partners (annexins) for the cell death signal phosphatidylserine and definition of their recognition motif.

PubMed

Rosenbaum, Sabrina; Kreft, Sandra; Etich, Julia; Frie, Christian; Stermann, Jacek; Grskovic, Ivan; Frey, Benjamin; Mielenz, Dirk; Pöschl, Ernst; Gaipl, Udo; Paulsson, Mats; Brachvogel, Bent

2011-02-18

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

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

Volakakis, Nikolaos; Joodmardi, Eliza; Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se

2009-12-25

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4Amore » NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.« less

Novel interactive partners of neuroligin 3: new aspects for pathogenesis of autism.

PubMed

Shen, Chen; Huo, Li-rong; Zhao, Xin-liang; Wang, Pei-rong; Zhong, Nanbert

2015-05-01

Autism is a neurodevelopmental disorder with a strong genetic predisposition. Neurolign 3 (NLGN3) as a postsynaptic transmembrane protein, functions in both neuron synaptogenesis and glia-neuron communications. Previously, a gain of function mutation (R451C) in NLGN3 was identified in autistic patients, which illustrates the involvement of NLGN3 in autism pathogenesis. As proper synaptic targeting and functioning are controlled by intracellular protein interactions, in the current study, we tried to discover the intracellular regulation network in which NLGN3 might be involved by a yeast two-hybrid-based interactor identification. Fifty-one protein candidate partners were identified after screening a human fetal complementary DNA (cDNA) library with an intracellular fragment of NLGN3. The interactions of NLGN3 with a subset of candidates, including EEF1A1, FLNA, ITPRIP, CYP11A1, MT-CO2, GPR175, ACOT2, and QPRT, were further validated in human neuroblastoma cells or brain tissues. Furthermore, our study suggested that NLGN3 was functioning in cytosolic calcium balance and participating in calcium-regulated cellular processes. Our findings of novel NLGN3 binding partners provide evidences of involvement of NLGN3 in multiple biological pathways, especially calcium regulating and mitochondrial function, thus suggesting further significance. This new data not only leads to a better understanding of the physiological functions of NLGN3, but also provide new aspects for pathogenesis of autism.

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis

PubMed Central

2018-01-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles. PMID:29518071

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

PubMed

Holland, David O; Johnson, Margaret E

2018-03-01

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that 'leftover' proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

PubMed

Suzuki, Atsushi; Niimi, Yuki; Shinmyozu, Kaori; Zhou, Zhi; Kiso, Makoto; Saga, Yumiko

2016-01-01

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development. © 2015 The Authors.

Multifarious Roles of Intrinsic Disorder in Proteins Illustrate Its Broad Impact on Plant Biology

PubMed Central

Sun, Xiaolin; Rikkerink, Erik H.A.; Jones, William T.; Uversky, Vladimir N.

2013-01-01

Intrinsically disordered proteins (IDPs) are highly abundant in eukaryotic proteomes. Plant IDPs play critical roles in plant biology and often act as integrators of signals from multiple plant regulatory and environmental inputs. Binding promiscuity and plasticity allow IDPs to interact with multiple partners in protein interaction networks and provide important functional advantages in molecular recognition through transient protein–protein interactions. Short interaction-prone segments within IDPs, termed molecular recognition features, represent potential binding sites that can undergo disorder-to-order transition upon binding to their partners. In this review, we summarize the evidence for the importance of IDPs in plant biology and evaluate the functions associated with intrinsic disorder in five different types of plant protein families experimentally confirmed as IDPs. Functional studies of these proteins illustrate the broad impact of disorder on many areas of plant biology, including abiotic stress, transcriptional regulation, light perception, and development. Based on the roles of disorder in the protein–protein interactions, we propose various modes of action for plant IDPs that may provide insight for future experimental approaches aimed at understanding the molecular basis of protein function within important plant pathways. PMID:23362206

Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

PubMed

Grondin, Julie M; Langelaan, David N; Smith, Steven P

2017-01-01

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1 H- 15 N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate binding partners, to quantify the dissociation constant (K d ) of any identified interactions, and to map the carbohydrate binding site on the structure of the protein. Here, we describe the titration of a family 32 carbohydrate binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction, and map the GalNAc binding site onto the structure of CpCBM32.

The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor (MITF) by binding to transcriptional repressor HINT1.

PubMed

Schöler, Jonas; Ferralli, Jacqueline; Thiry, Stéphane; Chiquet-Ehrismann, Ruth

2015-03-27

Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Because teneurin ICDs do not contain any intrinsic DNA binding sequences, interaction partners are required to affect transcription. Here, we identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast two-hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Directly comparing the transcriptomes of MITF versus TEN1-ICD-overexpressing BS149 cells revealed 42 co-regulated genes, including glycoprotein non-metastatic b (GPNMB). Using real-time quantitative PCR to detect endogenous GPNMB expression upon overexpression of MITF and HINT1 as well as promoter reporter assays using GPNMB promoter constructs, we could demonstrate that the teneurin-1 ICD binds HINT1, thus switching on MITF-dependent transcription of GPNMB. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Understand protein functions by comparing the similarity of local structural environments.

PubMed

Chen, Jiawen; Xie, Zhong-Ru; Wu, Yinghao

2017-02-01

The three-dimensional structures of proteins play an essential role in regulating binding between proteins and their partners, offering a direct relationship between structures and functions of proteins. It is widely accepted that the function of a protein can be determined if its structure is similar to other proteins whose functions are known. However, it is also observed that proteins with similar global structures do not necessarily correspond to the same function, while proteins with very different folds can share similar functions. This indicates that function similarity is originated from the local structural information of proteins instead of their global shapes. We assume that proteins with similar local environments prefer binding to similar types of molecular targets. In order to testify this assumption, we designed a new structural indicator to define the similarity of local environment between residues in different proteins. This indicator was further used to calculate the probability that a given residue binds to a specific type of structural neighbors, including DNA, RNA, small molecules and proteins. After applying the method to a large-scale non-redundant database of proteins, we show that the positive signal of binding probability calculated from the local structural indicator is statistically meaningful. In summary, our studies suggested that the local environment of residues in a protein is a good indicator to recognize specific binding partners of the protein. The new method could be a potential addition to a suite of existing template-based approaches for protein function prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM.

PubMed

Gonzalez, Walter G; Miksovska, Jaroslava

2014-09-01

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca(2+)DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS-DREAM complexes increases upon Ca(2+) association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca(2+)DREAM were determined to be 195±20μM and 62±4μM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

PubMed

Planelles-Herrero, Vicente José; Blanc, Florian; Sirigu, Serena; Sirkia, Helena; Clause, Jeffrey; Sourigues, Yannick; Johnsrud, Daniel O; Amigues, Beatrice; Cecchini, Marco; Gilbert, Susan P; Houdusse, Anne; Titus, Margaret A

2016-05-24

Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

Structure based re-design of the binding specificity of anti-apoptotic Bcl-xL

PubMed Central

Chen, T. Scott; Palacios, Hector; Keating, Amy E.

2012-01-01

Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Re-designed proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to re-engineer the multi-specific anti-apoptotic protein Bcl-xL to remove its interactions with many of its binding partners, making it a high affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-xL sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-xL variants, we successfully identified a protein with ~1,000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best re-designed protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. PMID:23154169

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains.

PubMed

Gao, Yong-Guang; Yan, Xian-Zhong; Song, Ai-Xin; Chang, Yong-Gang; Gao, Xue-Chao; Jiang, Nan; Zhang, Qi; Hu, Hong-Yu

2006-12-01

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.

Coevolution between positive reciprocity, punishment, and partner switching in repeated interactions.

PubMed

Wubs, Matthias; Bshary, Redouan; Lehmann, Laurent

2016-06-15

Cooperation based on mutual investments can occur between unrelated individuals when they are engaged in repeated interactions. Individuals then need to use a conditional strategy to deter their interaction partners from defecting. Responding to defection such that the future payoff of a defector is reduced relative to cooperating with it is called a partner control mechanism. Three main partner control mechanisms are (i) to switch from cooperation to defection when being defected ('positive reciprocity'), (ii) to actively reduce the payoff of a defecting partner ('punishment'), or (iii) to stop interacting and switch partner ('partner switching'). However, such mechanisms to stabilize cooperation are often studied in isolation from each other. In order to better understand the conditions under which each partner control mechanism tends to be favoured by selection, we here analyse by way of individual-based simulations the coevolution between positive reciprocity, punishment, and partner switching. We show that random interactions in an unstructured population and a high number of rounds increase the likelihood that selection favours partner switching. In contrast, interactions localized in small groups (without genetic structure) increase the likelihood that selection favours punishment and/or positive reciprocity. This study thus highlights the importance of comparing different control mechanisms for cooperation under different conditions. © 2016 The Author(s).

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

Small Maf proteins (MafF, MafG, MafK): History, structure and function.

PubMed

Katsuoka, Fumiki; Yamamoto, Masayuki

2016-07-25

The small Maf proteins (sMafs) are basic region leucine zipper (bZIP)-type transcription factors. The basic region of the Maf family is unique among the bZIP factors, and it contributes to the distinct DNA-binding mode of this class of proteins. MafF, MafG and MafK are the three vertebrate sMafs, and no functional differences have been observed among them in terms of their bZIP structures. sMafs form homodimers by themselves, and they form heterodimers with cap 'n' collar (CNC) proteins (p45 NF-E2, Nrf1, Nrf2, and Nrf3) and also with Bach proteins (Bach1 and Bach2). Because CNC and Bach proteins cannot bind to DNA as monomers, sMafs are indispensable partners that are required by CNC and Bach proteins to exert their functions. sMafs lack the transcriptional activation domain; hence, their homodimers act as transcriptional repressors. In contrast, sMafs participate in transcriptional activation or repression depending on their heterodimeric partner molecules and context. Mouse genetic analyses have revealed that various biological pathways are under the regulation of CNC-sMaf heterodimers. In this review, we summarize the history and current progress of sMaf studies in relation to their partners. Copyright © 2016 Elsevier B.V. All rights reserved.

The Molecular Chaperone TRiC/CCT Binds to the Trp-Asp 40 (WD40) Repeat Protein WDR68 and Promotes Its Folding, Protein Kinase DYRK1A Binding, and Nuclear Accumulation*

PubMed Central

Miyata, Yoshihiko; Shibata, Takeshi; Aoshima, Masato; Tsubata, Takuichi; Nishida, Eisuke

2014-01-01

Trp-Asp (WD) repeat protein 68 (WDR68) is an evolutionarily conserved WD40 repeat protein that binds to several proteins, including dual specificity tyrosine phosphorylation-regulated protein kinase (DYRK1A), MAPK/ERK kinase kinase 1 (MEKK1), and Cullin4-damage-specific DNA-binding protein 1 (CUL4-DDB1). WDR68 affects multiple and diverse physiological functions, such as controlling anthocyanin synthesis in plants, tissue growth in insects, and craniofacial development in vertebrates. However, the biochemical basis and the regulatory mechanism of WDR68 activity remain largely unknown. To better understand the cellular function of WDR68, here we have isolated and identified cellular WDR68 binding partners using a phosphoproteomic approach. More than 200 cellular proteins with wide varieties of biochemical functions were identified as WDR68-binding protein candidates. Eight T-complex protein 1 (TCP1) subunits comprising the molecular chaperone TCP1 ring complex/chaperonin-containing TCP1 (TRiC/CCT) were identified as major WDR68-binding proteins, and phosphorylation sites in both WDR68 and TRiC/CCT were identified. Co-immunoprecipitation experiments confirmed the binding between TRiC/CCT and WDR68. Computer-aided structural analysis suggested that WDR68 forms a seven-bladed β-propeller ring. Experiments with a series of deletion mutants in combination with the structural modeling showed that three of the seven β-propeller blades of WDR68 are essential and sufficient for TRiC/CCT binding. Knockdown of cellular TRiC/CCT by siRNA caused an abnormal WDR68 structure and led to reduction of its DYRK1A-binding activity. Concomitantly, nuclear accumulation of WDR68 was suppressed by the knockdown of TRiC/CCT, and WDR68 formed cellular aggregates when overexpressed in the TRiC/CCT-deficient cells. Altogether, our results demonstrate that the molecular chaperone TRiC/CCT is essential for correct protein folding, DYRK1A binding, and nuclear accumulation of WDR68. PMID:25342745

The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

PubMed Central

Münz, Márton; Hein, Jotun; Biggin, Philip C.

2012-01-01

In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595

Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer

PubMed Central

Glaser, Bryan T.; Bergendahl, Veit; Anthony, Larry C.; Olson, Brian; Burgess, Richard R.

2009-01-01

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function. PMID:19649256

Antagonistic Actions of HLH/bHLH Proteins Are Involved in Grain Length and Weight in Rice

PubMed Central

Heang, Dany; Sassa, Hidenori

2012-01-01

Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1. PMID:22363621

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

PubMed

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

26 CFR 1.732-3 - Corresponding adjustment to basis of assets of a distributed corporation controlled by a...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 8 2010-04-01 2010-04-01 false Corresponding adjustment to basis of assets of a distributed corporation controlled by a corporate partner. 1.732-3 Section 1.732-3 Internal Revenue INTERNAL... controlled by a corporate partner. The determination of whether a corporate partner has control of a...

Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

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

Magno, Aaron L.; Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009; Ingley, Evan

Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependentmore » stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.« less

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin*

PubMed Central

Stauch, Kelly; Kieken, Fabien; Sorgen, Paul

2012-01-01

In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca2+, pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis. PMID:22718765

Dynamin1 is a novel target for IRSp53 protein and works with mammalian enabled (Mena) protein and Eps8 to regulate filopodial dynamics.

PubMed

Chou, Ai Mei; Sem, Kai Ping; Wright, Graham Daniel; Sudhaharan, Thankiah; Ahmed, Sohail

2014-08-29

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and Characterization of FAM124B as a Novel Component of a CHD7 and CHD8 Containing Complex

PubMed Central

Batsukh, Tserendulam; Schulz, Yvonne; Wolf, Stephan; Rabe, Tamara I.; Oellerich, Thomas; Urlaub, Henning; Schaefer, Inga-Marie; Pauli, Silke

2012-01-01

Background Mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7) lead to CHARGE syndrome, an autosomal dominant multiple malformation disorder. Proteins involved in chromatin remodeling typically act in multiprotein complexes. We previously demonstrated that a part of human CHD7 interacts with a part of human CHD8, another chromodomain helicase DNA binding protein presumably being involved in the pathogenesis of neurodevelopmental (NDD) and autism spectrum disorders (ASD). Because identification of novel CHD7 and CHD8 interacting partners will provide further insights into the pathogenesis of CHARGE syndrome and ASD/NDD, we searched for additional associated polypeptides using the method of stable isotope labeling by amino acids in cell culture (SILAC) in combination with mass spectrometry. Principle findings The hitherto uncharacterized FAM124B (Family with sequence similarity 124B) was identified as a potential interaction partner of both CHD7 and CHD8. We confirmed the result by co-immunoprecipitation studies and showed a direct binding to the CHD8 part by direct yeast two hybrid experiments. Furthermore, we characterized FAM124B as a mainly nuclear localized protein with a widespread expression in embryonic and adult mouse tissues. Conclusion Our results demonstrate that FAM124B is a potential interacting partner of a CHD7 and CHD8 containing complex. From the overlapping expression pattern between Chd7 and Fam124B at murine embryonic day E12.5 and the high expression of Fam124B in the developing mouse brain, we conclude that Fam124B is a novel protein possibly involved in the pathogenesis of CHARGE syndrome and neurodevelopmental disorders. PMID:23285124

Searching for cellular partners of hantaviral nonstructural protein NSs: Y2H screening of mouse cDNA library and analysis of cellular interactome.

PubMed

Rönnberg, Tuomas; Jääskeläinen, Kirsi; Blot, Guillaume; Parviainen, Ville; Vaheri, Antti; Renkonen, Risto; Bouloy, Michele; Plyusnin, Alexander

2012-01-01

Hantaviruses (Bunyaviridae) are negative-strand RNA viruses with a tripartite genome. The small (S) segment encodes the nucleocapsid protein and, in some hantaviruses, also the nonstructural protein (NSs). The aim of this study was to find potential cellular partners for the hantaviral NSs protein. Toward this aim, yeast two-hybrid (Y2H) screening of mouse cDNA library was performed followed by a search for potential NSs protein counterparts via analyzing a cellular interactome. The resulting interaction network was shown to form logical, clustered structures. Furthermore, several potential binding partners for the NSs protein, for instance ACBD3, were identified and, to prove the principle, interaction between NSs and ACBD3 proteins was demonstrated biochemically.

FK506-Binding Proteins and Their Diverse Functions.

PubMed

Tong, Mingming; Jiang, Yu

2015-01-01

FK506 binding proteins (FKBPs) are a family of highly conserved proteins in eukaryotes. The prototype of this protein family, FKBP12, is the binding partner for immunosuppressive drugs FK506 and rapamycin. FKBP12 functions as a cis/trans peptidyl prolyl isomerase (PPIase) that catalyzes interconversion between prolyl cis/trans conformations. Members of the FKBP family contain one or several PPIase domains, which do not always exhibit PPIase activity yet are all essential for their function. FKBPs are involved in diverse cellular functions including protein folding, cellular signaling, apoptosis and transcription. They elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches. In this review, we provide a general summary for the structures and diverse functions of FKBPs found in mammalian cells.

Partners' controlling behaviors and intimate partner sexual violence among married women in Uganda.

PubMed

Wandera, Stephen Ojiambo; Kwagala, Betty; Ndugga, Patricia; Kabagenyi, Allen

2015-03-04

Studies on the association between partners' controlling behaviors and intimate partner sexual violence (IPSV) in Uganda are limited. The aim of this paper was to investigate the association between IPSV and partners' controlling behaviors among married women in Uganda. We used the 2011 Uganda Demographic and Health Survey (UDHS) data, and selected a weighted sample of 1,307 women who were in a union, out of those considered for the domestic violence module. We used chi-squared tests and multivariable logistic regressions to investigate the factors associated with IPSV, including partners' controlling behaviors. More than a quarter (27%) of women who were in a union in Uganda reported IPSV. The odds of reporting IPSV were higher among women whose partners were jealous if they talked with other men (OR = 1.81; 95% CI: 1.22-2.68), if their partners accused them of unfaithfulness (OR = 1.50; 95% CI: 1.03-2.19) and if their partners did not permit them to meet with female friends (OR = 1.63; 95% CI: 1.11-2.39). The odds of IPSV were also higher among women whose partners tried to limit contact with their family (OR = 1.73; 95% CI: 1.11-2.67) and often got drunk (OR = 1.80; 95% CI: 1.15-2.81). Finally, women who were sometimes or often afraid of their partners (OR = 1.78; 95% CI: 1.21-2.60 and OR = 1.56; 95% CI: 1.04-2.40 respectively) were more likely to report IPSV. In Uganda, women's socio-economic and demographic background and empowerment had no mitigating effect on IPSV in the face of their partners' dysfunctional behaviors. Interventions addressing IPSV should place more emphasis on reducing partners' controlling behaviors and the prevention of problem drinking.

Anger, Control, and Intimate Partner Violence in Young Adulthood

PubMed Central

Giordano, Peggy C.; Copp, Jennifer E.; Longmore, Monica A.; Manning, Wendy D.

2015-01-01

A common theme in the literature is that intimate partner violence (IPV) is not about anger, but about power and control. While prior research has focused either on respondents' or partners' controlling behaviors, an interactionist perspective provides the basis for hypothesizing that both respondent and partner control will be significantly related to the odds of reporting perpetration, and that emotional processes are a component of IPV experiences. Analyses rely on interview data collected at waves 1 and 5 of a longitudinal study (Toledo Adolescent Relationships Study; n = 928) of adolescent and young adult relationships. Results indicate that after controlling for traditional predictors, both respondent and partner control attempts and measures of anger (including a measure of relationship-based anger) contributed significantly to the odds of reporting perpetration. Further, these patterns did not differ by gender, indicating some areas of similarity in the relationship and emotional processes associated with variations in men and women's IPV reports. PMID:26924886

Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Production of bioinspired and rationally designed polymer hydrogels for controlled delivery of therapeutic proteins

NASA Astrophysics Data System (ADS)

Kim, Sung Hye

Hydrogel systems for controlled delivery therapeutic growth factors have been developed in a wide spectrum of strategies: these systems aim for the release of growth factors via a passive diffusion, electrostatic interaction, degradation of hydrogels, and responsiveness to external stimuli. Heparin, a highly sulfated glycosaminoglycan (GAG), was employed for a targeted delivery system of vascular endothelial growth factor (VEGF) to endothelial cells overexpressing a relevant receptor VEGFR-2. Addition of dimeric VEGF to 4-arm star-shaped poly(ethylene glycol) (PEG) immobilized with low-molecular weight heparin (LMWH) afforded a non-covalently assembled hydrogel via interaction between heparin and VEGF, with storage modulus 10 Pa. The release of VEGF and hydrogel erosion reached maximum 100 % at day 4 in the presence of VEGFR-2 overexpressing pocine aortic endothelial cell (PAE/KDR), while those of 80% were achieved via passive release at day 5 in the presence of PAE cell lacking VEGFR-2 or in the absence of cell, indicating that the release of VEGF was in targeted manner toward cell receptor. The proliferation of PAE/KDR in the presence of [PEG-LMWH/VEGF] hydrogel was greater by ca. 30% at day 4 compared to that of PAE, confirming that the release of VEGF was in response to the cellular demand. The phosphorylation fraction of VEGFR-2 on PAE/KDR was greater in the presence of [PEG-LMWH/VEGF] hydrogel, increasing from 0.568 at day 1 to 0.790 at day 4, whereas it was maintained at 0.230 at day 4 in the presence of [PEG-LMWH] hydrogel. This study has proven that this hydrogel, assembled via bio-inspired non-covalent interaction, liberating VEGFon celluar demand to target cell, eroding upon VEGF release, and triggering endothelial cell proliferation, could be used in multiple applications including targeted delivery and angiogenesis. Heparin has been widely exploited in growth factor delivery systems owing to its ability to bind many growth factors through the flexible patterns of functional groups. However, heterogeneity in the composition and in the polydispersity of heparin has been problematic in controlled delivery system and thus motivated the development of homogeneous heparin mimics. Peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in select applications. Studied was the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for VEGF; these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in select cases, isothermal titration calorimetry (ITC). The shortest peptide, SPa, showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SPa and PF4ZIP was indicated via SPR ( KD = 5.27 muM) and confirmed via ITC (KD = 8.09 muM). The binding by SPa of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications. Hydrogel consisting of SPa was formed via a covalent Michael Addition reaction between maleimide- and thiol-terminated multi-arm PEGs and Cys-SPa. The mechanical property of hydrogel was tunable from ca. 186 to 1940 Pa. by varing the cross-linking density, suggesting its flexible applications depending on matrix needs. The non-anti-coagulative property of SPa, assessed via activated partial thromboplastin time (APTT) and HeptestRTM in comparison to LMWH, implied its usefulness in applications without excessive bleeding. The VEGF released from [PEG-SPa] hydrogel showed up to ca. 400% greater bioactivity on proliferation of human umbilical vein endothelical cell (HUVEC) compared to the VEGF incubated in solution for the same period: this was significantly higher than that of [PEG] hydrogel (ca. 280%), suggesting the SPa may protect the bioactivity of VEGF when bound. The release of dual growth factor, i.e. VEGF and fibroblast growth factor-2 (FGF-2), were investigated on [PEG-SPa] hydrogel: the release of bFGF was lower than that of VEGF due to weaker binding affinity to matrix-bound SPa. The HUVEC culture on dual growth factor loaded [PEG-SPa] showed that the synergistic effects of dual system in select concentrations, suggesting the opportunity of manipulating cell responses. Given that sulfated peptides for various binding targets with desired affinity can be identified, applications are suggested in multiple growth factors delivery where an integrated action of multiple growth factors is required, such as angiogenesis.

Partner-delivered reflexology: effects on cancer pain and anxiety.

PubMed

Stephenson, Nancy L N; Swanson, Melvin; Dalton, Joann; Keefe, Frances J; Engelke, Martha

2007-01-01

To compare the effects of partner-delivered foot reflexology and usual care plus attention on patients' perceived pain and anxiety. The experimental pretest/post-test design included patient-partner dyads randomly assigned to an experimental or control group. Four hospitals in the southeastern United States. 42 experimental and 44 control subjects comprised 86 dyads of patients with metastatic cancer and their partners, representing 16 different types of cancer; 23% of patients had lung cancer, followed by breast, colorectal, and head and neck cancer and lymphoma. The subjects had a mean age of 58.3 years, 51% were female, 66% had a high school education or less, and 58% were Caucasian, 40% were African American, and 1% were Filipino. The intervention included a 15- to 30-minute teaching session on foot reflexology to the partner by a certified reflexologist, an optional 15- to 30-minute foot reflexology session for the partner, and a 30-minute, partner-delivered foot reflexology intervention for the patient. The control group received a 30-minute reading session from their partners. Pain and anxiety. Following the initial partner-delivered foot reflexology, patients experienced a significant decrease in pain intensity and anxiety. A nurse reflexologist taught partners how to perform reflexology on patients with metastatic cancer pain in the hospital, resulting in an immediate decrease in pain intensity and anxiety; minimal changes were seen in the control group, who received usual care plus attention. Hospitals could have qualified professionals offer reflexology as a complementary therapy and teach interested partners the modality.

p46Shc Inhibits Thiolase and Lipid Oxidation in Mitochondria*

PubMed Central

Tomilov, Alexey; Tomilova, Natalia; Shan, Yuxi; Hagopian, Kevork; Bettaieb, Ahmed; Kim, Kyoungmi; Pelicci, Pier Giuseppe; Haj, Fawaz; Ramsey, Jon; Cortopassi, Gino

2016-01-01

Although the p46Shc isoform has been known to be mitochondrially localized for 11 years, its function in mitochondria has been a mystery. We confirmed p46Shc to be mitochondrially localized and showed that the major mitochondrial partner of p46Shc is the lipid oxidation enzyme 3-ketoacylCoA thiolase ACAA2, to which p46Shc binds directly and with a strong affinity. Increasing p46Shc expression inhibits, and decreasing p46Shc stimulates enzymatic activity of thiolase in vitro. Thus, we suggest p46Shc to be a negative mitochondrial thiolase activity regulator, and reduction of p46Shc expression activates thiolase. This is the first demonstration of a protein that directly binds and controls thiolase activity. Thiolase was thought previously only to be regulated by metabolite balance and steady-state flux control. Thiolase is the last enzyme of the mitochondrial fatty acid beta-oxidation spiral, and thus is important for energy metabolism. Mice with reduction of p46Shc are lean, resist obesity, have higher lipid oxidation capacity, and increased thiolase activity. The thiolase-p46Shc connection shown here in vitro and in organello may be an important underlying mechanism explaining the metabolic phenotype of Shc-depleted mice in vivo. PMID:27059956

GuiTope: an application for mapping random-sequence peptides to protein sequences.

PubMed

Halperin, Rebecca F; Stafford, Phillip; Emery, Jack S; Navalkar, Krupa Arun; Johnston, Stephen Albert

2012-01-03

Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.

Physical interaction between the strawberry allergen Fra a 1 and an associated partner FaAP: Interaction of Fra a 1 proteins and FaAP.

PubMed

Franz-Oberdorf, Katrin; Langer, Andreas; Strasser, Ralf; Isono, Erika; Ranftl, Quirin L; Wunschel, Christian; Schwab, Wilfried

2017-10-01

The strawberry fruit allergens Fra a 1.01E, Fra a 1.02 and Fra a 1.03 belong to the group of pathogenesis-related 10 (PR-10) proteins and are homologs of the major birch pollen Bet v 1 and apple allergen Mal d 1. Bet v 1 related proteins are the most extensively studied allergens but their physiological function in planta remains elusive. Since Mal d 1-Associated Protein has been previously identified as interaction partner of Mal d 1 we studied the binding of the orthologous Fra a 1-Associated Protein (FaAP) to Fra a 1.01E/1.02/1.03. As the C-terminal sequence of FaAP showed strong auto-activation activity in yeast 2-hybrid analysis a novel time resolved DNA-switching system was successfully applied. Fra a 1.01E, Fra a 1.02, and Fra a 1.03 bind to FaAP with K D of 4.5 ± 1.1, 15 ± 3, and 11 ± 2 nM, respectively. Fra a 1.01E forms a dimer, whereas Fra a 1.02 and Fra a 1.03 bind as monomer. The results imply that PR-10 proteins might be integrated into a protein-interaction network and FaAP binding appears to be essential for the physiological function of the Fra a 1 proteins. © 2017 Wiley Periodicals, Inc.

Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

PubMed Central

Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

2013-01-01

A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma.

PubMed

Qin, Elizabeth Y; Cooper, Dominique D; Abbott, Keene L; Lennon, James; Nagaraja, Surya; Mackay, Alan; Jones, Chris; Vogel, Hannes; Jackson, Peter K; Monje, Michelle

2017-08-24

The lateral ventricle subventricular zone (SVZ) is a frequent and consequential site of pediatric and adult glioma spread, but the cellular and molecular mechanisms mediating this are poorly understood. We demonstrate that neural precursor cell (NPC):glioma cell communication underpins this propensity of glioma to colonize the SVZ through secretion of chemoattractant signals toward which glioma cells home. Biochemical, proteomic, and functional analyses of SVZ NPC-secreted factors revealed the neurite outgrowth-promoting factor pleiotrophin, along with required binding partners SPARC/SPARCL1 and HSP90B, as key mediators of this chemoattractant effect. Pleiotrophin expression is strongly enriched in the SVZ, and pleiotrophin knock down starkly reduced glioma invasion of the SVZ in the murine brain. Pleiotrophin, in complex with the binding partners, activated glioma Rho/ROCK signaling, and ROCK inhibition decreased invasion toward SVZ NPC-secreted factors. These findings demonstrate a pathogenic role for NPC:glioma interactions and potential therapeutic targets to limit glioma invasion. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

PubMed Central

Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Lucia, Maria Dell’Anna; Dell, Giulia 'Omo; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Giulia, Maria Rizzo; Gurtner, Aymone; Piaggio, Giulia

2017-01-01

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation. PMID:27793050

Romantic Partner Selection and Socialization during Early Adolescence

PubMed Central

Simon, Valerie A.; Aikins, Julie Wargo; Prinstein, Mitchell J.

2012-01-01

This prospective study examined romantic partner selection and socialization among a sample of 78 young adolescents (6th–8th graders). Independent assessments of adolescent and romantic partner adjustment were collected before and after relationships initiated via peer nomination and self-report. Prior to their relationship, adolescents and partners were significantly alike on popularity, physical attraction, and depressive symptoms. Controlling for initial similarity, partners' popularity, depressive symptoms, relational aggression and relational victimization significantly predicted changes in adolescents' functioning in these areas over time. However, the magnitude and direction of change varied according to adolescents' and partners' pre-relationship functioning. In general, adolescents who dated high-functioning partners changed more than those who dated low-functioning partners, and partner characteristics predicted greater change among low versus high-functioning adolescents. Results were consistent even when controlling for best friend characteristics. The current findings are among the first to demonstrate unique contributions of romantic partner characteristics to adolescents' psychosocial functioning. PMID:19037942

Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

PubMed Central

Uversky, Vladimir N.

2014-01-01

Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

Robust Multiple-Range Coherent Quantum State Transfer.

PubMed

Chen, Bing; Peng, Yan-Dong; Li, Yong; Qian, Xiao-Feng

2016-07-01

We propose a multiple-range quantum communication channel to realize coherent two-way quantum state transport with high fidelity. In our scheme, an information carrier (a qubit) and its remote partner are both adiabatically coupled to the same data bus, i.e., an N-site tight-binding chain that has a single defect at the center. At the weak interaction regime, our system is effectively equivalent to a three level system of which a coherent superposition of the two carrier states constitutes a dark state. The adiabatic coupling allows a well controllable information exchange timing via the dark state between the two carriers. Numerical results show that our scheme is robust and efficient under practically inevitable perturbative defects of the data bus as well as environmental dephasing noise.

Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton.

PubMed

Brdicková, N; Brdicka, T; Andera, L; Spicka, J; Angelisová, P; Milgram, S L; Horejsí, V

2001-10-26

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Autoinhibition of ankyrin-B/G membrane target bindings by intrinsically disordered segments from the tail regions

PubMed Central

Wang, Chao; Wei, Zhiyi

2017-01-01

Ankyrins together with their spectrin partners are the master organizers of micron-scale membrane domains in diverse tissues. The 24 ankyrin (ANK) repeats of ankyrins bind to numerous membrane proteins, linking them to spectrin-based cytoskeletons at specific membrane microdomains. The accessibility of the target binding groove of ANK repeats must be regulated to achieve spatially defined functions of ankyrins/target complexes in different tissues, though little is known in this regard. Here we systemically investigated the autoinhibition mechanism of ankyrin-B/G by combined biochemical, biophysical and structural biology approaches. We discovered that the entire ANK repeats are inhibited by combinatorial and quasi-independent bindings of multiple disordered segments located in the ankyrin-B/G linkers and tails, suggesting a mechanistic basis for differential regulations of membrane target bindings by ankyrins. In addition to elucidating the autoinhibition mechanisms of ankyrins, our study may also shed light on regulations on target bindings by other long repeat-containing proteins. PMID:28841137

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold*

PubMed Central

Tonkin, Michelle L.; Beck, Josh R.; Bradley, Peter J.; Boulanger, Martin J.

2014-01-01

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation. PMID:24675080

[Importance of binding of 2,3-diphosphoglycerate and ATP to hemoglobin for erythrocyte glycolysis: activation by 2,3-diphosphoglycerate of hexokinase at intracellular conditions].

PubMed

Geier, T; Glende, M; Reich, J G

1978-01-01

In a theoretical study the influence of hemoglobin and Mg-ions as binding partners of red cell 2,3-diphosphoglycerate and ATP was investigated. Free hemoglobin may be an efficient competitor of Mg2+ for the ligand ATP. At conditions which favour hemoglobin as binding partner (i.e. desoxygenation, low medium pH and incubation temperature, as in blood preservation) up to 95% of the whole cellular ATP (ca. 2mM in cell water) may be bound to hemoglobin (ca. 7 mM). This binding is largely prevented in the presence of physiological amounts of diphosphoglycerate (ca. 7 mM) which is in excess and has a higher binding affinity to hemoglobin. Therefore, diphosphoglycerate keeps ATP (MgATP) in cell water solution at conditions in which Hb would trop it in the presence of Mg2+ (ca. 3mM). It can be calculated that, by lack of free MgATP, the activity of hexokinase within the cell drops by a factor of greater than 10 when diphosphoglycerate is metabolized. This indirect activation by diphosphoglycerate of hexokinase is operative at free concentrations of DPG far below those which exert the well known excess inhibitory effect on hexokinase and phosphofructokinase. In a model study, the activation by diphosphoglycerate of the initial two-kinase stage was introduced into a simplified kinetic model of glycolysis. A pronounced hysteresis loop of the stationary concentrations of ATP and diphosphoglycerate was produced indicating the existence of several stationary states, one with high ATP and high diphosphoglycerate, the other one with low values. It is demonstrated that diphosphoglycerate, being a protector of glycolysis at physiological concentrations, triggers an autocatalytic breakdown of the energy state when permitted to drop to low values.

Defining the RNA-Protein Interactions in the Trypanosome Preribosomal Complex

PubMed Central

Wang, Lei; Ciganda, Martin

2013-01-01

In eukaryotes, 5S rRNA is transcribed in the nucleoplasm and requires the ribosomal protein L5 to deliver it to the nucleolus for ribosomal assembly. The trypanosome-specific proteins P34 and P37 form a novel preribosomal complex with the eukaryotic conserved L5-5S rRNA complex in the nucleoplasm. Previous results suggested that P34 acts together with L5 to bridge the interaction with 5S rRNA and thus to stabilize 5S rRNA, an important role in the early steps of ribosomal biogenesis. Here, we have delineated the domains of the two protein components, L5 and P34, and regions of the RNA partner, 5S rRNA, that are critical for protein-RNA interactions within the complex. We found that the L18 domain of L5 and the N terminus and RNA recognition motif of P34 bind 5S rRNA. We showed that Trypanosoma brucei L5 binds the β arm of 5S rRNA, while P34 binds loop A/stem V of 5S rRNA. We demonstrated that 5S rRNA is able to enhance the association between the protein components of the complex, L5 and P34. Both loop A/stem V and the β arm of 5S rRNA can separately enhance the protein-protein association, but their effects are neither additive nor synergistic. Domains in the two proteins for protein-protein and protein-RNA interactions overlap or are close to each other. This suggests that 5S rRNA binding might cause conformational changes in L5 and P34 and might also bridge the interactions, thus enhancing binding between the protein partners of this novel complex. PMID:23397568

Structural features of LC8-induced self-association of swallow.

PubMed

Kidane, Ariam I; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-09-03

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Structural Features of LC8-Induced Self Association of Swallow†

PubMed Central

Kidane, Ariam I.; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-01-01

Cell function depends on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than dynein and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the mechanistic and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, inter-monomer NOEs assigned to residues expected at interface positions and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled-coil. Secondary chemical shifts and 15N backbone relaxation identify the boundaries and distinguishing structural features of the coiled-coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled-coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled-coil is critical for bicoid mRNA localization because it could induce structural organization of Swallow, which except for the central LC8-promoted coiled-coil is primarily disordered. PMID:23914803

Small heterodimer partner blocks cardiac hypertrophy by interfering with GATA6 signaling.

PubMed

Nam, Yoon Seok; Kim, Yoojung; Joung, Hosouk; Kwon, Duk-Hwa; Choe, Nakwon; Min, Hyun-Ki; Kim, Yong Sook; Kim, Hyung-Seok; Kim, Don-Kyu; Cho, Young Kuk; Kim, Yong-Hoon; Nam, Kwang-Il; Choi, Hyoung Chul; Park, Dong Ho; Suk, Kyoungho; Lee, In-Kyu; Ahn, Youngkeun; Lee, Chul-Ho; Choi, Hueng-Sik; Eom, Gwang Hyeon; Kook, Hyun

2014-08-15

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated. We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy. The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice. These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response. © 2014 American Heart Association, Inc.

The CENP-T/-W complex is a binding partner of the histone chaperone FACT

PubMed Central

Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J.; Sullivan, Kevin F.; Almouzni, Geneviève

2016-01-01

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A–H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres. PMID:27284163

The CENP-T/-W complex is a binding partner of the histone chaperone FACT.

PubMed

Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J; Sullivan, Kevin F; Almouzni, Geneviève

2016-06-01

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres. © 2016 Prendergast et al.; Published by Cold Spring Harbor Laboratory Press.

Common structural features of cholesterol binding sites in crystallized soluble proteins

PubMed Central

Bukiya, Anna N.; Dopico, Alejandro M.

2017-01-01

Cholesterol-protein interactions are essential for the architectural organization of cell membranes and for lipid metabolism. While cholesterol-sensing motifs in transmembrane proteins have been identified, little is known about cholesterol recognition by soluble proteins. We reviewed the structural characteristics of binding sites for cholesterol and cholesterol sulfate from crystallographic structures available in the Protein Data Bank. This analysis unveiled key features of cholesterol-binding sites that are present in either all or the majority of sites: i) the cholesterol molecule is generally positioned between protein domains that have an organized secondary structure; ii) the cholesterol hydroxyl/sulfo group is often partnered by Asn, Gln, and/or Tyr, while the hydrophobic part of cholesterol interacts with Leu, Ile, Val, and/or Phe; iii) cholesterol hydrogen-bonding partners are often found on α-helices, while amino acids that interact with cholesterol’s hydrophobic core have a slight preference for β-strands and secondary structure-lacking protein areas; iv) the steroid’s C21 and C26 constitute the “hot spots” most often seen for steroid-protein hydrophobic interactions; v) common “cold spots” are C8–C10, C13, and C17, at which contacts with the proteins were not detected. Several common features we identified for soluble protein-steroid interaction appear evolutionarily conserved. PMID:28420706

A critical role for the transcription factor Scl in platelet production during stress thrombopoiesis.

PubMed

McCormack, Matthew P; Hall, Mark A; Schoenwaelder, Simone M; Zhao, Quan; Ellis, Sarah; Prentice, Julia A; Clarke, Ashleigh J; Slater, Nicholas J; Salmon, Jessica M; Jackson, Shaun P; Jane, Stephen M; Curtis, David J

2006-10-01

The generation of platelets from megakaryocytes in the steady state is regulated by a variety of cytokines and transcription factors, including thrombopoietin (TPO), GATA-1, and NF-E2. Less is known about platelet production in the setting of stress thrombopoiesis, a pivotal event in the context of cytotoxic chemotherapy. Here we show in mice that the transcription factor Scl is critical for platelet production after chemotherapy and in thrombopoiesis induced by administration of TPO. Megakaryocytes from these mice showed appropriate increases in number and ploidy but failed to shed platelets. Ultrastructural examination of Scl-null megakaryocytes revealed a disorganized demarcation membrane and reduction in platelet granules. Quantitative real-time polymerase chain reaction showed that Scl-null platelets lacked NF-E2, and chromatin immunoprecipitation analysis demonstrated Scl binding to the NF-E2 promoter in the human megakaryoblastic-cell line Meg-01, along with its binding partners E47, Lmo2, and the cofactors Ldb1 and GATA-2. These findings suggest that Scl acts up-stream of NF-E2 expression to control megakaryocyte development and platelet release in settings of thrombopoietic stress.

Association of Cell Adhesion Molecules Contactin-6 and Latrophilin-1 Regulates Neuronal Apoptosis

PubMed Central

Zuko, Amila; Oguro-Ando, Asami; Post, Harm; Taggenbrock, Renske L. R. E.; van Dijk, Roland E.; Altelaar, A. F. Maarten; Heck, Albert J. R.; Petrenko, Alexander G.; van der Zwaag, Bert; Shimoda, Yasushi; Pasterkamp, R. Jeroen; Burbach, J. Peter H.

2016-01-01

In view of important neurobiological functions of the cell adhesion molecule contactin-6 (Cntn6) that have emerged from studies on null-mutant mice and autism spectrum disorders patients, we set out to examine pathways underlying functions of Cntn6 using a proteomics approach. We identified the cell adhesion GPCR latrophilin-1 (Lphn1, a.k.a. CIRL1/CL, ADGRL1) as a binding partner for Cntn6 forming together a heteromeric cis-complex. Lphn1 expression in cultured neurons caused reduction in neurite outgrowth and increase in apoptosis, which was rescued by coexpression of Cntn6. In cultured neurons derived from Cntn6-/- mice, Lphn1 knockdown reduced apoptosis, suggesting that the observed apoptosis was Lphn1-dependent. In line with these data, the number of apoptotic cells was increased in the cortex of Cntn6-/- mice compared to wild-type littermate controls. These results show that Cntn6 can modulate the activity of Lphn1 by direct binding and suggests that Cntn6 may prevent apoptosis thereby impinging on neurodevelopment. PMID:28018171

Loss-of-function mutation in GATA4 causes anomalies of human testicular development

PubMed Central

Lourenço, Diana; Brauner, Raja; Rybczyńska, Magda; Nihoul-Fékété, Claire; McElreavey, Ken; Bashamboo, Anu

2011-01-01

Approximately 1 of every 250 newborns has some abnormality of genital and/or gonadal development. However, a specific molecular cause is identified in only 20% of these cases of disorder of sex development (DSD). We identified a family of French origin presenting with 46,XY DSD and congenital heart disease. Sequencing of the ORF of GATA4 identified a heterozygous missense mutation (p.Gly221Arg) in the conserved N-terminal zinc finger of GATA4. This mutation was not observed in 450 ancestry-matched control individuals. The mutation compromised the ability of the protein to bind to and transactivate the anti-Müllerian hormone (AMH) promoter. The mutation does not interfere with the direct protein–protein interaction, but it disrupts synergistic activation of the AMH promoter by GATA4 and NR5A1. The p.Gly221Arg mutant protein also failed to bind to a known protein partner FOG2 that is essential for gonad formation. Our data demonstrate the key role of GATA4 in human testicular development. PMID:21220346

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

A case-control study evaluating the role of internet meet-up sites and mobile telephone applications in influencing a syphilis outbreak: Multnomah County, Oregon, USA 2014.

PubMed

DeSilva, Malini; Hedberg, Katrina; Robinson, Byron; Toevs, Kim; Neblett-Fanfair, Robyn; Petrosky, Emiko; Hariri, Susan; Schafer, Sean

2016-08-01

Early syphilis in Multnomah County, Oregon, USA, increased 16-fold during 2007-2013. Cases predominantly occurred among men who have sex with men (MSM); 55% were HIV coinfected. We conducted a case-control study to evaluate the association between meeting sex partners online and early syphilis. Cases subjects (cases) were Multnomah County resident, English speaking, MSM, aged ≥18 years with laboratory-confirmed early syphilis reported 1 January to 31 December 2013. We recruited two MSM controls subjects (controls) per case, frequency matched by HIV status and age. Participants completed self-administered questionnaires. We performed multivariable logistic regression. Seventy per cent (40/57) of cases and 42% (50/119) of controls met partners online (p<0.001). Cases more frequently met partners online (adjusted OR (aOR)=3.0; 95% CI 1.2 to 6.7), controlling for presumptive confounders. Cases reported more partners than controls (medians 5, 2; p<0.001). When including number of partners, aOR decreased to 1.4 (95% CI 0.5 to 3.9). Early syphilis was associated with meeting partners online. We believe this association may be related to number of sex partners acting as an intermediate variable between use of online resources to meet sex partners and early syphilis. Online meet-up sites might represent areas for public health interventions targeting at-risk individuals. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Warts phosphorylates Mud to promote Pins-mediated mitotic spindle orientation in Drosophila independent of Yorkie

PubMed Central

Dewey, Evan B.; Sanchez, Desiree; Johnston, Christopher A.

2015-01-01

SUMMARY Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily-conserved cell proliferation pathway. PMID:26592339

The Diverse Roles of Arrestin Scaffolds in G Protein-Coupled Receptor Signaling.

PubMed

Peterson, Yuri K; Luttrell, Louis M

2017-07-01

The visual/ β -arrestins, a small family of proteins originally described for their role in the desensitization and intracellular trafficking of G protein-coupled receptors (GPCRs), have emerged as key regulators of multiple signaling pathways. Evolutionarily related to a larger group of regulatory scaffolds that share a common arrestin fold, the visual/ β -arrestins acquired the capacity to detect and bind activated GPCRs on the plasma membrane, which enables them to control GPCR desensitization, internalization, and intracellular trafficking. By acting as scaffolds that bind key pathway intermediates, visual/ β -arrestins both influence the tonic level of pathway activity in cells and, in some cases, serve as ligand-regulated scaffolds for GPCR-mediated signaling. Growing evidence supports the physiologic and pathophysiologic roles of arrestins and underscores their potential as therapeutic targets. Circumventing arrestin-dependent GPCR desensitization may alleviate the problem of tachyphylaxis to drugs that target GPCRs, and find application in the management of chronic pain, asthma, and psychiatric illness. As signaling scaffolds, arrestins are also central regulators of pathways controlling cell growth, migration, and survival, suggesting that manipulating their scaffolding functions may be beneficial in inflammatory diseases, fibrosis, and cancer. In this review we examine the structure-function relationships that enable arrestins to perform their diverse roles, addressing arrestin structure at the molecular level, the relationship between arrestin conformation and function, and sites of interaction between arrestins, GPCRs, and nonreceptor-binding partners. We conclude with a discussion of arrestins as therapeutic targets and the settings in which manipulating arrestin function might be of clinical benefit. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Warts phosphorylates mud to promote pins-mediated mitotic spindle orientation in Drosophila, independent of Yorkie.

PubMed

Dewey, Evan B; Sanchez, Desiree; Johnston, Christopher A

2015-11-02

Multicellular animals have evolved conserved signaling pathways that translate cell polarity cues into mitotic spindle positioning to control the orientation of cell division within complex tissue structures. These oriented cell divisions are essential for the development of cell diversity and the maintenance of tissue homeostasis. Despite intense efforts, the molecular mechanisms that control spindle orientation remain incompletely defined. Here, we describe a role for the Hippo (Hpo) kinase complex in promoting Partner of Inscuteable (Pins)-mediated spindle orientation. Knockdown of Hpo, Salvador (Sav), or Warts (Wts) each result in a partial loss of spindle orientation, a phenotype previously described following loss of the Pins-binding protein Mushroom body defect (Mud). Similar to orthologs spanning yeast to mammals, Wts kinase localizes to mitotic spindle poles, a prominent site of Mud localization. Wts directly phosphorylates Mud in vitro within its C-terminal coiled-coil domain. This Mud coiled-coil domain directly binds the adjacent Pins-binding domain to dampen the Pins/Mud interaction, and Wts-mediated phosphorylation uncouples this intramolecular Mud interaction. Loss of Wts prevents cortical Pins/Mud association without affecting Mud accumulation at spindle poles, suggesting phosphorylation acts as a molecular switch to specifically activate cortical Mud function. Finally, loss of Wts in Drosophila imaginal disc epithelial cells results in diminished cortical Mud and defective planar spindle orientation. Our results provide new insights into the molecular basis for dynamic regulation of the cortical Pins/Mud spindle positioning complex and highlight a novel link with an essential, evolutionarily conserved cell proliferation pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional importance of EAK1 tyrosine phosphorylation in vivo

USDA-ARS?s Scientific Manuscript database

The plant receptor kinase BRASSINOSTEROID ASSOCIATED KINASE 1 (BAK1) is known as a partner of several ligand-binding leucine-rich repeat receptor kinases, including BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLS2. Autophosphorylation of receptor kinases is recognized to be an i...

Controlling behavior, power relations within intimate relationships and intimate partner physical and sexual violence against women in Nigeria

PubMed Central

2011-01-01

Background Controlling behavior is more common and can be equally or more threatening than physical or sexual violence. This study sought to determine the role of husband/partner controlling behavior and power relations within intimate relationships in the lifetime risk of physical and sexual violence in Nigeria. Methods This study used secondary data from a cross-sectional nationally-representative survey collected by face-to-face interviews from women aged 15 - 49 years in the 2008 Nigeria Demographic and Health Survey. Utilizing a stratified two-stage cluster sample design, data was collected frrm 19 216 eligible with the DHS domestic violence module, which is based on the Conflict Tactics Scale (CTS). Multivariate logistic regression analysis was used to determine the role of husband/partner controlling behavior in the risk of ever experiencing physical and sexual violence among 2877 women aged 15 - 49 years who were currently or formerly married or cohabiting with a male partner. Results Women who reported controlling behavior by husband/partner had a higher likelihood of experiencing physical violence (RR = 3.04; 95% CI: 2.50 - 3.69), and women resident in rural areas and working in low status occupations had increased likelihood of experiencing physical IPV. Controlling behavior by husband/partner was associated with higher likelihood of experiencing physical violence (RR = 4.01; 95% CI: 2.54 - 6.34). In addition, women who justified wife beating and earned more than their husband/partner were at higher likelihood of experiencing physical and sexual violence. In contrast, women who had decision-making autonomy had lower likelihood of experiencing physical and sexual violence. Conclusion Controlling behavior by husband/partner significantly increases the likelihood of physical and sexual IPV, thus acting as a precursor to violence. Findings emphasize the need to adopt a proactive integrated approach to controlling behavior and intimate partner violence within the society. PMID:21714854

Partnership for a Healthier America: Creating Change Through Private Sector Partnerships.

PubMed

Simon, Caitlin; Kocot, S Lawrence; Dietz, William H

2017-06-01

This review provides background on the formation of the Partnership for a Healthier America (PHA), that was created in conjunction with the Let's Move! initiative, and an overview of its work to date. To encourage industry to offer and promote healthier options, PHA partners with the private sector. Principles that guide PHA partnerships include ensuring that partnerships represent meaningful change, partners sign a legally binding contract and progress is monitored and publicly reported. Since 2010, PHA has established private sector partnerships in an effort to transform the marketplace to ensure that every child has the chance to grow up at a healthy weight. Many agreements between PHA and its industry partners align with the White House Task Force Report on Childhood Obesity. The reach and impact of over 200 partnerships attest to the success of this initiative.

WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac.

PubMed

Miki, Hiroaki; Takenawa, Tadaomi

2002-04-26

We previously reported that IRSp53 binds both Rac and WAVE2, inducing formation of Rac/IRSp53/WAVE2 complex that is important for membrane ruffling. However, recent reports noted a specific interaction between IRSp53 and Cdc42 but not Rac, which led us to re-examine the binding of IRSp53 to Rac. Immunoprecipitation analysis and pull-down assay reveal that full-length IRSp53 binds Rac much less efficiently than the N-terminal fragment, which may be caused by intramolecular interaction. Interestingly, the intramolecular interaction is interrupted by the binding of WAVE2 and full-length IRSp53 associates with Rac in the presence of WAVE2. We also report that IRSp53 induces spreading and neurite formation of N1E-115 cells, which presumably reflect functional cooperation with Rac.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

PubMed Central

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I.; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R.; Oren, Moshe; Croce, Carlo M.; Bernassola, Francesca; Melino, Gerry

2007-01-01

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin–protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73α, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73α for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73α and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1−/− cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73α and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. PMID:17592138

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

Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang

Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, amore » vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding partner, RXR{alpha}.« less

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms.

PubMed

Fulcher, Luke J; Bozatzi, Polyxeni; Tachie-Menson, Theresa; Wu, Kevin Z L; Cummins, Timothy D; Bufton, Joshua C; Pinkas, Daniel M; Dunbar, Karen; Shrestha, Sabin; Wood, Nicola T; Weidlich, Simone; Macartney, Thomas J; Varghese, Joby; Gourlay, Robert; Campbell, David G; Dingwell, Kevin S; Smith, James C; Bullock, Alex N; Sapkota, Gopal P

2018-05-22

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Insight into the Role of Ca2+-Binding Protein 5 in Vesicle Exocytosis

PubMed Central

Sokal, Izabela

2011-01-01

Purpose. CaBP5 is a neuronal calmodulin-like Ca2+-binding protein that is expressed in the retina and in the cochlea. Although CaBP5 knockout mice displayed reduced sensitivity of retinal ganglion cell light responses, the function of CaBP5 in vivo is still unknown. To gain further insight into CaBP5 function, the authors screened for CaBP5-interacting partners. Methods. Potential retinal interacting partners for CaBP5 were identified using affinity chromatography followed by mass spectrometry and by yeast two-hybrid screening of a bovine retina cDNA library. Interacting partners were further analyzed using coimmunoprecipitation. Immunohistochemistry and subcellular fractionation were performed to determine their colocalization in the retina. The effect of CaBP5 on dopamine release and neurite outgrowth of PC12 cells was analyzed using ELISA and fluorescent labeling. Results. Using affinity chromatography, the authors identified Munc18–1 and myosin VI as interacting partners for CaBP5. Munc18–1 was also identified using the yeast two-hybrid system. Colocalization and coimmunoprecipitation of CaBP5 with these two proteins in retinal tissue further established their physiological interactions. Furthermore, CaBP5 expression in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis. Conclusions. This study shows that CaBP5 interacts with Munc18–1 and myosin VI, two proteins involved in the synaptic vesicle cycle. Together with the effect of CaBP5 in stimulating neurite outgrowth and vesicle exocytosis in PC12 cells, these results suggest that CaBP5 plays a role in neurotransmitter release. PMID:22039235

The Social Contract and the African American Elderly.

ERIC Educational Resources Information Center

Madison, Anna

1992-01-01

Explores whether or not vulnerable African-American elderly have a basic right to protection under the social contract that binds members of society as equal partners in a compact guaranteeing all members basic rights. A social justice perspective places these citizens among those who qualify for public support. (SLD)

NFI Transcription Factors Interact with FOXA1 to Regulate Prostate-Specific Gene Expression

PubMed Central

Elliott, Amicia D.; DeGraff, David J.; Anderson, Philip D.; Anumanthan, Govindaraj; Yamashita, Hironobu; Sun, Qian; Friedman, David B.; Hachey, David L.; Yu, Xiuping; Sheehan, Jonathan H.; Ahn, Jung-Mo; Raj, Ganesh V.; Piston, David W.; Gronostajski, Richard M.; Matusik, Robert J.

2014-01-01

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression. PMID:24801505

Caregiving tasks and caregiver burden; effects of an psycho-educational intervention in partners of patients with post-operative heart failure.

PubMed

Ågren, Susanna; Strömberg, Anna; Jaarsma, Tiny; Luttik, Marie Louise A

2015-01-01

To evaluate the effects of a psycho-educational intervention on caregiver burden in partners of patients with postoperative heart failure. Since partners of cardiac surgery patients play a significant role in the patient's recovery, it is important to address their needs during hospitalization and after discharge. Forty-two patients with postoperative heart failure and their partners participated in a randomized controlled pilot study. Dyads in the intervention group received psycho-educational support from a multidisciplinary team. Dyads in the control group received usual care. No significant differences were found in the performance of caregiving tasks and perceived caregiver burden in the control versus the intervention group. A pilot study exploring the effects of a psycho-educational intervention in patients and their partners did not reveal significant effects with regard to reduced feelings of burden in partners. Alleviating caregiver burden in partners may need a more intense or specific approach. Copyright © 2015 Elsevier Inc. All rights reserved.

Does Endometriosis Affect Sexual Activity and Satisfaction of the Man Partner? A Comparison of Partners From Women Diagnosed With Endometriosis and Controls.

PubMed

Hämmerli, Silvan; Kohl Schwartz, Alexandra Sabrina; Geraedts, Kirsten; Imesch, Patrick; Rauchfuss, Martina; Wölfler, Monika Maria; Haeberlin, Felix; von Orelli, Stefanie; Eberhard, Markus; Imthurn, Bruno; Leeners, Brigitte

2018-06-01

Endometriosis-associated pain and dyspareunia influence female sexuality, but little is known about men's experiences in affected couples. To investigate how men partners experience sexuality in partnership with women with endometriosis. A multi-center case-control study was performed between 2010 and 2015 in Switzerland, Germany, and Austria. 236 Partners of endometriosis patients and 236 partners of age-matched control women without endometriosis with a similar ethnic background were asked to answer selected, relevant questions of the Brief Index of Sexual Functioning and the Global Sexual Functioning questionnaire, as well as some investigator-derived questions. We sought to evaluate sexual satisfaction of men partners of endometriosis patients, investigate differences in sexual activities between men partners of women with and without endometriosis, and identify options to improve partnership sexuality in couples affected by endometriosis. Many partners of endometriosis patients reported changes in sexuality (75%). A majority of both groups was (very) satisfied with their sexual relationship (73.8% vs 58.1%, P = .002). Nevertheless, more partners of women diagnosed with endometriosis were not satisfied (P = .002) and their sexual problems more strongly interfered with relationship happiness (P = .001) than in partners of control women. Frequencies of sexual intercourse (P < .001) and all other partnered sexual activities (oral sex, petting) were significantly higher in the control group. The wish for an increased frequency of sexual activity (P = .387) and sexual desire (P = .919) did not differ statistically between both groups. There is a need to evaluate qualitative factors that influence sexual satisfaction in endometriosis patients. This is one of the first studies to investigate male sexuality affected by endometriosis. The meticulous verification of diagnosis and disease stage according to operation reports and histology allows for a high reliability of diagnosis. Our men's response rate of almost 50% is higher compared to other studies. Recruiting men through their woman partner may have caused selection bias. The adjustment to the specific situation in endometriosis by selecting questions from the Brief Index of Sexual Functioning and Global Sexual Functioning and adding investigator-derived questions likely influenced the validity of the questionnaires. Despite the fact that both partners of endometriosis patients and of control women largely reported high sexual satisfaction, there are challenges for some couples that arise in the context of a sexual relationship when one partner has endometriosis. Challenges such as sexuality-related pain or a reduced frequency of sexual activities should be addressed by health care professionals to ameliorate any current difficulties and to prevent the development or aggravation of sexual dysfunction. Hämmerli S, Kohl Schwartz AS, Geraedts K, et al. Does Endometriosis Affect Sexual Activity and Satisfaction of the Man Partner? A Comparison of Partners From Women Diagnosed With Endometriosis and Controls. J Sex Med 2018;15:853-865. Copyright © 2018 International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.

Protocols Utilizing Constant pH Molecular Dynamics to Compute pH-Dependent Binding Free Energies

PubMed Central

2015-01-01

In protein–ligand binding, the electrostatic environments of the two binding partners may vary significantly in bound and unbound states, which may lead to protonation changes upon binding. In cases where ligand binding results in a net uptake or release of protons, the free energy of binding is pH-dependent. Nevertheless, conventional free energy calculations and molecular docking protocols typically do not rigorously account for changes in protonation that may occur upon ligand binding. To address these shortcomings, we present a simple methodology based on Wyman’s binding polynomial formalism to account for the pH dependence of binding free energies and demonstrate its use on cucurbit[7]uril (CB[7]) host–guest systems. Using constant pH molecular dynamics and a reference binding free energy that is taken either from experiment or from thermodynamic integration computations, the pH-dependent binding free energy is determined. This computational protocol accurately captures the large pKa shifts observed experimentally upon CB[7]:guest association and reproduces experimental binding free energies at different levels of pH. We show that incorrect assignment of fixed protonation states in free energy computations can give errors of >2 kcal/mol in these host–guest systems. Use of the methods presented here avoids such errors, thus suggesting their utility in computing proton-linked binding free energies for protein–ligand complexes. PMID:25134690

Preferences and Ratings of Partner Traits in Female Survivors of Childhood Abuse With PTSD and Healthy Controls.

PubMed

Lieberz, Klara A; Müller-Engelmann, Meike; Priebe, Kathlen; Friedmann, Franziska; Görg, Nora; Herzog, Julia Isabell; Steil, Regina

2017-11-01

There is growing empirical evidence for an association between childhood abuse (CA) and intimate partner violence (IPV) in adulthood. We tested whether revictimized survivors of severe to extreme severities of child sexual abuse (CSA) and severe severities of child physical abuse (CPA) differed from nonvictimized healthy controls in their trait preferences in intimate partners and their current mate choice. In a sample of 52 revictimized female patients with posttraumatic stress disorder (PTSD) after CSA/CPA and 52 female healthy controls, the validated Intimate Partner Preferences Questionnaire (IPPQ) was used to assess (a) the desirability of tenderness, dominance, and aggression traits in potential partners, and (b) the presence of these traits in their current intimate partners. Factors potentially associated with partner preference and mate choice, for example, chronicity of traumatic events and lower self-esteem, were explored. Our results showed that, in general, revictimized PTSD patients did not have a preference for dominant or aggressive partners. However, revictimized women displayed a significantly larger discrepancy than did healthy controls between their preferences for tenderness traits and their ratings of the presence of tenderness traits in their current partners. Our results indicated that revictimized patients had lower self-esteem values; however, these values were associated with higher demands for tenderness traits. Furthermore, our results revealed that compared with patients who experienced early-onset childhood abuse (CA), those who experienced later onset CA were more accepting of dominant traits in potential partners. Women who had experienced IPV rated their current partners to be overly dominant. A higher tolerance of dominance traits might increase the risk of IPV in a specific subgroup of abused women (women with a later onset of abuse experiences and experiences of IPV).

Spouses' involvement in their partners' diabetes management: associations with spouse stress and perceived marital quality.

PubMed

August, Kristin J; Rook, Karen S; Franks, Melissa M; Parris Stephens, Mary Ann

2013-10-01

Spouses frequently attempt to influence (control) or support their chronically ill partners' adherence behaviors. Studies have documented effects of spousal control and support on chronically ill individuals, but little is known about how these two forms of involvement in a partner's disease management may be associated with spouses' stress or the quality of their interactions with their ill partners. The current study sought to address this gap by examining spouses' day-to-day involvement in their marital partner's management of type 2 diabetes (n = 129). Multilevel analyses of daily diary data revealed that on days when spouses exerted control, they reported more stress and more tense marital interactions, although these associations were more pronounced when patients exhibited poor adherence, had been ill for a longer period of time, and had more comorbid health conditions. On days when spouses provided support, in contrast, they reported less stress and more enjoyable marital interactions. The findings from the current study suggest that spouses' day-to-day stress and quality of interactions with their partners are associated with spouses' involvement in their partners' disease management, with health-related social control and support exhibiting distinctive associations.

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

PubMed Central

Nallamsetty, Sreedevi; Waugh, David S.

2007-01-01

Certain highly soluble proteins, such as Escherichia coli maltose-binding protein (MBP), have the ability to enhance the solubility of their fusion partners, making them attractive vehicles for the production of recombinant proteins, yet the mechanism of solubility enhancement remains poorly understood. Here, we report that the solubility-enhancing properties of MBP are dramatically affected by amino acid substitutions that alter the equilibrium between its “open” and “closed” conformations. Our findings indicate that the solubility-enhancing activity of MBP is mediated by its open conformation and point to a likely role for the ligand-binding cleft in the mechanism of solubility enhancement. PMID:17964542

The potential role of CacyBP/SIP in tumorigenesis.

PubMed

Ning, Xiaoxuan; Chen, Yang; Wang, Xiaosu; Li, Qiaoneng; Sun, Shiren

2016-08-01

Calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) was initially described as a binding partner of S100A6 in the Ehrlich ascites tumor cells and later as a Siah-1-interacting protein. This 30 kDa protein includes three domains and is involved in cell proliferation, differentiation, cytoskeletal rearrangement, and transcriptional regulation via binding to various proteins. Studies have also shown that the CacyBP/SIP is a critical protein in tumorigenesis. But, its promotion or suppression of cancer progression may depend on the cell type. In this review, the biological characteristics and target proteins of CacyBP/SIP have been described. Moreover, the exact role of CacyBP/SIP in various cancers is discussed.

Atomic resolution model of the antibody Fc interaction with the complement C1q component.

PubMed

Schneider, Sebastian; Zacharias, Martin

2012-05-01

The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q-Fc complex is known so far. Based on systematic protein-protein docking searches and Molecular Dynamics simulations a structural model of the C1q-IgG1-Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1-Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge-charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures. Copyright © 2012 Elsevier Ltd. All rights reserved.

Dissecting partner recognition by an intrinsically disordered protein using descriptive random mutagenesis.

PubMed

Gruet, Antoine; Dosnon, Marion; Vassena, Andrea; Lombard, Vincent; Gerlier, Denis; Bignon, Christophe; Longhi, Sonia

2013-09-23

In view of getting insights into the molecular determinants of the binding efficiency of intrinsically disordered proteins (IDPs), we used random mutagenesis. As a proof of concept, we chose the interaction between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein and assessed how amino acid substitutions introduced at random within NTAIL affect partner recognition. In contrast with directed evolution approaches, we did not apply any selection and used the gene library approach not for production purposes but for achieving a better understanding of the NTAIL/XD interaction. For that reason, and to differentiate our approach from similar approaches that make use of systematic (i.e., targeted) mutagenesis, we propose to call it "descriptive random mutagenesis" (DRM). NTAIL variants generated by error-prone PCR were picked at random in the absence of selection pressure and were characterized in terms of sequence and binding abilities toward XD. DRM not only identified determinants of NTAIL/XD interaction that were in good agreement with previous work but also provided new insights. In particular, we discovered that the primary interaction site is poorly evolvable in terms of binding abilities toward XD. We also identified a critical NTAIL residue whose role in stabilizing the NTAIL/XD complex had previously escaped detection, and we identified NTAIL regulatory sites that dampen the interaction while being located outside the primary interaction site. Results show that DRM is a valuable approach to study binding abilities of IDPs. © 2013 Elsevier Ltd. All rights reserved.

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells.

PubMed

Wakayama, Tomohiko; Sai, Yoshimichi; Ito, Akihiko; Kato, Yukio; Kurobo, Miho; Murakami, Yoshinori; Nakashima, Emi; Tsuji, Akira; Kitamura, Yukihiko; Iseki, Shoichi

2007-06-01

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

A single arginine residue is required for the interaction of the electron transferring flavoprotein (ETF) with three of its dehydrogenase partners.

PubMed

Parker, Antony R

2003-12-01

The interaction of several dehydrogenases with the electron transferring flavoprotein (ETF) is a crucial step required for the successful transfer of electrons into the electron transport chain. The exact determinants regarding the interaction of ETF with its dehydrogenase partners are still unknown. Chemical modification of ETF with arginine-specific reagents resulted in the loss, to varying degrees, of activity with medium chain acyl-coenzyme A dehydrogenase (MCAD). The kinetic profiles showed the inactivations followed pseudo-first-order kinetics for all reagents used. For activity with MCAD, maximum inactivation of ETF was accomplished by 2,3-butanedione (4% residual activity after 120 min) and it was shown that modification of one arginine residue was responsible for the inactivation. Almost 100% restoration of this ETF activity was achieved upon incubation with free arginine. However, the same 2,3-butanedione modified ETF only possessed decreased activity with dimethylglycine-(DMGDH, 44%) and sarcosine- (SDH, 27%) dehydrogenases unlike the abolition with MCAD. Full protection of ETF from arginine modification by 2,3-butanedione was achieved using substrate-protected DMGDH, MCAD and SDH respectively. Cross-protection studies of ETF with the three dehydrogenases implied use of the same single arginine residue in the binding of all three dehydrogenases. These results lead us to conclude that this single arginine residue is essential in the binding of the ETF to MCAD, but only contributes partially to the binding of ETF to SDH and DMGDH and thus, the determinants of the dehydrogenase binding sites overlap but are not identical.

Interolog interfaces in protein–protein docking

PubMed Central

Alsop, James D.

2015-01-01

ABSTRACT Proteins are essential elements of biological systems, and their function typically relies on their ability to successfully bind to specific partners. Recently, an emphasis of study into protein interactions has been on hot spots, or residues in the binding interface that make a significant contribution to the binding energetics. In this study, we investigate how conservation of hot spots can be used to guide docking prediction. We show that the use of evolutionary data combined with hot spot prediction highlights near‐native structures across a range of benchmark examples. Our approach explores various strategies for using hot spots and evolutionary data to score protein complexes, using both absolute and chemical definitions of conservation along with refinements to these strategies that look at windowed conservation and filtering to ensure a minimum number of hot spots in each binding partner. Finally, structure‐based models of orthologs were generated for comparison with sequence‐based scoring. Using two data sets of 22 and 85 examples, a high rate of top 10 and top 1 predictions are observed, with up to 82% of examples returning a top 10 hit and 35% returning top 1 hit depending on the data set and strategy applied; upon inclusion of the native structure among the decoys, up to 55% of examples yielded a top 1 hit. The 20 common examples between data sets show that more carefully curated interolog data yields better predictions, particularly in achieving top 1 hits. Proteins 2015; 83:1940–1946. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25740680

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

PubMed

Mu, Hong; Geacintov, Nicholas E; Min, Jung-Hyun; Zhang, Yingkai; Broyde, Suse

2017-06-19

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N 2 -dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the "pre-flipped" base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [ Mu , H. , ( 2015 ) Biochemistry , 54 ( 34 ), 5263 - 7 ]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

PubMed Central

2017-01-01

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54(34), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER. PMID:28460163

Bovine Leukemia Virus SU Protein Interacts with Zinc, and Mutations within Two Interacting Regions Differently Affect Viral Fusion and Infectivity In Vivo

PubMed Central

Gatot, Jean-Stéphane; Callebaut, Isabelle; Van Lint, Carine; Demonté, Dominique; Kerkhofs, Pierre; Portetelle, Daniel; Burny, Arsène; Willems, Luc; Kettmann, Richard

2002-01-01

Bovine leukemia virus (BLV) and human T-cell lymphotropic virus type 1 (HTLV-1) belong to the genus of deltaretroviruses. Their entry into the host cell is supposed to be mediated by interactions of the extracellular (SU) envelope glycoproteins with cellular receptors. To gain insight into the mechanisms governing this process, we investigated the ability of SU proteins to interact with specific ligands. In particular, by affinity chromatography, we have shown that BLV SU protein specifically interacted with zinc ions. To identify the protein domains involved in binding, 16 peptides distributed along the sequence were tested. Two of them appeared to be able to interact with zinc. To unravel the role of these SU regions in the biology of the virus, mutations were introduced into the env gene of a BLV molecular clone in order to modify residues potentially interacting with zinc. The fusogenic capacity of envelope mutated within the first zinc-binding region (104 to 123) was completely abolished. Furthermore, the integrity of this domain was also required for in vivo infectivity. In contrast, mutations within the second zinc-binding region (218 to 237) did not hamper the fusogenic capacity; indeed, the syncytia were even larger. In sheep, mutations in region 218 to 237 did not alter infectivity or viral spread. Finally, we demonstrated that the envelope of the related HTLV-1 was also able to bind zinc. Interestingly, zinc ions were found to be associated with the receptor-binding domain (RBD) of Friend murine leukemia virus (Fr-MLV) SU glycoprotein, further supporting their relevance in SU structure. Based on the sequence similarities shared with the Fr-MLV RBD, whose three-dimensional structure has been experimentally determined, we located the BLV zinc-binding peptide 104-123 on the opposite side of the potential receptor-binding surface. This observation supports the hypothesis that zinc ions could mediate interactions of the SU RBD either with the C-terminal part of SU, thereby contributing to the SU structural integrity, or with a partner(s) different from the receptor. PMID:12134000

Sex Differences in Intimate Partner Violence and the Use of Coercive Control as a Motivational Factor for Intimate Partner Violence

ERIC Educational Resources Information Center

Tanha, Marieh; Beck, Connie J. A.; Figueredo, Aurelio Jose; Raghavan, Chitra

2010-01-01

Research argues that coercive control (CC) is a special case of intimate partner violence (IPV). The present study hypothesized that instead CC is the "motivator" for other types of IPV, with control of the victim as the goal. When CC fails, physical types of IPV are used. This hypothesized relationship was tested using a large matched sample of…

Psychoeducational support to post cardiac surgery heart failure patients and their partners--a randomised pilot study.

PubMed

Ågren, Susanna; Berg, Sören; Svedjeholm, Rolf; Strömberg, Anna

2015-02-01

Postoperative heart failure is a serious complication that changes the lives of both the person who is critically ill and family in many ways. The purpose of this study was to evaluate the effects of an intervention in postoperative heart failure patient-partner dyads regarding health, symptoms of depression and perceived control. Pilot study with a randomised controlled design evaluating psychosocial support and education from an interdisciplinary team. Patients with postoperative heart failure and their partners. SF-36, Beck Depression Inventory, Perceived Control at baseline, 3 and 12 months. A total of 42 patient-partner completed baseline assessment. Partners in the intervention group increased health in the role emotional and mental health dimensions and patients increased health in vitality, social function and mental health dimensions compared with the control group. Patients' perceived control improved significantly in the intervention group over time. Psychoeducational support to post cardiac surgery heart failure dyads improved health in both patients and partners at short term follow-up and improved patients' perceived control at both short and long-term follow-up. Psychoeducational support appears to be a promising intervention but the results need to be confirmed in larger studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ties that bind: implications of social support for rural, partnered African American women's health functioning.

PubMed

Black, Angela R; Cook, Jennifer L; Murry, Velma McBride; Cutrona, Carolyn E

2005-01-01

Ecological theory was used to explore the pathways through which intimate relationship quality influenced health functioning among rural, partnered African American women. Structural equation modeling was used to analyze data from 349 women in Georgia and Iowa. Women's intimate relationship quality was positively associated with their psychological and physical health functioning. Support from community residents moderated this link, which was strongest for women who felt most connected with their neighbors and for women who believed their neighborhood to have a sense of communal responsibility. Future research should identify other factors salient to health functioning among members of this population.

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

PubMed

Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Design, synthesis and characterization of peptidomimetic conjugate of BODIPY targeting HER2 protein extracellular domain

PubMed Central

Banappagari, Sashikanth; McCall, Alecia; Fontenot, Krystal; Vicente, M. Graca H.; Gujar, Amit; Satyanarayanajois, Seetharama

2013-01-01

Among the EGFRs, HER2 is a major heterodimer partner and also has important implications in the formation of particular tumors. Interaction of HER2 protein with other EGFR proteins can be modulated by small molecule ligands and, hence, these protein-protein interactions play a key role in biochemical reactions related to control of cell growth. A peptidomimetic (compound 5-1) that binds to HER2 protein extracellular domain and inhibits protein-protein interactions of EGFRs was conjugated with BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene). Conjugation of BODIPY to the peptidomimetic was investigated by different approaches. The conjugate was characterized for its ability to bind to HER2 overexpressing SKBR-3 and BT-474 cells. Furthermore, cellular uptake of conjugate of BODIPY was studied in the presence of membrane tracker and Lyso tracker using confocal microscopy. Our results suggested that fluorescently labeled compound 5-7 binds to the extracellular domain and stays in the membrane for nearly 24 h. After 24 h there is an indication of internalization of the conjugate. Inhibition of protein-protein interaction and downstream signaling effect of compound 5-1 was also studied by proximity ligation assay and western blot analysis. Results suggested that compound 5-1 inhibits protein-protein interactions of HER2-HER3 and phosphorylation of HER2 in a time-dependent manner. PMID:23688700

Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial Heparin-Binding Hemagglutinin.

PubMed

Huang, Teng-Yi; Irene, Deli; Zulueta, Medel Manuel L; Tai, Tzu-Jui; Lain, Shih-Han; Cheng, Cheng-Po; Tsai, Ping-Xi; Lin, Shu-Yi; Chen, Zhi-Geng; Ku, Chiao-Chu; Hsiao, Chwan-Deng; Chyan, Chia-Lin; Hung, Shang-Cheng

2017-04-03

Heparin-binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell-surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly 13 C- and 15 N-labeled HS octasaccharide and a uniformly 13 C- and 15 N-labeled form of HBHA were prepared. Residues 180-195 at the C-terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

T-lymphoid, megakaryocyte, and granulocyte development are sensitive to decreases in CBFβ dosage.

PubMed Central

Talebian, Laleh; Li, Zhe; Guo, Yalin; Gaudet, Justin; Speck, Maren E.; Sugiyama, Daisuke; Kaur, Prabhjot; Pear, Warren S.; Maillard, Ivan; Speck, Nancy A.

2007-01-01

The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non–DNA-binding partner CBFβ. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBFβ levels by 3- or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBFβ levels, and were virtually absent when CBFβ levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4−CD8− double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBFβ threshold for normal T-cell development, and situate an essential role for core-binding factors during the earliest stages of T-cell development. PMID:16940420

Non-disclosure of HIV-positive status to a partner and mother-to-child transmission of HIV: Evidence from a case–control study conducted in a rural county in Kenya

PubMed Central

2017-01-01

Background Many factors contribute to an enhanced risk of infant HIV acquisition, two of which may include failure of a mother to disclose her HIV-positive status to her partner and exclusion of male partners in preventing mother-to-child transmission of HIV (PMTCT) interventions. To justify why HIV programmes need to integrate male partner involvement and partner disclosure, we need to establish an association between the two factors and infant HIV acquisition. Objective To determine whether failure to disclose an HIV-positive status to a male partner is associated with increased risk of infant HIV acquisition, and whether part of the association is explained by exclusion of male partner in PMTCT programmes. Methods Using a case–control study design, we identified a total of 180 mother–baby pairs with HIV-exposed infants. Thirty-six pairs with HIV-positive babies (cases) were compared to 144 pairs with HIV-negative babies (controls) on whether the mothers had disclosed their HIV status to their partner in order to determine whether a disclosure or lack of it contributed to increased risk of mother-to-child transmission of HIV (MTCT). Each case pair was matched to four control pairs from the same facility. Results Overall, 16.7% of mothers had not disclosed their HIV status to their partners, the proportion being significantly more among cases (52.8% vs. 7.6%, p < 0.001). Non-disclosure was significantly associated with infant HIV acquisition (aOR 9.8 (3.0–26.3); p < 0.001) and male partner involvement partially mediated the effect of non-disclosure on infant HIV acquisition (indirect coefficient = 0.17, p < 0.005). Conclusions Failure of an HIV-positive woman to disclose her status to her male partner and exclusion of male partners in PMTCT programmes are two social factors that may curtail success of interventions towards the goal of eliminating MTCT. PMID:29568627

Effect of Screening for Partner Violence on Women's Quality of Life

PubMed Central

Klevens, Joanne; Kee, Romina; Trick, William; Garcia, Diana; Angulo, Francisco R.; Jones, Robin; Sadowski, Laura S.

2015-01-01

Context Although partner violence screening has been endorsed by many health organizations, there is insufficient evidence that it has beneficial health outcomes. Objective To determine the effect of computerized screening for partner violence plus provision of a partner violence resource list vs provision of a partner violence list only on women's health in primary care settings, compared with a control group. Design, Setting, and Participants A 3-group blinded randomized controlled trial at 10 primary health care centers in Cook County, Illinois. Participants were enrolled from May 2009–April 2010 and reinterviewed 1 year (range, 48–56 weeks) later. Participants were English- or Spanish-speaking women meeting specific inclusion criteria and seeking clinical services at study sites. Of 3537 women approached, 2727 were eligible, 2708 were randomized (99%), and 2364 (87%) were recontacted 1 year later. Mean age of participants was 39 years. Participants were predominantly non-Latina African American (55%) or Latina (37%), had a high school education or less (57%), and were uninsured (57%). Intervention Randomization into 3 intervention groups: (1) partner violence screen (using the Partner Violence Screen instrument) plus a list of local partner violence resources if screening was positive (n=909); (2) partner violence resource list only without screen (n=893); and (3) no-screen, no-partner violence list control group (n=898). Main Outcome Measures Quality of life (QOL, physical and mental health components) was the primary outcome, measured on the 12-item Short Form (scale range 0–100, mean of 50 for US population). Results At 1-year follow-up, there were no significant differences in the QOL physical health component between the screen plus partner violence resource list group (n=801; mean score, 46.8; 95% CI, 46.1–47.4), the partner violence resource list only group (n=772; mean score, 46.4; 95% CI, 45.8–47.1), and the control group (n=791; mean score, 47.2; 95% CI, 46.5–47.8), or in the mental health component (screen plus partner violence resource list group [mean score, 48.3; 95% CI, 47.5–49.1], the partner violence resource list only group [mean score, 48.0; 95% CI, 47.2–48.9], and the control group [mean score, 47.8; 95% CI, 47.0–48.6]). There were also no differences between groups in days unable to work or complete housework; number of hospitalizations, emergency department, or ambulatory care visits; proportion who contacted a partner violence agency; or recurrence of partner violence. Conclusions Among women receiving care in primary care clinics, providing a partner violence resource list with or without screening did not result in improved health. Trial Registration clinicaltrials.gov Identifier: NCT00526994 PMID:22893165

New Insights into Ribosome Structure and Function.

PubMed

Jobe, Amy; Liu, Zheng; Gutierrez-Vargas, Cristina; Frank, Joachim

2018-06-14

In the past 4 years, because of the advent of new cameras, many ribosome structures have been solved by cryoelectron microscopy (cryo-EM) at high, often near-atomic resolution, bringing new mechanistic insights into the processes of translation initiation, peptide elongation, termination, and recycling. Thus, cryo-EM has joined X-ray crystallography as a powerful technique in structural studies of translation. The significance of this new development is that structures of ribosomes in complex with their functional binding partners can now be determined to high resolution in multiple states as they perform their work. The aim of this article is to provide an overview of these new studies and assess the contributions they have made toward an understanding of translation and translational control. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Robust Multiple-Range Coherent Quantum State Transfer

PubMed Central

Chen, Bing; Peng, Yan-Dong; Li, Yong; Qian, Xiao-Feng

2016-01-01

We propose a multiple-range quantum communication channel to realize coherent two-way quantum state transport with high fidelity. In our scheme, an information carrier (a qubit) and its remote partner are both adiabatically coupled to the same data bus, i.e., an N-site tight-binding chain that has a single defect at the center. At the weak interaction regime, our system is effectively equivalent to a three level system of which a coherent superposition of the two carrier states constitutes a dark state. The adiabatic coupling allows a well controllable information exchange timing via the dark state between the two carriers. Numerical results show that our scheme is robust and efficient under practically inevitable perturbative defects of the data bus as well as environmental dephasing noise. PMID:27364891

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking

PubMed Central

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045

A Shared Docking Motif in TRF1 and TRF2 Used for Differential Recruitment of Telomeric Proteins

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

Chen, Yong; Yang, Yuting; van Overbeek, Megan

2008-05-01

Mammalian telomeres are protected by a six-protein complex: shelterin. Shelterin contains two closely related proteins (TRF1 and TRF2), which recruit various proteins to telomeres. We dissect the interactions of TRF1 and TRF2 with their shared binding partner (TIN2) and other shelterin accessory factors. TRF1 recognizes TIN2 using a conserved molecular surface in its TRF homology (TRFH) domain. However, this same surface does not act as a TIN2 binding site in TRF2, and TIN2 binding to TRF2 is mediated by a region outside the TRFH domain. Instead, the TRFH docking site of TRF2 binds a shelterin accessory factor (Apollo), which doesmore » not interact with the TRFH domain of TRF1. Conversely, the TRFH domain of TRF1, but not of TRF2, interacts with another shelterin-associated factor: PinX1.« less

Poly(A)-binding proteins and mRNA localization: who rules the roost?

PubMed

Gray, Nicola K; Hrabálková, Lenka; Scanlon, Jessica P; Smith, Richard W P

2015-12-01

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. © 2015 Authors; published by Portland Press Limited.

AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2

PubMed Central

Wang, Wei; Schwemmers, Sven; Hexner, Elizabeth O.

2010-01-01

The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2V617F mutation. Although NF-E2 levels correlate with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2V617F. In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-β significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients. PMID:20339092

Vasectomy, cigarette smoking, and age at first sexual intercourse as risk factors for prostate cancer in middle-aged men.

PubMed Central

Honda, G. D.; Bernstein, L.; Ross, R. K.; Greenland, S.; Gerkins, V.; Henderson, B. E.

1988-01-01

A population-based case-control study was conducted in men aged 60 or less to assess the risk of prostate cancer associated with vasectomy and other factors. Data were obtained from 216 case-control pairs by telephone interviews; this number represented 55% of all eligible cases. The matched pairs relative risk (RR) for vasectomy in ever married men was 1.4 with a 95% confidence interval (CI) of 0.9-2.3. There was a positive association between the number of years since vasectomy and prostate cancer risk (1-sided P = 0.01). Early age at first sexual intercourse was associated with increased prostate cancer risk (age less than 17 vs. 21+, RR = 2.3, 95% CI = 1.3, 4.0) but there were no consistent associations with number of sexual partners or frequency of sexual intercourse. Cigarette smoking was also associated with increased risk of prostate cancer (RR = 1.9, 95% CI = 1.2, 3.0) and there was a positive dose-response relationship with years of smoking (1-sided P = 0.001). We discuss the possible implication of the low response rate on each of these findings. To determine whether the association with vasectomy might have a hormonal basis, we compared levels of testosterone (T) and testosterone binding globulin-binding capacity (TeBG-bc) in 33 of the vasectomized control men with levels in 33 non-vasectomized controls of the same age, weight and height. T levels were higher in vasectomized than in non-vasectomized controls (1-sided P = 0.06). The ratio of T to TeBG-bc (an index of bioavailable T) was 13.5% higher in vasectomized men (1-sided P = 0.03). PMID:3355774

Do men with prostate cancer and their partners receive the information they need for optimal illness self-management in the immediate post-diagnostic phase?

PubMed

Levesque, Janelle V; Lambert, Sylvie D; Girgis, Afaf; Turner, Jane; McElduff, Patrick; Kayser, Karen

2015-01-01

To (a) determine whether the information provided to men with prostate cancer and their partners in the immediate postdiagnostic phase met their needs; and (b) examine patient and partner satisfaction with the information received. Pre-intervention survey data from a pilot randomized controlled trial of a self-directed coping skills intervention involving 42 patients with prostate cancer, and their partners were collected to examine their psychosocial concerns/needs. The main concerns for patients and partners were psychosocial in nature such as managing emotions, concern about the future, and losing control. Overall, patients and partners received most information about tests and treatment options. Partners reported receiving significantly less information about support services ( P = 0.03) and self-care strategies ( P = 0.03) compared to patients. Partners also reported being significantly less satisfied with the information they received ( P = 0.007). Whereas medical information is routinely given, patients and partners may benefit from greater information about psychosocial issues arising from cancer. Despite increased recognition of partner's information needs these still remain unmet.

Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis.

PubMed

Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-12

The emergence of novel binding proteins or antibody mimetics capable of binding to ligand analytes in a manner analogous to that of the antigen-antibody interaction has spurred increased interest in the biotechnology and bioanalytical communities. The goal is to produce antibody mimetics designed to outperform antibodies with regard to binding affinities, cellular and tumor penetration, large-scale production, and temperature and pH stability. The generation of antibody mimetics with tailored characteristics involves the identification of a naturally occurring protein scaffold as a template that binds to a desired ligand. This scaffold is then engineered to create a superior binder by first creating a library that is then subjected to a series of selection steps. Antibody mimetics have been successfully used in the development of binding assays for the detection of analytes in biological samples, as well as in separation methods, cancer therapy, targeted drug delivery, and in vivo imaging. This review describes recent advances in the field of antibody mimetics and their applications in bioanalytical chemistry, specifically in diagnostics and other analytical methods.

Kinetic rate constant prediction supports the conformational selection mechanism of protein binding.

PubMed

Moal, Iain H; Bates, Paul A

2012-01-01

The prediction of protein-protein kinetic rate constants provides a fundamental test of our understanding of molecular recognition, and will play an important role in the modeling of complex biological systems. In this paper, a feature selection and regression algorithm is applied to mine a large set of molecular descriptors and construct simple models for association and dissociation rate constants using empirical data. Using separate test data for validation, the predicted rate constants can be combined to calculate binding affinity with accuracy matching that of state of the art empirical free energy functions. The models show that the rate of association is linearly related to the proportion of unbound proteins in the bound conformational ensemble relative to the unbound conformational ensemble, indicating that the binding partners must adopt a geometry near to that of the bound prior to binding. Mirroring the conformational selection and population shift mechanism of protein binding, the models provide a strong separate line of evidence for the preponderance of this mechanism in protein-protein binding, complementing structural and theoretical studies.

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

PubMed

Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

2015-01-01

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

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

Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction ofmore » A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.« less

Evidence for dynamically organized modularity in the yeast protein-protein interaction network

NASA Astrophysics Data System (ADS)

Han, Jing-Dong J.; Bertin, Nicolas; Hao, Tong; Goldberg, Debra S.; Berriz, Gabriel F.; Zhang, Lan V.; Dupuy, Denis; Walhout, Albertha J. M.; Cusick, Michael E.; Roth, Frederick P.; Vidal, Marc

2004-07-01

In apparently scale-free protein-protein interaction networks, or `interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the `hubs', interact with many partners. Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs. Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space. We uncovered two types of hub: `party' hubs, which interact with most of their partners simultaneously, and `date' hubs, which bind their different partners at different times or locations. Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes-or modules -to each other, whereas party hubs function inside modules.

Intrinsic disorder in spondins and some of their interacting partners

PubMed Central

Alowolodu, Oluwole; Johnson, Gbemisola; Addou, Iqbal; Zhdanova, Irina V.; Uversky, Vladimir N.

2016-01-01

ABSTRACT Spondins, which are proteins that inhibit and promote adherence of embryonic cells so as to aid axonal growth are part of the thrombospondin-1 family. Spondins function in several important biological processes, such as apoptosis, angiogenesis, etc. Spondins constitute a thrombospondin subfamily that includes F-spondin, a protein that interacts with Aβ precursor protein and inhibits its proteolytic processing; R-spondin, a 4-membered group of proteins that regulates Wnt pathway and have other functions, such as regulation of kidney proliferation, induction of epithelial proliferation, the tumor suppressant action; M-spondin that mediates mechanical linkage between the muscles and apodemes; and the SCO-spondin, a protein important for neuronal development. In this study, we investigated intrinsic disorder status of human spondins and their interacting partners, such as members of the LRP family, LGR family, Frizzled family, and several other binding partners in order to establish the existence and importance of disordered regions in spondins and their interacting partners by conducting a detailed analysis of their sequences, finding disordered regions, and establishing a correlation between their structure and biological functions. PMID:28232900

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

PubMed

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-09-01

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design.

MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.

PubMed

Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R

2016-07-08

Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

The cell fate determinant Scribble is required for maintenance of hematopoietic stem cell function.

PubMed

Mohr, Juliane; Dash, Banaja P; Schnoeder, Tina M; Wolleschak, Denise; Herzog, Carolin; Tubio Santamaria, Nuria; Weinert, Sönke; Godavarthy, Sonika; Zanetti, Costanza; Naumann, Michael; Hartleben, Björn; Huber, Tobias B; Krause, Daniela S; Kähne, Thilo; Bullinger, Lars; Heidel, Florian H

2018-05-01

Cell fate determinants influence self-renewal potential of hematopoietic stem cells. Scribble and Llgl1 belong to the Scribble polarity complex and reveal tumor-suppressor function in drosophila. In hematopoietic cells, genetic inactivation of Llgl1 leads to expansion of the stem cell pool and increases self-renewal capacity without conferring malignant transformation. Here we show that genetic inactivation of its putative complex partner Scribble results in functional impairment of hematopoietic stem cells (HSC) over serial transplantation and during stress. Although loss of Scribble deregulates transcriptional downstream effectors involved in stem cell proliferation, cell signaling, and cell motility, these effectors do not overlap with transcriptional targets of Llgl1. Binding partner analysis of Scribble in hematopoietic cells using affinity purification followed by mass spectometry confirms its role in cell signaling and motility but not for binding to polarity modules described in drosophila. Finally, requirement of Scribble for self-renewal capacity also affects leukemia stem cell function. Thus, Scribble is a regulator of adult HSCs, essential for maintenance of HSCs during phases of cell stress.

SKI promotes Smad3 linker phosphorylations associated with the tumor-promoting trait of TGFbeta.

PubMed

Lin, Qiushi; Chen, Dahu; Timchenko, Nikolai A; Medrano, Estela E

2010-05-01

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

The nuclear DEK interactome supports multi-functionality.

PubMed

Smith, Eric A; Krumpelbeck, Eric F; Jegga, Anil G; Prell, Malte; Matrka, Marie M; Kappes, Ferdinand; Greis, Kenneth D; Ali, Abdullah M; Meetei, Amom R; Wells, Susanne I

2018-01-01

DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry. With this approach, we identify IMPDH2, DDX21, and RPL7a as novel DEK binding partners, hinting at new roles for the oncogene in de novo nucleotide biosynthesis and ribosome formation. Additionally, a hydroxyurea-specific interaction with replication protein A (RPA) was observed, suggesting that a DEK-RPA complex may form in response to DNA replication fork stalling. Taken together, these findings highlight diverse activities for DEK across cellular pathways and support a model wherein this molecule performs a plethora of functions. © 2017 Wiley Periodicals, Inc.

"Hit-and-Run" leaves its mark: catalyst transcription factors and chromatin modification.

PubMed

Varala, Kranthi; Li, Ying; Marshall-Colón, Amy; Para, Alessia; Coruzzi, Gloria M

2015-08-01

Understanding how transcription factor (TF) binding is related to gene regulation is a moving target. We recently uncovered genome-wide evidence for a "Hit-and-Run" model of transcription. In this model, a master TF "hits" a target promoter to initiate a rapid response to a signal. As the "hit" is transient, the model invokes recruitment of partner TFs to sustain transcription over time. Following the "run", the master TF "hits" other targets to propagate the response genome-wide. As such, a TF may act as a "catalyst" to mount a broad and acute response in cells that first sense the signal, while the recruited TF partners promote long-term adaptive behavior in the whole organism. This "Hit-and-Run" model likely has broad relevance, as TF perturbation studies across eukaryotes show small overlaps between TF-regulated and TF-bound genes, implicating transient TF-target binding. Here, we explore this "Hit-and-Run" model to suggest molecular mechanisms and its biological relevance. © 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.

“Hit‐and‐Run” leaves its mark: Catalyst transcription factors and chromatin modification

PubMed Central

Varala, Kranthi; Li, Ying; Marshall‐Colón, Amy; Para, Alessia

2015-01-01

Understanding how transcription factor (TF) binding is related to gene regulation is a moving target. We recently uncovered genome‐wide evidence for a “Hit‐and‐Run” model of transcription. In this model, a master TF “hits” a target promoter to initiate a rapid response to a signal. As the “hit” is transient, the model invokes recruitment of partner TFs to sustain transcription over time. Following the “run”, the master TF “hits” other targets to propagate the response genome‐wide. As such, a TF may act as a “catalyst” to mount a broad and acute response in cells that first sense the signal, while the recruited TF partners promote long‐term adaptive behavior in the whole organism. This “Hit‐and‐Run” model likely has broad relevance, as TF perturbation studies across eukaryotes show small overlaps between TF‐regulated and TF‐bound genes, implicating transient TF‐target binding. Here, we explore this “Hit‐and‐Run” model to suggest molecular mechanisms and its biological relevance. PMID:26108710

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.

PubMed

Tsuchiya, Megumi; Karim, M Rezaul; Matsumoto, Taro; Ogawa, Hidesato; Taniguchi, Hiroaki

2017-01-24

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase

PubMed Central

Mittag, Tanja; Marsh, Joseph; Grishaev, Alexander; Orlicky, Stephen; Lin, Hong; Sicheri, Frank; Tyers, Mike; Forman-Kay, Julie D.

2010-01-01

Summary Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations utilizing experimental NMR and small-angle x-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated. PMID:20399186

Regulation of ROCK Activity in Cancer

PubMed Central

Morgan-Fisher, Marie; Wewer, Ulla M.

2013-01-01

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)–loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

Characterization of diverse internal binding specificities of PDZ domains by yeast two-hybrid screening of a special peptide library.

PubMed

Mu, Yi; Cai, Pengfei; Hu, Siqi; Ma, Sucan; Gao, Youhe

2014-01-01

Protein-protein interactions (PPIs) are essential events to play important roles in a series of biological processes. There are probably more ways of PPIs than we currently realized. Structural and functional investigations of weak PPIs have lagged behind those of strong PPIs due to technical difficulties. Weak PPIs are often short-lived, which may result in more dynamic signals with important biological roles within and/or between cells. For example, the characteristics of PSD-95/Dlg/ZO-1 (PDZ) domain binding to internal sequences, which are primarily weak interactions, have not yet been systematically explored. In the present study, we constructed a nearly random octapeptide yeast two-hybrid library. A total of 24 PDZ domains were used as baits for screening the library. Fourteen of these domains were able to bind internal PDZ-domain binding motifs (PBMs), and PBMs screened for nine PDZ domains exhibited strong preferences. Among 11 PDZ domains that have not been reported their internal PBM binding ability, six were confirmed to bind internal PBMs. The first PDZ domain of LNX2, which has not been reported to bind C-terminal PBMs, was found to bind internal PBMs. These results suggest that the internal PBMs binding ability of PDZ domains may have been underestimated. The data provided diverse internal binding properties for several PDZ domains that may help identify their novel binding partners.

Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach.

PubMed

Tenzer, Stefan; Moro, Albertomaria; Kuharev, Jörg; Francis, Ashwanth Christopher; Vidalino, Laura; Provenzani, Alessandro; Macchi, Paolo

2013-06-07

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.

C/EBPγ Is a Critical Regulator of Cellular Stress Response Networks through Heterodimerization with ATF4

PubMed Central

Huggins, Christopher J.; Mayekar, Manasi K.; Martin, Nancy; Saylor, Karen L.; Gonit, Mesfin; Jailwala, Parthav; Kasoji, Manjula; Haines, Diana C.; Quiñones, Octavio A.

2015-01-01

The integrated stress response (ISR) controls cellular adaptations to nutrient deprivation, redox imbalances, and endoplasmic reticulum (ER) stress. ISR genes are upregulated in stressed cells, primarily by the bZIP transcription factor ATF4 through its recruitment to cis-regulatory C/EBP:ATF response elements (CAREs) together with a dimeric partner of uncertain identity. Here, we show that C/EBPγ:ATF4 heterodimers, but not C/EBPβ:ATF4 dimers, are the predominant CARE-binding species in stressed cells. C/EBPγ and ATF4 associate with genomic CAREs in a mutually dependent manner and coregulate many ISR genes. In contrast, the C/EBP family members C/EBPβ and C/EBP homologous protein (CHOP) were largely dispensable for induction of stress genes. Cebpg−/− mouse embryonic fibroblasts (MEFs) proliferate poorly and exhibit oxidative stress due to reduced glutathione levels and impaired expression of several glutathione biosynthesis pathway genes. Cebpg−/− mice (C57BL/6 background) display reduced body size and microphthalmia, similar to ATF4-null animals. In addition, C/EBPγ-deficient newborns die from atelectasis and respiratory failure, which can be mitigated by in utero exposure to the antioxidant, N-acetyl-cysteine. Cebpg−/− mice on a mixed strain background showed improved viability but, upon aging, developed significantly fewer malignant solid tumors than WT animals. Our findings identify C/EBPγ as a novel antioxidant regulator and an obligatory ATF4 partner that controls redox homeostasis in normal and cancerous cells. PMID:26667036

Informal Social Control of Intimate Partner Violence against Women: Results from a Concept Mapping Study of Urban Neighborhoods

ERIC Educational Resources Information Center

Frye, Victoria; Paul, Margaret M.; Todd, Mary-Justine; Lewis, Veronica; Cupid, Malik; Coleman, Jane; Salmon, Christina; O'Campo, Patricia

2012-01-01

How the neighborhood environment relates to intimate partner violence against women has been studied using theories applied originally to general violence. Extending social disorganization and collective efficacy theories, they apply a traditional measure informal social control that does not reflect behaviors specific to partner violence. We…

Exploring the binding pathways of the 14-3-3ζ protein: Structural and free-energy profiles revealed by Hamiltonian replica exchange molecular dynamics with distancefield distance restraints

PubMed Central

Nagy, Gabor; Oostenbrink, Chris; Hritz, Jozef

2017-01-01

The 14-3-3 protein family performs regulatory functions in eukaryotic organisms by binding to a large number of phosphorylated protein partners. Whilst the binding mode of the phosphopeptides within the primary 14-3-3 binding site is well established based on the crystal structures of their complexes, little is known about the binding process itself. We present a computational study of the process by which phosphopeptides bind to the 14-3-3ζ protein. Applying a novel scheme combining Hamiltonian replica exchange molecular dynamics and distancefield restraints allowed us to map and compare the most likely phosphopeptide-binding pathways to the 14-3-3ζ protein. The most important structural changes to the protein and peptides involved in the binding process were identified. In order to bind phosphopeptides to the primary interaction site, the 14-3-3ζ adopted a newly found wide-opened conformation. Based on our findings we additionally propose a secondary interaction site on the inner surface of the 14-3-3ζ dimer, and a direct interference on the binding process by the flexible C-terminal tail. A minimalistic model was designed to allow for the efficient calculation of absolute binding affinities. Binding affinities calculated from the potential of mean force along the binding pathway are in line with the available experimental estimates for two of the studied systems. PMID:28727767

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015.

PubMed

Falconer, Robert J

2016-10-01

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x-ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion-π and π-π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Reactions to a Partner-Assisted Emotional Disclosure Intervention: Direct Observation and Self-Report of Patient and Partner Communication

ERIC Educational Resources Information Center

Porter, Laura S.; Baucom, Donald H.; Keefe, Francis J.; Patterson, Emily S.

2012-01-01

Partner-assisted emotional disclosure is a couple-based intervention designed to help patients disclose cancer-related concerns to their spouses-partners. We previously found that, compared with an education/support control condition, partner-assisted emotional disclosure led to significant improvements in relationship quality and intimacy for…

Contribution of partner switching and SpoIIAA cycling to regulation of sigmaF activity in sporulating Bacillus subtilis.

PubMed

Magnin, T; Lord, M; Yudkin, M D

1997-06-01

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of sigmaF. We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigmaF.

The Contribution of Emotional Partners to Sexual Risk Taking and Violence among Female Sex Workers in Mombasa, Kenya: A Cohort Study

PubMed Central

Bosire, Wilkister; Nelson, Gill; Kingola, Nzioki; Zhang, Xu-Dong; Temmerman, Marleen; Chersich, Matthew F.

2013-01-01

Objectives To assess sexual risk-taking of female sex workers (FSWs) with emotional partners (boyfriends and husbands), compared to regular and casual clients. Experiences of violence and the degree of relationship control that FSWs have with emotional partners are also described. Design Cohort study with quarterly follow-up visit over 12-months. Methods Four hundred HIV-uninfected FSWs older than 16 years were recruited from their homes and guesthouses in Mombasa, Kenya. A structured questionnaire assessed participant characteristics and study outcomes at each visit, and women received risk-reduction counselling, male and female condoms, and HIV testing. Results Four or more unprotected sex acts in the past week were reported by 21.3% of women during sex with emotional partners, compared to 5.8% with regular and 4.8% with casual clients (P<0.001). Total number of unprotected sex acts per week was 5–6-fold higher with emotional partners (603 acts with 259 partners) than with regular or casual clients (125 acts with 456, and 98 acts with 632 clients, respectively; P<0.001). Mostly, perceptions of “trust” underscored unprotected sex with emotional partners. Low control over these relationships, common to many women (36.9%), was linked with higher partner numbers, inconsistent condom use, and being physically forced to have sex by their emotional partners. Half experienced sexual or physical violence in the past year, similarly associated with partner numbers and inconsistent condom use. Conclusions High-risk sexual behaviour, low control and frequent violence in relationships with emotional partners heighten FSWs' vulnerability and high HIV risk, requiring targeted interventions that also encompass emotional partners. PMID:23950879

Self-Control and Emotional and Verbal Aggression in Dating Relationships: A Dyadic Understanding.

PubMed

Baker, Elizabeth A; Klipfel, Katherine M; van Dulmen, Manfred H M

2016-08-01

Guided by the dynamic developmental systems perspective, this study extends past research by examining the association between self-control and emotional and verbal aggression (EVA) using a dyadic multi-method design. Guided by empirical research and the dynamic developmental systems perspective, we hypothesized that (a) there would be a negative association between one's own self-control and one's own perpetration of EVA and (b) there would also be a negative association between one's partner's self-control and one's own perpetration of EVA. One hundred twenty heterosexual dating couples (ages 18-25 years) provided data on self-control (Grasmick et al.'s Low Self-Control Scale; reverse scored for ease of interpretation), self-reported perpetration of EVA (Emotional and Verbal Abuse subscale of the Conflict in Adolescent Dating Relationships Inventory), and observationally assessed perpetration of EVA. Data were analyzed using path analyses within the Actor-Partner Interdependence Model (APIM) framework. Consistent with previous findings, we found that self-control was negatively associated with the perpetration of EVA. Furthermore, we found partner effects, such that female-but not male-self-control predicted partner-observed perpetration of EVA. These findings highlight the importance of examining risk factors for EVA of both partners. Our findings also suggest that the association between self-control and EVA is partially a function of whether EVA is assessed through self-report or observational methodology. This highlights the need to conduct multi-method assessments in future research. As discussed in the article, our findings have implications for theories on intimate partner violence, study designs, and couple interventions.

Genetic Dissection of DivIVA Functions in Listeria monocytogenes.

PubMed

Kaval, Karan Gautam; Hauf, Samuel; Rismondo, Jeanine; Hahn, Birgitt; Halbedel, Sven

2017-12-15

DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future. IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins. Copyright © 2017 American Society for Microbiology.

Health, social and economic consequences of hypersomnia: a controlled national study from a national registry evaluating the societal effect on patients and their partners.

PubMed

Jennum, Poul; Ibsen, Rikke; Avlund, Kirsten; Kjellberg, Jakob

2014-04-01

Hypersomnia causes significant socioeconomic burden, but there is insufficient information about the time course and the effect on the partner. The aim of this study was to estimate the factual direct and productivity costs of hypersomnia in a controlled study including all national patients and their partners. Using records from the Danish National Patient Registry (1997-2009), we identified all patients with a diagnosis of hypersomnia and compared these patients and their partners with randomly chosen controls matched for age, gender, geographic area and marital status. Direct and productivity costs, including frequencies of primary and sector contacts and procedures, medication, labour supply and social transfer payments were extracted from the national databases. A total of 2,855 national patients was compared to 11,382 controls. About 70 % of patients and controls were married or cohabiting. Patients with hypersomnia had significantly higher rates of health-related contact, medication use and socioeconomic cost. Furthermore, they had slightly lower employment rates, and those in employment had a lower income level than control subjects. The annual mean excess health-related cost including social transfers was 3,498 for patients with hypersomnia and 3,851 for their partners. The social and health-related consequences could be identified up to 11 years before the first diagnosis among both the patients and their partners and became more pronounced as the disease advanced. The health effects were present in all age groups and in both genders. On the basis of this retrospective controlled study in the Danish population, symptoms and findings of hypersomnia are associated with major socioeconomic consequences for patients, their partners and society.

Intrinsic disorder in scaffold proteins: Getting more from less

PubMed Central

Cortese, Marc S.; Uversky, Vladimir N.; Dunker, A. Keith

2008-01-01

Regulation, recognition and cell signaling involve the coordinated actions of many players. Signaling scaffolds, with their ability to bring together proteins belonging to common and/or interlinked pathways, play crucial roles in orchestrating numerous events by coordinating specific interactions among signaling proteins. This review examines the roles of intrinsic disorder (ID) in signaling scaffold protein function. Several well-characterized scaffold proteins with structurally and functionally characterized ID regions are used here to illustrate the importance of ID for scaffolding function. These examples include scaffolds that are mostly disordered, only partially disordered or those in which the ID resides in a scaffold partner. Specific scaffolds discussed include RNase, voltage-activated potassium channels, axin, BRCA1, GSK-3β, p53, Ste5, titin, Fus3, BRCA1, Titin, MAP2, D-AKAP2 and AKAP250. Among the mechanisms discussed are: molecular recognition features, fly-casting, ease of encounter complex formation, structural isolation of partners, modulation of interactions between bound partners, masking of intramolecular interaction sites, maximized interaction surface per residue, toleration of high evolutionary rates, binding site overlap, allosteric modification, palindromic binding, reduced constraints for alternative splicing, efficient regulation via posttranslational modification, efficient regulation via rapid degradation, protection of normally solvent-exposed sites, enhancing the plasticity of interaction and molecular crowding. We conclude that ID can enhance scaffold function by a diverse array of mechanisms. In other words, scaffold proteins utilize several ID-facilitated mechanisms to enhance function, and by doing so, get more functionality from less structure. PMID:18619997

The role of synaptotagmin I C2A calcium-binding domain in synaptic vesicle clustering during synapse formation

PubMed Central

Gardzinski, Peter; Lee, David W K; Fei, Guang-He; Hui, Kwokyin; Huang, Guan J; Sun, Hong-Shuo; Feng, Zhong-Ping

2007-01-01

Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis. Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I. PMID:17317745

Contribution of EGFR and ErbB-3 Heterodimerization to the EGFR Mutation-Induced Gefitinib- and Erlotinib-Resistance in Non-Small-Cell Lung Carcinoma Treatments.

PubMed

Wang, Debby D; Ma, Lichun; Wong, Maria P; Lee, Victor H F; Yan, Hong

2015-01-01

EGFR mutation-induced drug resistance has become a major threat to the treatment of non-small-cell lung carcinoma. Essentially, the resistance mechanism involves modifications of the intracellular signaling pathways. In our work, we separately investigated the EGFR and ErbB-3 heterodimerization, regarded as the origin of intracellular signaling pathways. On one hand, we combined the molecular interaction in EGFR heterodimerization with that between the EGFR tyrosine kinase and its inhibitor. For 168 clinical subjects, we characterized their corresponding EGFR mutations using molecular interactions, with three potential dimerization partners (ErbB-2, IGF-1R and c-Met) of EGFR and two of its small molecule inhibitors (gefitinib and erlotinib). Based on molecular dynamics simulations and structural analysis, we modeled these mutant-partner or mutant-inhibitor interactions using binding free energy and its components. As a consequence, the mutant-partner interactions are amplified for mutants L858R and L858R_T790M, compared to the wild type EGFR. Mutant delL747_P753insS represents the largest difference between the mutant-IGF-1R interaction and the mutant-inhibitor interaction, which explains the shorter progression-free survival of an inhibitor to this mutant type. Besides, feature sets including different energy components were constructed, and efficient regression trees were applied to map these features to the progression-free survival of an inhibitor. On the other hand, we comparably examined the interactions between ErbB-3 and its partners (EGFR mutants, IGF-1R, ErbB-2 and c-Met). Compared to others, c-Met shows a remarkably-strong binding with ErbB-3, implying its significant role in regulating ErbB-3 signaling. Moreover, EGFR mutants corresponding to poor clinical outcomes, such as L858R_T790M, possess lower binding affinities with ErbB-3 than c-Met does. This may promote the communication between ErbB-3 and c-Met in these cancer cells. The analysis verified the important contribution of IGF-1R or c-Met in the drug resistance mechanism developed in lung cancer treatments, which may bring many benefits to specialized therapy design and innovative drug discovery.

15 CFR 806.12 - Partnerships.

Code of Federal Regulations, 2011 CFR

2011-01-01

... ECONOMIC ANALYSIS, DEPARTMENT OF COMMERCE DIRECT INVESTMENT SURVEYS § 806.12 Partnerships. Limited partners... control exercised by, the general partner(s), although the latter may differ from the financial interest...

Partner Influence in Diet and Exercise Behaviors: Testing Behavior Modeling, Social Control, and Normative Body Size.

PubMed

Perry, Brea; Ciciurkaite, Gabriele; Brady, Christy Freadreacea; Garcia, Justin

2016-01-01

Previous research has documented social contagion in obesity and related health behaviors, but less is known about the social processes underlying these patterns. Focusing on married or cohabitating couples, we simultaneously explore three potential social mechanisms influencing obesity: normative body size, social control, and behavior modeling. We analyze the association between partner characteristics and the obesity-related health behaviors of focal respondents, comparing the effects of partners' body type, partners' attempts to manage respondents' eating behaviors, and partners' own health behaviors on respondents' health behaviors (physical activity, fruit and vegetable consumption, and fast food consumption). Data on 215 partners are extracted from a larger study of social mechanisms of obesity in family and community contexts conducted in 2011 in the United States. Negative binomial regression models indicate that partner behavior is significantly related to respondent behavior (p < .001), net of controls. These results are suggestive of a behavior modeling mechanism in obesity-related patterns of consumption and physical activity. In contrast, we find little support for the influence of normative body size or partner social control in this sample, though generalizations about the relevance of these processes may be inappropriate. These results underscore the importance of policies and interventions that target dyads and social groups, suggesting that adoption of exercise or diet modifications in one individual is likely to spread to others, creating a social environment characterized by mutual reinforcement of healthy behavior.

A Case-Control Study on Intimate Partner Violence during Pregnancy and Low Birth Weight, Southeast Ethiopia

PubMed Central

Demelash, Habtamu; Nigatu, Dabere; Gashaw, Ketema

2015-01-01

Introduction. Violence against women has serious consequences for their reproductive and sexual health including birth outcomes. In Ethiopia, though the average parity of pregnant women is much higher than in other African countries, the link between intimate partner violence with low birth weight is unknown. Objective. The aim of this study was to examine the association between intimate partner violence and low birth weight among pregnant women. Method. Hospital based case-control study was conducted among 387 mothers (129 cases and 258 controls). Anthropometric measurements were taken both from mothers and their live births. The association between intimate partner violence and birth weight was computed through bivariable and multivariable logistic regression analyses and statistical significance was declared at P < 0.05. Result. Out of 387 interviewed mothers, 100 (25.8%) had experienced intimate partner violence during their index pregnancy period. Relatively more mothers of low birth weight infants were abused (48%) compared with controls (16.4%). Those mothers who suffered acts of any type of intimate partner violence during pregnancy were three times more likely to have a newborn with low birth weight (95% CI; (1.57 to 7.18)). The association between overall intimate partner violence and LBW was adjusted for potential confounder variables. Conclusion. This research result gives insight for health professional about the importance of screening for intimate partner violence during pregnancy. Health care providers should consider violence in their practice and try to identify women at risk. PMID:26798345

Heterodimers of Retinoic Acid Receptors and Thyroid Hormone Receptors Display Unique Combinatorial Regulatory Properties

PubMed Central

Lee, Sangho; Privalsky, Martin L.

2009-01-01

Nuclear receptors are ligand-regulated transcription factors that regulate key aspects of metazoan development, differentiation, and homeostasis. Nuclear receptors recognize target genes by binding to specific DNA recognition sequences, denoted hormone response elements (HREs). Many nuclear receptors can recognize HREs as either homodimers or heterodimers. Retinoid X receptors (RXRs), in particular, serve as important heterodimer partners for many other nuclear receptors, including thyroid hormone receptors (TRs), and RXR/TR heterodimers have been proposed to be the primary mediators of target gene regulation by T3 hormone. Here, we report that the retinoic acid receptors (RARs), a distinct class of nuclear receptors, are also efficient heterodimer partners for TRs. These RAR/TR heterodimers form with similar affinities as RXR/TR heterodimers on an assortment of consensus and natural HREs, and preferentially assemble with the RAR partner 5′ of the TR moiety. The corepressor and coactivator recruitment properties of these RAR/TR heterodimers and their transcriptional activities in vivo are distinct from those observed with the corresponding RXR heterodimers. Our studies indicate that RXRs are not unique in their ability to partner with TRs, and that RARs can also serve as robust heterodimer partners and combinatorial regulators of T3-modulated gene expression. PMID:15650024

Protein partners in the life history of activated fibroblast growth factor receptors.

PubMed

Vecchione, Anna; Cooper, Helen J; Trim, Kimberley J; Akbarzadeh, Shiva; Heath, John K; Wheldon, Lee M

2007-12-01

Fibroblast growth factor receptors (FGFRs) are a family of four transmembrane (TM) receptor tyrosine kinases (RTKs) which bind to a large family of fibroblast growth factor (FGF) ligands with varying affinity and specificity. FGFR signaling regulates many physiological and pathological processes in development and tissue homeostasis. Understanding FGFR signaling processes requires the identification of partner proteins which regulate receptor function and biological outputs. In this study, we employ an epitope-tagged, covalently dimerized, and constitutively activated form of FGFR1 to identify potential protein partners by MS. By this approach, we sample candidate FGFR effectors throughout the life history of the receptor. Functional classification of the partners identified revealed specific subclasses involved in protein biosynthesis and folding; structural and regulatory components of the cytoskeleton; known signaling effectors and small GTPases implicated in endocytosis and vesicular trafficking. The kinase dependency of the interaction was determined for a subset of previously unrecognized partners by coimmunoprecipitation, Western blotting, and immunocytochemistry. From this group, the small GTPase Rab5 was selected for functional interrogation. We show that short hairpin (sh) RNA-mediated depletion of Rab5 attenuates the activation of the extracellular-regulated kinase (ERK) 1/2 pathway by FGFR signaling. The strategic approach adopted in this study has revealed bona fide novel effectors of the FGFR signaling pathway.

Does the Internet assist clients to carry out contact tracing? A randomized controlled trial using web-based information.

PubMed

Tomnay, J E; Pitts, M K; Kuo, T C; Fairley, C K

2006-06-01

The primary outcome was to determine the acceptability of the Internet, specifically a website for use in standard partner notification. A secondary objective was to determine if a website would enhance the outcomes of currently used partner notification methods. In a randomized control trial, 105 participants diagnosed with chlamydia and non-gonoccocal urethritis (NGU) were randomized and 97 completed the study. About 30% of participants were provided a standard partner letter and 70% were provided the standard partner letter and website. All participants reported that their partners had no objections to the website (0%, 95% confidence interval [CI] 0-5%). There were no complaints received from any partner. The odds ratio for contacting partners was not significantly different between the two groups of participants. The results of this study indicate that providing a website with specific information about the infection to which a partner has been exposed is not harmful.

Moderating Effect of Negative Peer Group Climate on the Relation Between Men's Locus of Control and Aggression Toward Intimate Partners.

PubMed

Schmidt, Megan R; Lisco, Claire G; Parrott, Dominic J; Tharp, Andra T

2016-03-01

The present study sought to examine the interactive effects of an external locus of control and interaction in a negative peer group climate on men's perpetration of physical aggression and infliction of injury toward their female intimate partners. Participants were 206 heterosexual males recruited from the metro-Atlanta community who completed self-report measures of external locus of control, involvement in a negative peer group climate, and physical aggression and infliction of injury against intimate partners during the past 12 months. Negative peer group climate was conceptualized as a peer group that displays behavior which may instigate aggressive norms, attitudes, and behaviors. Results indicated that men with an external locus of control were more likely to perpetrate physical aggression toward and inflict injury on their intimate partners if they reported high, but not low, involvement in a negative peer group climate. These results extend current research suggesting external locus of control as a risk factor for intimate partner aggression by highlighting the impact of negative peer groups. Implications and future intervention research are discussed. © The Author(s) 2014.

Exploring 3D structural influences of aliphatic and aromatic chemicals on α-cyclodextrin binding.

PubMed

Linden, Lukas; Goss, Kai-Uwe; Endo, Satoshi

2016-04-15

Binding of solutes to macromolecules is often influenced by steric effects caused by the 3D structures of both binding partners. In this study, the 1:1 α-cyclodextrin (αCD) binding constants (Ka1) for 70 organic chemicals were determined to explore the solute-structural effects on the αCD binding. Ka1 was measured using a three-part partitioning system with either a headspace or a passive sampler serving as the reference phase. The Ka1 values ranged from 1.08 to 4.97 log units. The results show that longer linear aliphatic chemicals form more stable complexes than shorter ones, and that the position of the functional group has a strong influence on Ka1, even stronger than the type of the functional group. Comparison of linear and variously branched aliphatic chemicals indicates that having a sterically unhindered alkyl chain is favorable for binding. These results suggest that only one alkyl chain can enter the binding cavity. Relatively small aromatic chemicals such as 1,3-dichlorobenzene bind to αCD well, while larger ones like tetrachlorobenzene and 3-ring aromatic chemicals show only a weak interaction with αCD, which can be explained by cavity exclusion. The findings of this study help interpret cyclodextrin binding data and facilitate the understanding of binding processes to macromolecules. Copyright © 2016 Elsevier Inc. All rights reserved.

Intimate Partner and General Aggression Perpetration among Combat Veterans Presenting to a Posttraumatic Stress Disorder Clinic

PubMed Central

Taft, Casey T.; Weatherill, Robin P.; Woodward, Halley E.; Pinto, Lavinia A.; Watkins, Laura E.; Miller, Mark W.; Dekel, Rachel

2013-01-01

This study examined rates and correlates of intimate partner and general aggression perpetration among 236 male combat veterans seeking services in a VA PTSD clinic. Approximately 33% of those in an intimate relationship reported perpetrating partner physical aggression in the previous year, and 91% reported partner psychological aggression. Comparable rates were found for general aggression perpetration among partnered and non-partnered veterans. PTSD symptoms as well as symptoms of depression were associated with aggression across subgroups and forms of aggression, and PTSD symptoms reflecting arousal and lack of control were generally the strongest predictor of aggression. Findings indicate a need for additional aggression screening and intervention development for this population, and highlight the targeting of heightened arousal and lack of behavioral control in aggression interventions. PMID:20099937

Discrepant Alcohol Use, Intimate Partner Violence, and Relationship Adjustment among Lesbian Women and their Relationship Partners.

PubMed

Kelley, Michelle L; Lewis, Robin J; Mason, Tyler B

2015-11-01

This study examined the association between relationship adjustment and discrepant alcohol use among lesbian women and their same-sex intimate partners after controlling for verbal and physical aggression. Lesbian women ( N = 819) who were members of online marketing research panels completed an online survey in which they reported both their own and same-sex intimate partner's alcohol use, their relationship adjustment, and their own and their partner's physical aggression and psychological aggression (i.e., verbal aggression and dominance/isolation). Partners' alcohol use was moderately correlated. Discrepancy in alcohol use was associated with poorer relationship adjustment after controlling for psychological aggression and physical aggression. Results are discussed in terms of the similarity and differences with previous literature primarily focused on heterosexual couples.

FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition.

PubMed

Simpson, Gordon G; Dijkwel, Paul P; Quesada, Victor; Henderson, Ian; Dean, Caroline

2003-06-13

The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development. FCA contains a WW protein interaction domain that is essential for FCA function. We have identified FY as a protein partner for this domain. FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p. FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation. FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site. The FCA/FY interaction is also required for the downregulation of the floral repressor FLC. We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing.

Bacterial Sigma Factors and Anti-Sigma Factors: Structure, Function and Distribution

PubMed Central

Paget, Mark S.

2015-01-01

Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoters as well as the initial steps in RNA synthesis. This review focuses on the structure and function of the major sigma-70 class that includes the housekeeping sigma factor (Group 1) that directs the bulk of transcription during active growth, and structurally-related alternative sigma factors (Groups 2–4) that control a wide variety of adaptive responses such as morphological development and the management of stress. A recurring theme in sigma factor control is their sequestration by anti-sigma factors that occlude their RNAP-binding determinants. Sigma factors are then released through a wide variety of mechanisms, often involving branched signal transduction pathways that allow the integration of distinct signals. Three major strategies for sigma release are discussed: regulated proteolysis, partner-switching, and direct sensing by the anti-sigma factor. PMID:26131973

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

PubMed

Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R; Weissmann, Florian; Ordureau, Alban; Wu, Kuen-Phon; Zhang, Wei; Yu, Shanshan; Mercredi, Peter Y; Harrison, Joseph S; Davidson, Iain F; Qiao, Renping; Lu, Ying; Dube, Prakash; Brunner, Michael R; Grace, Christy R R; Miller, Darcie J; Haselbach, David; Jarvis, Marc A; Yamaguchi, Masaya; Yanishevski, David; Petzold, Georg; Sidhu, Sachdev S; Kuhlman, Brian; Kirschner, Marc W; Harper, J Wade; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

2016-06-02

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic analysis identifies a novel function for galectin-3 in the cell entry of parvovirus.

PubMed

Garcin, Pierre; Cohen, Sarah; Terpstra, Sanne; Kelly, Isabelle; Foster, Leonard J; Panté, Nelly

2013-02-21

Cellular factors associated with the parvovirus minute virus of mice (MVM) during infection are thought to play important roles in the MVM life cycle but only a few of these have been identified. Here we used a proteomic-based approach in order to identify host-binding partners of MVM. Using purified MVM as bait for immunoprecipitation assays, a total of 150 proteins were identified in MVM immunoprecipitates by quantitative liquid chromatography-tandem mass spectrometry. Galectin-3 was one of six proteins showing a statistically significant enrichment across replicates. Small interfering RNA depletion studies revealed an important role for galectin-3 in MVM endocytosis and infectivity in LA9 mouse fibroblast cells. Galectin-3-depleted cells were less susceptible to MVM infection than control cells and showed a significant reduction of MVM cellular uptake, but not of MVM binding to the cell surface. Our results indicate an important role for galectin-3 in the cellular uptake of MVM. We propose that galectin-3 facilitates the access of MVM to its receptor(s) at the plasma membrane and in this way promotes MVM endocytosis. Copyright © 2012 Elsevier B.V. All rights reserved.

The RING domain of the scaffold protein Ste5 adopts a molten globular character with high thermal and chemical stability.

PubMed

Walczak, Michal J; Samatanga, Brighton; van Drogen, Frank; Peter, Matthias; Jelesarov, Ilian; Wider, Gerhard

2014-01-27

Ste5 is a scaffold protein that controls the pheromone response of the MAP-kinase cascade in yeast cells. Upon pheromone stimulation, Ste5 (through its RING-H2 domain) interacts with the β and γ subunits of an activated heterodimeric G protein and promotes activation of the MAP-kinase cascade. With structural and biophysical studies, we show that the Ste5 RING-H2 domain exists as a molten globule under native buffer conditions, in yeast extracts, and even in denaturing conditions containing urea (7 M). Furthermore, it exhibits high thermal stability in native conditions. Binding of the Ste5 RING-H2 domain to the physiological Gβ/γ (Ste4/Ste18) ligand is accompanied by a conformational transition into a better folded, more globular structure. This study reveals novel insights into the folding mechanism and recruitment of binding partners by the Ste5 RING-H2 domain. We speculate that many RING domains may share a similar mechanism of substrate recognition and molten-globule-like character. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of glutamate receptor internalization by the spine cytoskeleton is mediated by its PKA-dependent association with CPG2

PubMed Central

Loebrich, Sven; Djukic, Biljana; Tong, Zachary J.; Cottrell, Jeffrey R.; Turrigiano, Gina G.; Nedivi, Elly

2013-01-01

A key neuronal mechanism for adjusting excitatory synaptic strength is clathrin-mediated endocytosis of postsynaptic glutamate receptors (GluRs). The actin cytoskeleton is critical for clathrin-mediated endocytosis, yet we lack a mechanistic understanding of its interaction with the endocytic process and how it may be regulated. Here we show that F-actin in dendritic spines physically binds the synaptic nuclear envelope 1 gene product candidate plasticity gene 2 (CPG2) in a PKA-dependent manner, and that this association is required for synaptic GluR internalization. Mutating two PKA sites on CPG2 disrupts its cytoskeletal association, attenuating GluR endocytosis and affecting the efficacy of synaptic transmission in vivo. These results identify CPG2 as an F-actin binding partner that functionally mediates interaction of the spine cytoskeleton with postsynaptic endocytosis. Further, the regulation of CPG2/F-actin association by PKA provides a gateway for cellular control of synaptic receptor internalization through second messenger signaling pathways. Recent identification of human synaptic nuclear envelope 1 as a risk locus for bipolar disorder suggests that CPG2 could play a role in synaptic dysfunction underlying neuropsychiatric disease. PMID:24191017

BCL-2 family proteins: changing partners in the dance towards death.

PubMed

Kale, Justin; Osterlund, Elizabeth J; Andrews, David W

2018-01-01

The BCL-2 family of proteins controls cell death primarily by direct binding interactions that regulate mitochondrial outer membrane permeabilization (MOMP) leading to the irreversible release of intermembrane space proteins, subsequent caspase activation and apoptosis. The affinities and relative abundance of the BCL-2 family proteins dictate the predominate interactions between anti-apoptotic and pro-apoptotic BCL-2 family proteins that regulate MOMP. We highlight the core mechanisms of BCL-2 family regulation of MOMP with an emphasis on how the interactions between the BCL-2 family proteins govern cell fate. We address the critical importance of both the concentration and affinities of BCL-2 family proteins and show how differences in either can greatly change the outcome. Further, we explain the importance of using full-length BCL-2 family proteins (versus truncated versions or peptides) to parse out the core mechanisms of MOMP regulation by the BCL-2 family. Finally, we discuss how post-translational modifications and differing intracellular localizations alter the mechanisms of apoptosis regulation by BCL-2 family proteins. Successful therapeutic intervention of MOMP regulation in human disease requires an understanding of the factors that mediate the major binding interactions between BCL-2 family proteins in cells.

Molecular partners of hNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, suggest its involvement in distinct cellular processes relevant to congenital disorders of glycosylation, cancer, neurodegeneration and a variety of further pathologies.

PubMed

Hacker, Benedikt; Schultheiß, Christoph; Döring, Michael; Kurzik-Dumke, Ursula

2018-06-01

This study provides first insights into the involvement of hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID and yeast ALG3 gene, in various putative molecular networks. HNOT/ALG3 encodes two translated transcripts encoding precursor proteins differing in their N-terminus and showing 33% identity with the yeast asparagine-linked glycosylation 3 (ALG3) protein. Experimental evidence for the functional homology of the proteins of fly and man in the N-glycosylation has still to be provided. In this study, using the yeast two-hybrid technique we identify 17 molecular partners of hNOT-1/ALG3-1. We disclose the building of hNOT/ALG3 homodimers and provide experimental evidence for its in vivo interaction with the functionally linked proteins OSBP, OSBPL9 and LRP1, the SYPL1 protein and the transcription factor CREB3. Regarding the latter, we show that the 55 kDa N-glycosylated hNOT-1/ALG3-1 molecule binds the N-glycosylated CREB3 precursor but does not interact with CREB3's proteolytic products specific to the endoplasmic reticulum and to the nucleus. The interaction between the two partners is a prerequisite for the proteolytic activation of CREB3. In case of the further binding partners, our data suggest that hNOT-1/ALG3-1 interacts with both OSBPs and with their direct targets LRP1 and VAMP/VAP-A. Moreover, our results show that various partners of hNOT-1/ALG3-1 interact with its diverse post translationally processed products destined to distinct cellular compartments. Generally, our data suggest the involvement of hNOT-1/ALG3-1 in various molecular contexts determining essential processes associated with distinct cellular machineries and related to various pathologies, such as cancer, viral infections, neuronal and immunological disorders and CDG.

Dynamics of the Ternary Complex Formed by c-Myc Interactor JPO2, Transcriptional Co-activator LEDGF/p75, and Chromatin*

PubMed Central

Hendrix, Jelle; van Heertum, Bart; Vanstreels, Els; Daelemans, Dirk; De Rijck, Jan

2014-01-01

Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator involved in targeting human immunodeficiency virus (HIV) integration and the development of MLL fusion-mediated acute leukemia. A previous study revealed that LEDGF/p75 dynamically scans the chromatin, and upon interaction with HIV-1 integrase, their complex is locked on chromatin. At present, it is not known whether LEDGF/p75-mediated chromatin locking is typical for interacting proteins. Here, we employed continuous photobleaching and fluorescence correlation and cross-correlation spectroscopy to investigate in vivo chromatin binding of JPO2, a LEDGF/p75- and c-Myc-interacting protein involved in transcriptional regulation. In the absence of LEDGF/p75, JPO2 performs chromatin scanning inherent to transcription factors. However, whereas the dynamics of JPO2 chromatin binding are decelerated upon interaction with LEDGF/p75, very strong locking of their complex onto chromatin is absent. Similar results were obtained with the domesticated transposase PogZ, another cellular interaction partner of LEDGF/p75. We furthermore show that diffusive JPO2 can oligomerize; that JPO2 and LEDGF/p75 interact directly and specifically in vivo through the specific interaction domain of JPO2 and the C-terminal domain of LEDGF/p75, comprising the integrase-binding domain; and that modulation of JPO2 dynamics requires a functional PWWP domain in LEDGF/p75. Our results suggest that the dynamics of the LEDGF/p75-chromatin interaction depend on the specific partner and that strong chromatin locking is not a property of all LEDGF/p75-binding proteins. PMID:24634210

3D model for Cancerous Inhibitor of Protein Phosphatase 2A armadillo domain unveils highly conserved protein-protein interaction characteristics.

PubMed

Dahlström, Käthe M; Salminen, Tiina A

2015-12-07

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43.

PubMed

Boogerd, Kees-Jan; Wong, L Y Elaine; Christoffels, Vincent M; Klarenbeek, Meinke; Ruijter, Jan M; Moorman, Antoon F M; Barnett, Phil

2008-06-01

T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 (Cx43) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs. Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3. Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

PubMed

Schmidt, Mirko H H; Broll, Rainer; Bruch, Hans-Peter; Bögler, Oliver; Duchrow, Michael

2003-01-01

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase. Copyright 2002 John Wiley & Sons, Ltd.

Mate Value Discrepancy and Mate Retention Behaviors of Self and Partner.

PubMed

Sela, Yael; Mogilski, Justin K; Shackelford, Todd K; Zeigler-Hill, Virgil; Fink, Bernhard

2017-10-01

This study investigated the relationship between perceived mate value discrepancy (i.e., the difference between an individual's mate value and their partner's mate value) and perceived frequency of mate retention performed by an individual relative to his or her partner. In two studies, participants in long-term, exclusive, sexual, heterosexual relationships reported their own, and their partner's, mate value and mate retention. Samples included 899 community members (Study 1) and 941 students and community members (Study 2). In Study 1, we documented that individuals with higher self-perceived short-term mate value, and who perceive their partner to have lower (vs. higher) short-term mate value, perform less frequent Benefit-Provisioning mate retention, controlling for the partner's Benefit-Provisioning mate retention. In Study 2, we documented that individuals who perceive that they could less easily replace their partner, and who perceive their partner could more (vs. less) easily replace them, perform more frequent mate retention (Benefit-Provisioning and Cost-Inflicting), controlling for the partner's mate retention. These results highlight the importance of assessing perceived discrepancies in mate value (notably, regarding the replaceability of self and partner with another long-term mate) and perceived mate retention behaviors of self, relative to partner, between men and women in long-term relationships. © 2016 Wiley Periodicals, Inc.

37 CFR 2.193 - Trademark correspondence and signature requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Director. (d) Signatory must be identified. The name of the person who signs a document in connection with... behalf of an owner is: (i) A person with legal authority to bind the owner (e.g., a corporate officer or... the owner (e.g., a corporate officer or general partner of a partnership), or a practitioner qualified...

37 CFR 2.193 - Trademark correspondence and signature requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Director. (d) Signatory must be identified. The name of the person who signs a document in connection with... behalf of an owner is: (i) A person with legal authority to bind the owner (e.g., a corporate officer or... the owner (e.g., a corporate officer or general partner of a partnership), or a practitioner qualified...

37 CFR 2.193 - Trademark correspondence and signature requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Director. (d) Signatory must be identified. The name of the person who signs a document in connection with... behalf of an owner is: (i) A person with legal authority to bind the owner (e.g., a corporate officer or... the owner (e.g., a corporate officer or general partner of a partnership), or a practitioner qualified...

An Investigation on Social Representations: Inanimate Agent Can Mislead Dogs (Canis familiaris) in a Food Choice Task.

PubMed

Abdai, Judit; Gergely, Anna; Petró, Eszter; Topál, József; Miklósi, Ádám

2015-01-01

The nature of mental representation of others plays a crucial role in social interactions. Dogs present an ideal model species for the investigation of such mental representations because they develop social ties with both conspecifics and heterospecifics. Former studies found that dogs' preference for larger food quantity could be reversed by humans who indicate the smaller quantity. The question is whether this social bias is restricted to human partners. We suggest that after a short positive social experience, an unfamiliar moving inanimate agent (UMO) can also change dogs' choice between two food quantities. We tested four groups of dogs with different partners: In the (1) Helper UMO and (2) Helper UMO Control groups the partner was an interactive remote control car that helped the dog to obtain an otherwise unreachable food. In the (3) Non-helper UMO and (4) Human partner groups dogs had restricted interaction with the remote control car and the unfamiliar human partners. In the Human partner, Helper UMO and Helper UMO Control groups the partners were able to revert dogs' choice for the small amount by indicating the small one, but the Non-helper UMO was not. We suggest that dogs are able to generalize their wide range of experiences with humans to another type of agent as well, based on the recognition of similarities in simple behavioural patterns.

An Investigation on Social Representations: Inanimate Agent Can Mislead Dogs (Canis familiaris) in a Food Choice Task

PubMed Central

Abdai, Judit; Gergely, Anna; Petró, Eszter; Topál, József; Miklósi, Ádám

2015-01-01

The nature of mental representation of others plays a crucial role in social interactions. Dogs present an ideal model species for the investigation of such mental representations because they develop social ties with both conspecifics and heterospecifics. Former studies found that dogs’ preference for larger food quantity could be reversed by humans who indicate the smaller quantity. The question is whether this social bias is restricted to human partners. We suggest that after a short positive social experience, an unfamiliar moving inanimate agent (UMO) can also change dogs’ choice between two food quantities. We tested four groups of dogs with different partners: In the (1) Helper UMO and (2) Helper UMO Control groups the partner was an interactive remote control car that helped the dog to obtain an otherwise unreachable food. In the (3) Non-helper UMO and (4) Human partner groups dogs had restricted interaction with the remote control car and the unfamiliar human partners. In the Human partner, Helper UMO and Helper UMO Control groups the partners were able to revert dogs’ choice for the small amount by indicating the small one, but the Non-helper UMO was not. We suggest that dogs are able to generalize their wide range of experiences with humans to another type of agent as well, based on the recognition of similarities in simple behavioural patterns. PMID:26241747

Network organization of the human autophagy system.

PubMed

Behrends, Christian; Sowa, Mathew E; Gygi, Steven P; Harper, J Wade

2010-07-01

Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

The role of overprotection by the partner in coping with diabetes: a moderated mediation model.

PubMed

Schokker, Marike C; Links, Thera P; Bouma, Jelte; Keers, Joost C; Sanderman, Robbert; Wolffenbuttel, Bruce H R; Hagedoorn, Mariët

2011-01-01

This study examined whether diabetes-specific self-efficacy mediates the association between overprotection and distress and whether this mediation depends on glycemic control and gender. The research sample of 215 individuals with diabetes and their partners completed a measure of partners' overprotective behaviours towards the patient. Patients also completed measures of diabetes-specific self-efficacy and diabetes-related distress. Further, HbA1c values were obtained as an indication of glycemic control. Diabetes-specific self-efficacy mediated the association between overprotection by the partner and diabetes-related distress especially when glycemic control was relatively poor. Furthermore, diabetes-specific self-efficacy mediated the association between overprotection and diabetes-related distress more strongly in female than in male patients. The findings underscore the importance of studying both moderators and mediators in the association between partner behaviour and distress in patients.

Individual and Partner Correlates of Sexual Satisfaction and Relationship Happiness in Midlife Couples: Dyadic Analysis of the International Survey of Relationships.

PubMed

Fisher, William A; Donahue, Kelly L; Long, J Scott; Heiman, Julia R; Rosen, Raymond C; Sand, Michael S

2015-08-01

The current research reports a dyadic analysis of sexual satisfaction, relationship happiness, and correlates of these couple outcomes in a large multinational dataset consisting of 1,009 midlife heterosexual couples (2,018 individuals) recruited in Japan, Brazil, Germany, Spain, and the United States (Heiman et al., 2011). Actor-Partner Interdependence Models (Kenny, Kashy, & Cook, 2006) identified correlates of sexual satisfaction that included individuals' reports of good health; frequent kissing, cuddling, and caressing; frequent recent sexual activity; attaching importance to one's own and one's partner's orgasm; better sexual functioning; and greater relationship happiness. Even after controlling for individual-level effects, partners' reports of good health; frequent kissing, cuddling, and caressing; frequent recent sexual activity; attaching importance to one's own and one's partner's orgasm; better sexual functioning; and greater relationship happiness contributed significantly to predicting and understanding individuals' sexual satisfaction. Correlates of relationship happiness included individuals' reports of good health; frequent kissing, cuddling, and caressing; frequent recent sexual activity; attaching importance to one's own and one's partner's orgasm; better sexual functioning; and greater sexual satisfaction, and once again, even after controlling for individual-level effects, partners' reports of each of these correlates contributed significantly to predicting and understanding individuals' relationship happiness. Interactions of individual and partner effects with participant gender are also reported. Current results demonstrate empirically that the partner "matters" to an individual's sexual satisfaction and relationship happiness and indicate that a comprehensive understanding of factors contributing to these couple outcomes requires a couple-level research strategy. Partner effects, even when controlling for individual effects, were consistently observed, and explanation of sexual satisfaction and relationship happiness always depended on identifying and understanding mutual and concurrent individual and partner influences.

Prevalence and correlates of intimate partner violence in HIV-positive women engaged in transactional sex in Mombasa, Kenya

PubMed Central

Wilson, Kate S; Deya, Ruth; Masese, Linnet; Simoni, Jane M; Stoep, Ann Vander; Shafi, Juma; Jaoko, Walter; Hughes, James P; McClelland, R Scott

2016-01-01

We evaluated the prevalence and correlates of intimate partner violence in the past year by a regular male partner in HIV-positive female sex workers in Mombasa, Kenya. This cross-sectional study included HIV-positive women ≥ 18 years old who reported engagement in transactional sex at the time of enrolment in the parent cohort. We asked 13 questions adapted from the World Health Organization survey on violence against women about physical, sexual, or emotional violence in the past year by the current or most recent emotional partner (index partner). We used standardised instruments to assess socio-demographic and behavioural characteristics as possible correlates of intimate partner violence. Associations between intimate partner violence and these correlates were evaluated using univariate and multivariate logistic regression. Overall, 286/357 women (80.4%) had an index partner, and 52/357 (14.6%, 95% confidence interval 10.9%–18.2%) reported intimate partner violence by that partner in the past year. In multivariate analysis, women with severe alcohol problems (adjusted odds ratio 4.39, 1.16–16.61) and those experiencing controlling behaviours by the index partner (adjusted odds ratio 4.98, 2.31–10.74) were significantly more likely to report recent intimate partner violence. Recent intimate partner violence was common in HIV-positive female sex workers. Interventions targeting risk factors for intimate partner violence, including alcohol problems and partner controlling behaviours, could help to reduce recurrent violence and negative health outcomes in this key population. PMID:26464502

Intimate Partner Violence and Its Association With Physical and Mental Health Symptoms Among Older Women in Germany.

PubMed

Stöckl, Heidi; Penhale, Bridget

2015-10-01

Intimate partner violence is a commonly acknowledged health care issue. While numerous studies established the health implications of physical and/or sexual intimate partner violence among women of reproductive age, the evidence is scarce for older women and for other forms of intimate partner violence. This study, therefore, investigates the prevalence of intimate partner violence in its different forms and its association with physical and mental health symptoms of older women, using women of reproductive age as a reference group. This study is a cross-sectional study, utilizing data from a national representative survey of 10,264 German women aged 16 to 86 years. Rates of physical and sexual intimate partner violence in the last year decreased from 8% to 3% and 1% among women aged 16 to 49 years, 50 to 65 years, and 66 to 86 years, respectively. The prevalence of emotional and economic abuse and controlling behavior by partners remained nearly the same. All forms of intimate partner violence had significant associations with women's health symptoms, such as gastrointestinal, psychosomatic and psychological symptoms, and pelvic problems. Controlling behavior was most consistently associated with most health symptoms. Health and care professionals who screen women for intimate partner violence should, therefore, consider incorporating questions about controlling behavior as well, because this form of violence is not only frequent but also has multiple health outcomes among women across all ages. © The Author(s) 2014.

People Are More Generous to a Partner Who Pays Attention to Them.

PubMed

Ohtsubo, Yohsuke; Yamaguchi, Chiaki

2017-01-01

People use relatively low-cost signals to maintain close relationships, in which they engage in costlier exchanges of tangible support. Paying attention to a partner allows an individual to communicate his or her interest in the relationship with the partner. Previous studies have revealed that when Person A pays attention to Person B, B's feeling of intimacy toward A increases. If social attention strengthens the bond between A and B, it is predicted that A's attention will also increase B's generous behavior toward A. This study tested this prediction. Participants first engaged in a collaborative task using computers. In the task, the putative partner (a computer program) either paid or did not pay attention to participants (high attention condition vs. low attention condition). In the control condition, the partner could not choose when to pay attention to participants. They then played three rounds of the dictator game with the partner. Confirming the previous finding, perceived intimacy was highest in the high attention condition, in the middle in the control condition, and lowest in the low attention condition. More importantly, participants in the high attention condition decided to give more resources to their partner than those in the low attention condition (but the difference between the high attention condition and the control condition was not significant). In addition, self-reported intimacy was positively correlated with the resource allocated to the partner. The results of this study demonstrated that social attention fosters a partner's generosity.

Women bar drinkers' discussions about birth control and risky sexual behavior.

PubMed

Levonyan-Radloff, Kristine; Parks, Kathleen A; Collins, R Lorraine

2012-08-01

This study evaluated how birth control discussions prior to sexual activity affected condom use in a sample of 225 young women bar drinkers. The use of alcohol and other drugs (AOD) and partner type also were assessed. Data were collected through daily reports and qualitative interviews over 12 weeks. A total of 1671 sexual events were reported (M = 7.4 events per participant), of which 64.7% (n = 1081) did not involve condom use. Discussions of birth control occurred prior to 10.9% (n = 183) of all sexual events. These discussions were more likely to be initiated by the woman and to occur when AOD had been used. Thematic analysis revealed four common themes: confirmation of the need to use condoms, confirmation of oral/hormonal contraceptive use, use of sufficient birth control, and discrepant condom use. The discussions reduced rates of risky sex when the sexual partner was more intimate (i.e., boyfriend/dating partner), regardless of AOD use and when the sexual partner was a friend or ex-partner and no AOD were used. The thematic analysis suggested that pregnancy prevention was a stronger motivation for discussing condom use, rather than risk of contracting an STI. Given the substantial rate of high risk partners, history of STIs, and limited amount of time women reported knowing "regular" partners, we suggest that interventions designed to reduce risky sex should be tailored to increase women's awareness of STI and pregnancy risk when under the influence of AOD, and promote condom use regardless of partner type.

Women Bar Drinkers’ Discussions about Birth Control and Risky Sexual Behavior

PubMed Central

Levonyan-Radloff, Kristine; Parks, Kathleen A.; Collins, R. Lorraine

2011-01-01

This study evaluated how birth control discussions prior to sexual activity affected condom use in a sample of 225 young women bar drinkers. The use of alcohol and other drugs (AOD) and partner type also were assessed. Data were collected through daily reports and qualitative interviews over 12 weeks. A total of 1671 sexual events were reported (M = 7.4 events per participant), of which 64.7% (n = 1081) did not involve condom use. Discussions of birth control occurred prior to 10.9% (n = 183) of all sexual events. These discussions were more likely to be initiated by the woman and to occur when AOD had been used. Thematic analysis revealed four common themes: confirmation of the need to use condoms, confirmation of oral/hormonal contraceptive use, use of sufficient birth control, and discrepant condom use. The discussions reduced rates of risky sex when the sexual partner was more intimate (i.e., boyfriend/dating partner), regardless of AOD use and when the sexual partner was a friend or ex-partner and no AOD were used. The thematic analysis suggested that pregnancy prevention was a stronger motivation for discussing condom use, rather than risk of contracting an STI. Given the substantial rate of high risk partners, history of STIs, and limited amount of time women reported knowing “regular” partners, we suggest that interventions designed to reduce risky sex should be tailored to increase women’s awareness of STI and pregnancy risk when under the influence of AOD, and promote condom use regardless of partner type. PMID:21892691

Both trust and self-control are necessary to prevent intrusive behaviors: evidence from a longitudinal study of married couples.

PubMed

Buyukcan-Tetik, Asuman; Finkenauer, Catrin; Kuppens, Sofie; Vohs, Kathleen D

2013-08-01

Many people engage in intrusive behaviors in close relationships. Existing research links intrusive behaviors to a lack of trust and an imbalance between self- and partner-interest. The authors tested the novel hypothesis that people need self-control to regulate intrusive behaviors. Self-control enables people to forgo their self-interests (reassurance or closeness) for the sake of their partner or the relationship. Specifically, we predicted that people need both trust and self-control to refrain from intrusive behavior. One-hundred-eighty-nine couples participated in a prospective longitudinal study with three waves. Consistent with predictions, multilevel analyses revealed an interaction between trust and self-control on intrusive behaviors cross-sectionally as well as longitudinally (albeit marginally). These results provide support for our hypothesis that neither trust in the partner nor self-control is sufficient to forestall intrusive behaviors, but rather both are necessary to refrain from intruding into one's partner's privacy. © 2013 American Psychological Association

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Optically degradable dendrons for temporary adhesion of proteins to DNA.

PubMed

Kostiainen, Mauri A; Kotimaa, Juha; Laukkanen, Marja-Leena; Pavan, Giovanni M

2010-06-18

Experimental studies and molecular dynamics modeling demonstrate that multivalent dendrons can be used to temporarily glue proteins and DNA together with high affinity. We describe N-maleimide-cored polyamine dendrons that can be conjugated with free cysteine residues on protein surfaces through 1,4-conjugate addition to give one-to-one protein-polymer conjugates. We used a genetically engineered cysteine mutant of class II hydrophobin (HFBI) and a single-chain Fragment variable (scFv) antibody as model proteins for the conjugation reactions. The binding affinity of the protein-dendron conjugates towards DNA was experimentally assessed by using the ethidium bromide displacement assay. The binding was found to depend on the generation of the dendron, with the second generation having a stronger affinity than the first generation. Thermodynamic parameters of the binding were obtained from molecular dynamics modeling, which showed that the high binding affinity for each system is almost completely driven by a strong favorable binding enthalpy that is opposed by unfavorable binding entropy. A short exposure to UV (lambda approximately 350 nm) can cleave the photolabile o-nitrobenzyl-linked binding ligands from the surface of the dendron, which results in loss of the multivalent binding interactions and triggers the release of the DNA and protein. The timescale of the release is very rapid and the binding partners can be efficiently released after 3 min of UV exposure.

Cooperative Binding of Cyclodextrin Dimers to Isoflavone Analogues Elucidated by Free Energy Calculations.

PubMed

Zhang, Haiyang; Tan, Tianwei; Hetényi, Csaba; Lv, Yongqin; van der Spoel, David

2014-04-03

Dimerization of cyclodextrin (CD) molecules is an elementary step in the construction of CD-based nanostructured materials. Cooperative binding of CD cavities to guest molecules facilitates the dimerization process and, consequently, the overall stability and assembly of CD nanostructures. In the present study, all three dimerization modes (head-to-head, head-to-tail, and tail-to-tail) of β-CD molecules and their binding to three isoflavone drug analogues (puerarin, daidzin, and daidzein) were investigated in explicit water surrounding using molecular dynamics simulations. Total and individual contributions from the binding partners and solvent environment to the thermodynamics of these binding reactions are quantified in detail using free energy calculations. Cooperative drug binding to two CD cavities gives an enhanced binding strength for daidzin and daidzein, whereas for puerarin no obvious enhancement is observed. Head-to-head dimerization yields the most stable complexes for inclusion of the tested isoflavones (templates) and may be a promising building block for construction of template-stabilized CD nanostructures. Compared to the case of CD monomers, the desolvation of CD dimers and entropy changes upon complexation prove to be influential factors of cooperative binding. Our results shed light on key points of the design of CD-based supramolecular assemblies. We also show that structure-based calculation of binding thermodynamics can quantify stabilization caused by cooperative effects in building blocks of nanostructured materials.

From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces

PubMed Central

Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

2011-01-01

Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

PubMed

Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-31

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

PubMed Central

Claveria-Gimeno, Rafael; Lanuza, Pilar M.; Morales-Chueca, Ignacio; Jorge-Torres, Olga C.; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-01

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities. PMID:28139759

A general mechanism for competitor-induced dissociation of molecular complexes

PubMed Central

Paramanathan, Thayaparan; Reeves, Daniel; Friedman, Larry J.; Kondev, Jane; Gelles, Jeff

2014-01-01

The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will in general accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor–induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily implyternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatio temporal resolution. PMID:25342513

Impaired bile acid handling and aggravated liver injury in mice expressing a hepatocyte-specific RXRα variant lacking the DNA-binding domain.

PubMed

Kosters, Astrid; Felix, Julio C; Desai, Moreshwar S; Karpen, Saul J

2014-02-01

Retinoid X Receptor α (RXRα) is the principal heterodimerization partner of class II Nuclear Receptors (NRs), and a major regulator of gene expression of numerous hepatic processes, including bile acid (BA) homeostasis through multiple partners. Specific contributions of hepatic RXRα domains in heterodimer function in response to either BA load or ductular cholestasis are not fully characterized. Wild-type (WT) mice and mice expressing a hepatocyte-specific RXRα lacking the DNA-Binding-Domain (hs-RxrαΔex4(-/-)), which retains partial ability to heterodimerize with its partners, were fed a 1% cholic acid (CA) diet for 5 days, a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 3 weeks, or control diet. Serum ALT (6.5-fold; p<0.05), AST (9.3-fold; p=0.06) and BA (2.8-fold; p<0.05) were increased in CA-fed hs-RxαΔex4(-/-) mice compared to CA-fed WT mice, but were equally induced between genotypes by DDC-feeding. CA-feeding elevated total (4.4-fold; p=0.06) and unconjugated (2.2-fold; p<0.02) bilirubin levels in hs-RxrαΔex4(-/-) mice compared to WT mice, but not in DDC-fed hs-RxrαΔex4(-/-) mice. Increased necrosis and inflammation was observed in CA-fed, but not in DDC-fed hs-RxrαΔex4(-/-) mice. Apoptotic markers DR5, CK8, CK18 RNA were increased in CA- and DDC-fed hs-RxrαΔex4(-/-) mice. Cleaved caspase 3, CK18 and p-JNK protein were elevated in CA-fed but not in DDC-fed hs-RxrαΔex4(-/-) mice. Induction of Ostβ and Cyp2b10 RNA was impaired in CA-fed and DDC-fed hs-RxrαΔex4(-/-) mice. Surprisingly, DDC-fed hs-RxrαΔex4(-/-) mice showed attenuated fibrosis compared to DDC-fed WT mice. These two models of cholestasis identify common and injury-specific roles for RXRα heterodimers and the functional relevance of an intact RXRα-DBD in the hepatocytic adaptive cholestatic response. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Characterizing the burden of premature ejaculation from a patient and partner perspective: a multi-country qualitative analysis

PubMed Central

Revicki, Dennis; Howard, Kellee; Hanlon, Jennifer; Mannix, Sally; Greene, Alison; Rothman, Margaret

2008-01-01

Background Premature ejaculation (PE) is a common sexual dysfunction among men which affects men and their partners. Little qualitative data are available to characterize the impact of PE on men and their partners about ejaculatory control, sexual satisfaction, emotional distress and relationships. The objective of this study was to assess the impact of PE from the perspective of men with PE and the female partners of men with PE on their sexual experience, distress and relationships. Methods Qualitative data were collected through 14 focus groups in the US and through one-on-one interviews in the US, UK, Italy, France, Germany, and Poland. Study participants included heterosexual men with PE and female partners of males with PE. All participants were asked about how PE affects their daily life, including emotional impacts. One-on-one interviews also included obtaining feedback on the male and female versions of 4-single item measures of PE focusing on ejaculatory control, satisfaction with intercourse, interpersonal distress, and relationship difficulty. Results Participants included 172 males with PE and 67 female partners of men with PE. Lack of control over ejaculation and dissatisfaction with intercourse emerged as central themes of PE. Lack of ejaculatory control resulted in greater dissatisfaction and greater emotional distress, including feelings of inadequacy, disappointment, and anxiety. Continued PE ultimately leads to greater problems with partners and often disrupts partner relationships. Participants indicated that PE was keeping them from attaining complete intimacy in their relationships even when their partners were generally satisfied with sexual intercourse. Impacts of PE on sexual satisfaction, emotional distress and partner relationships were consistent across countries. Feedback on the single-item PE measures confirmed relevance of the item content and further confirmed major themes identified from the qualitative data. Conclusion This qualitative study provides valuable insights on the substantial psychosocial burden of PE in the US and the Europe. Lack of control over ejaculation resulted in dissatisfaction with intercourse and increased emotional distress, and wide-ranging impact for both men with PE and their partners of men with PE. Data collected in this study may help inform the content of new patient reported measures for use in PE research. PMID:18474090

A novel method for the study of molecular interaction by using microscale thermophoresis.

PubMed

Mao, Yexuan; Yu, Lanlan; Yang, Ran; Qu, Ling-bo; Harrington, Perter de B

2015-01-01

The fundamental studies for the binding events of protein and its partner are crucial in drug development. In this study, a novel technology named microscale thermophoresis (MST) was applied in the investigation of molecular interaction between an organic dye fluorescein isothiocyanate (FITC) and bovine serum albumin (BSA), and the results were compared with those obtained from conventional fluorescence spectroscopy. The MST data demonstrated that with a short interaction time, FITC showed a high binding affinity for BSA by weak interaction instead of labeling the protein. By using competitive strategies in which warfarin and ibuprofen acted as the site markers of BSA, FITC was proven to mainly bind to the hydrophobic pocket of site II of BSA compared to site I of BSA. Except for the binding affinity, MST also provided additional information with respect to the aggregation of BSA and the binding of FITC to BSA aggregates, which is unobtainable by fluorescence spectroscopy. This work proves that MST as a new approach is powerful and reliable for investigation of protein-small molecule interaction. Copyright © 2014 Elsevier B.V. All rights reserved.

Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

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

Jenson, Justin M.; Ryan, Jeremy A.; Grant, Robert A.

Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry,more » relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors.« less

Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

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

M Mitchell; J Smith; M Mason

The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that ismore » directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.« less

Epistatic mutations in PUMA BH3 drive an alternate binding mode to potently and selectively inhibit anti-apoptotic Bfl-1

PubMed Central

Jenson, Justin M; Ryan, Jeremy A; Grant, Robert A; Letai, Anthony; Keating, Amy E

2017-01-01

Overexpression of anti-apoptotic Bcl-2 family proteins contributes to cancer progression and confers resistance to chemotherapy. Small molecules that target Bcl-2 are used in the clinic to treat leukemia, but tight and selective inhibitors are not available for Bcl-2 paralog Bfl-1. Guided by computational analysis, we designed variants of the native BH3 motif PUMA that are > 150-fold selective for Bfl-1 binding. The designed peptides potently trigger disruption of the mitochondrial outer membrane in cells dependent on Bfl-1, but not in cells dependent on other anti-apoptotic homologs. High-resolution crystal structures show that designed peptide FS2 binds Bfl-1 in a shifted geometry, relative to PUMA and other binding partners, due to a set of epistatic mutations. FS2 modified with an electrophile reacts with a cysteine near the peptide-binding groove to augment specificity. Designed Bfl-1 binders provide reagents for cellular profiling and leads for developing enhanced and cell-permeable peptide or small-molecule inhibitors. DOI: http://dx.doi.org/10.7554/eLife.25541.001 PMID:28594323

Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

PubMed

Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

2017-01-01

Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

27 CFR 31.104 - Withdrawal of one or more partners.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Withdrawal of one or more partners. 31.104 Section 31.104 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... this part, the remaining partner, or partners, must register the change in control by filing an amended...

27 CFR 31.104 - Withdrawal of one or more partners.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Withdrawal of one or more partners. 31.104 Section 31.104 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE... this part, the remaining partner, or partners, must register the change in control by filing an amended...

Prayer and forgiveness: Beyond relationship quality and extension to marriage.

PubMed

Fincham, Frank D; May, Ross W

2017-09-01

The majority of the world population profess religious/spiritual beliefs and prayer is a form of spiritual activity common across numerous religious/spiritual belief systems. Three studies therefore examined the role of prayer in romantic relationships. Study 1 (n = 91) showed that prayer for a dating partner predicted lower aggressive tendencies and greater forgiveness of partner transgressions, independently of relationship closeness. Study 2 (n = 89 married couples) is among the first to examine the prayer-forgiveness association using dyadic data. Controlling for relationship satisfaction in the actor-partner interdependence model, prayer for the spouse predicted own forgiveness but not partner's reports of their own forgiveness. To obviate the problem of obtaining all the data from the same reporter, Study 3 (n = 91 married couples) used partner reports of the spouse's forgiveness in an actor-partner interdependence model analysis. Controlling for religiosity, the results showed that prayer for the partner predicted partner reports of the prayer's forgiveness. The implications of these results are then discussed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

The influence of partner drug use and relationship power on treatment engagement.

PubMed

Riehman, Kara S; Iguchi, Martin Y; Zeller, Michelle; Morral, Andrew R

2003-05-01

Substance-using intimate partners negatively influence individuals' substance abuse treatment engagement and drug use, but little else is known about effects of intimate relationships on treatment. We examine how relationship dynamics (power, control, dependence, insecurity and decision-making power) influence treatment engagement, and whether this differs by gender and partner drug use. Sixty-four heroin users (42 men, 22 women) receiving methadone detoxification treatment in Los Angeles were interviewed at treatment entry and submitted daily diaries of drug use throughout the 21-day treatment. Total number of reported heroin-free days in the first eight treatment days was the dependent variable. Bivariate analyses revealed, that compared to men, women were more likely to have substance-using partners, reported greater power over a partner and greater household decision-making power in their relationships. Multivariate analysis indicated that individuals whose partners had more control over them reported fewer days abstinent. Among individuals with heroin-using partners, greater household decision-making power was associated with more days abstinent, but there was no association for individuals with non-using partners. Relationship power dynamics may be important influences on the treatment process, and some dimensions of power may interact with partner drug use status.

A Dyadic Growth Approach to Partner Regulation Attempts on Changes in Drinking and Negative Alcohol-Related Consequences.

PubMed

Rodriguez, Lindsey M

2016-12-05

Many individuals engage in regulation attempts to manage or reduce their partner's alcohol use. Research on partner social control behaviors has shown that regulation attempts generally factor into negative (i.e., punishing) and positive (i.e., rewarding) dimensions. In the alcohol domain, partner drinking has been associated with poorer relationship functioning through punishment. This research applied a dyadic growth model approach to investigate changes in alcohol consumption and negative alcohol-related consequences over 6 months, and evaluated whether partner regulation attempts (punishment and reward) were influential (i.e., successful) in these changes. Married couples (N = 123 dyads) completed web-based measures of partner regulation attempts, alcohol consumption, and alcohol-related consequences three times over a 6-month period. Results from dyadic growth curve analyses showed that partner punishment was significantly associated with increases in alcohol-related consequences-and marginally associated with increases in alcohol consumption-over the 6-month period. Partner reward was associated with decreases in consumption over the study period. These effects were not different for husbands and wives. Conclusion/Importance: Results support previous research demonstrating deleterious impact of partner punishing control strategies and provide important implications for future interventions and treatment.

Silenced suffering: the need for a better understanding of partner sexual violence.

PubMed

Logan, T K; Walker, Robert; Cole, Jennifer

2015-04-01

This article has two overall goals. First, to examine the current state of sexual violence research to highlight several shortcomings in the knowledge on partner sexual violence. Second, to describe several factors to consider in future research to facilitate a more in-depth understanding of partner sexual violence. Shortcomings of the research on partner sexual violence include (1) overreliance on dichotomous yes/no representations of sexual violence experiences; (2) lack of, or inadequate documentation of the scope and nature of partner sexual violence; (3) inadequate ways to account for impairment of consent under different circumstances; (4) difficulties in discriminating unwanted from nonconsensual sexual activities; and (5) limited information about the role sexual violence plays in the larger context of coercive control. In order to facilitate a more in-depth understanding of partner sexual assault, there is a need (1) to better understand the scope and nature of partner sexual assault and (2) to better understand the role partner sexual violence plays in coercive control. By improving the measurement of this phenomenon, victims, researchers, practitioners, and those involved in the justice system might be better equipped to respond to sexual violence among intimate partners. © The Author(s) 2013.

Expression of human PQBP-1 in Drosophila impairs long-term memory and induces abnormal courtship.

PubMed

Yoshimura, Natsue; Horiuchi, Daisuke; Shibata, Masao; Saitoe, Minoru; Qi, Mei-Ling; Okazawa, Hitoshi

2006-04-17

Frame shift mutations of the polyglutamine binding protein-1 (PQBP1) gene lead to total or partial truncation of the C-terminal domain (CTD) and cause mental retardation in human patients. Interestingly, normal Drosophila homologue of PQBP-1 lacks CTD. As a model to analyze the molecular network of PQBP-1 affecting intelligence, we generated transgenic flies expressing human PQBP-1 with CTD. Pavlovian olfactory conditioning revealed that the transgenic flies showed disturbance of long-term memory. In addition, they showed abnormal courtship that male flies follow male flies. Abnormal functions of PQBP-1 or its binding partner might be linked to these symptoms.

Every which way--nanos gene regulation in echinoderms.

PubMed

Oulhen, Nathalie; Wessel, Gary M

2014-03-01

Nanos is an essential factor of germ line success in all animals tested. This gene encodes a Zn-finger RNA-binding protein that in complex with its partner pumilio binds to and changes the fate of several known transcripts. We summarize here the documented functions of Nanos in several key organisms, and then emphasize echinoderms as a working model for how nanos expression is regulated. Nanos presence outside of the target cells is often detrimental to the animal, and in sea urchins, nanos expression appears to be regulated at every step of transcription, and post-transcriptional activity, making this gene product exciting, every which way. Copyright © 2013 Wiley Periodicals, Inc.

Every which way – nanos gene regulation in echinoderms

PubMed Central

Oulhen, Nathalie; Wessel, Gary M.

2014-01-01

Nanos is an essential factor of germ line success in all animals tested. This gene encodes a Zn-finger RNA-binding protein that in complex with its partner pumilio, binds to and changes the fate of several known transcripts. We summarize here the documented functions of nanos in several key organisms, and then emphasize echinoderms as a working model for how nanos expression is regulated. Nanos presence outside of the target cells is often detrimental to the animal, and in sea urchins, nanos expression appears to be regulated at every step of transcription, and post-transcriptional activity, making this gene product exciting, every which way. PMID:24376110

The measles virus N(TAIL)-XD complex: an illustrative example of fuzziness.

PubMed

Longhi, Sonia

2012-01-01

In this chapter, I focus on the biochemical and structural characterization of the complex between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) and the C-terminal X domain (XD) of the viral phosphoprotein (P). I summarize the main experimental data available so far pointing out the prevalently disordered nature of N(TAIL) even after complex formation and the role of the flexible C-terminal appendage in the binding reaction. I finally discuss the possible functional role of these residual disordered regions within the complex in terms of their ability to capture other regulatory, binding partners.

Synthetic lipopeptide inhibitors of RAS oncoproteins | NCI Technology Transfer Center | TTC

Cancer.gov

It is well known that overactive Ras signaling is linked to many forms of cancer, and despite intensive efforts worldwide to develop effective inhibitors of Ras, to date there is no anti-Ras inhibitor in clinical use. Researchers at the NCI’s Cancer and Inflammation Program, in collaboration with scientists at Vanderbilt University and the University of Illinois in Chicago, have identified a number of small peptidomimetic compounds that bind to Ras proteins with nanomolar affinity. NCI’s Cancer and Inflammation Program seeks partners interested in licensing or co-development of synthetic, highly potent cell-permeable inhibitors of Ras that bind to the protein directly.

Electrostatic forces govern the binding mechanism of intrinsically disordered histone chaperones

PubMed Central

Liu, Chuanbo; Wang, Tianshu; Bai, Yawen; Wang, Jin

2017-01-01

A unified picture to understand the protein recognition and function must include the native binding complex structure ensembles and the underlying binding mechanisms involved in specific biological processes. However, quantifications of both binding complex structures and dynamical mechanisms are still challenging for IDP. In this study, we have investigated the underlying molecular mechanism of the chaperone Chz1 and histone H2A.Z-H2B association by equilibrium and kinetic stopped-flow fluorescence spectroscopy. The dependence of free energy and kinetic rate constant on electrolyte mean activity coefficient and urea concentration are uncovered. Our results indicate a previous unseen binding kinetic intermediate. An initial conformation selection step of Chz1 is also revealed before the formation of this intermediate state. Based on these observations, a mixed mechanism of three steps including both conformation selection and induced fit is proposed. By combination of the ion- and denaturant-induced experiments, we demonstrate that electrostatic forces play a dominant role in the recognition of bipolar charged intrinsically disordered protein Chz1 to its preferred partner H2A.Z-H2B. Both the intra-chain and inter-chain electrostatic interactions have direct impacts on the native collapsed structure and binding mechanism. PMID:28552960

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

PubMed Central

Neumüller, Klaus G.; Elsayad, Kareem; Reisecker, Johannes M.; Waxham, M. Neal; Heinze, Katrin G.

2010-01-01

Background Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. Methodology/Principal Findings We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner. PMID:21124984

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

PubMed

Meyer, Sharon A; Jirtle, Randy L

2013-05-07

The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

PubMed

Morgan, Sarah V; Garwood, Claire J; Jennings, Luke; Simpson, Julie E; Castelli, Lydia M; Heath, Paul R; Mihaylov, Simeon R; Vaquéz-Villaseñor, Irina; Minshull, Thomas C; Ince, Paul G; Dickman, Mark J; Hautbergue, Guillaume M; Wharton, Stephen B

2018-05-08

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states. © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Crystal structure of the Agrobacterium virulence complex VirE1-VirE2 reveals a flexible protein that can accommodate different partners.

PubMed

Dym, Orly; Albeck, Shira; Unger, Tamar; Jacobovitch, Jossef; Branzburg, Anna; Michael, Yigal; Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Elbaum, Michael

2008-08-12

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.

Expression, purification and characterization of a full-length recombinant HIV-1 Vpu from inclusion bodies.

PubMed

Njengele, Zikhona; Kleynhans, Ronel; Sayed, Yasien; Mosebi, Salerwe

2016-12-01

Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli. Copyright © 2016 Elsevier Inc. All rights reserved.

MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication.

PubMed

Kumagai, Akiko; Dunphy, William G

2017-11-01

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation. © 2017 Kumagai and Dunphy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Affinity Map of Bromodomain Protein 4 (BRD4) Interactions with the Histone H4 Tail and the Small Molecule Inhibitor JQ1*

PubMed Central

Jung, Marie; Philpott, Martin; Müller, Susanne; Schulze, Jessica; Badock, Volker; Eberspächer, Uwe; Moosmayer, Dieter; Bader, Benjamin; Schmees, Norbert; Fernández-Montalván, Amaury; Haendler, Bernard

2014-01-01

Bromodomain protein 4 (BRD4) is a member of the bromodomain and extra-terminal domain (BET) protein family. It binds to acetylated histone tails via its tandem bromodomains BD1 and BD2 and forms a complex with the positive transcription elongation factor b, which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. We analyzed the binding of BRD4 BD1 and BD2 to different partners and showed that the strongest interactions took place with di- and tetra-acetylated peptides derived from the histone 4 N-terminal tail. We also found that several histone 4 residues neighboring the acetylated lysines significantly influenced binding. We generated 10 different BRD4 BD1 mutants and analyzed their affinities to acetylated histone tails and to the BET inhibitor JQ1 using several complementary biochemical and biophysical methods. The impact of these mutations was confirmed in a cellular environment. Altogether, the results show that Trp-81, Tyr-97, Asn-140, and Met-149 play similarly important roles in the recognition of acetylated histones and JQ1. Pro-82, Leu-94, Asp-145, and Ile-146 have a more differentiated role, suggesting that different kinds of interactions take place and that resistance mutations compatible with BRD4 function are possible. Our study extends the knowledge on the contribution of individual BRD4 amino acids to histone and JQ1 binding and may help in the design of new BET antagonists with improved pharmacological properties. PMID:24497639

Anandamide Revisited: How Cholesterol and Ceramides Control Receptor-Dependent and Receptor-Independent Signal Transmission Pathways of a Lipid Neurotransmitter.

PubMed

Di Scala, Coralie; Fantini, Jacques; Yahi, Nouara; Barrantes, Francisco J; Chahinian, Henri

2018-05-22

Anandamide is a lipid neurotransmitter derived from arachidonic acid, a polyunsaturated fatty acid. The chemical differences between anandamide and arachidonic acid result in a slightly enhanced solubility in water and absence of an ionisable group for the neurotransmitter compared with the fatty acid. In this review, we first analyze the conformational flexibility of anandamide in aqueous and membrane phases. We next study the interaction of the neurotransmitter with membrane lipids and discuss the molecular basis of the unexpected selectivity of anandamide for cholesterol and ceramide from among other membrane lipids. We show that cholesterol behaves as a binding partner for anandamide, and that following an initial interaction mediated by the establishment of a hydrogen bond, anandamide is attracted towards the membrane interior, where it forms a molecular complex with cholesterol after a functional conformation adaptation to the apolar membrane milieu. The complex is then directed to the anandamide cannabinoid receptor (CB1) which displays a high affinity binding pocket for anandamide. We propose that cholesterol may regulate the entry and exit of anandamide in and out of CB1 by interacting with low affinity cholesterol recognition sites (CARC and CRAC) located in transmembrane helices. The mirror topology of cholesterol binding sites in the seventh transmembrane domain is consistent with the delivery, extraction and flip-flop of anandamide through a coordinated cholesterol-dependent mechanism. The binding of anandamide to ceramide illustrates another key function of membrane lipids which may occur independently of protein receptors. Interestingly, ceramide forms a tight complex with anandamide which blocks the degradation pathway of both lipids and could be exploited for anti-cancer therapies.

Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

PubMed Central

Jahangiri, Arman; Nguyen, Alan; Sidorov, Maxim K.; Yagnik, Garima; Rick, Jonathan; Han, Sung Won; Chen, William; Flanigan, Patrick M.; Schneidman-Duhovny, Dina; Mascharak, Smita; De Lay, Michael; Imber, Brandon; Park, Catherine C.; Matsumoto, Kunio; Lu, Kan; Bergers, Gabriele; Sali, Andrej; Weiss, William A.

2017-01-01

The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes. PMID:28973887

Backbone conformational preferences of an intrinsically disordered protein in solution.

PubMed

Espinoza-Fonseca, L Michel; Ilizaliturri-Flores, Ian; Correa-Basurto, José

2012-06-01

We have performed a 4-μs molecular dynamics simulation to investigate the native conformational preferences of the intrinsically disordered kinase-inducible domain (KID) of the transcription factor CREB in solution. There is solid experimental evidence showing that KID does not possess a bound-like structure in solution; however, it has been proposed that coil-to-helix transitions upon binding to its binding partner (CBP) are template-driven. While these studies indicate that IDPs possess a bias towards the bound structure, they do not provide direct evidence on the time-dependent conformational preferences of IDPs in atomic detail. Our simulation captured intrinsic conformational characteristics of KID that are in good agreement with experimental data such as a very small percentage of helical structure in its segment α(B) and structural disorder in solution. We used dihedral principal component analysis dPCA to map the conformations of KID in the microsecond timescale. By using principal components as reaction coordinates, we further constructed dPCA-based free energy landscapes of KID. Analysis of the free energy landscapes showed that KID is best characterized as a conformational ensemble of rapidly interconverting conformations. Interestingly, we found that despite the conformational heterogeneity of the backbone and the absence of substantial secondary structure, KID does not randomly sample the conformational space in solution: analysis of the (Φ, Ψ) dihedral angles showed that several individual residues of KID possess a strong bias toward the helical region of the Ramachandran plot. We suggest that the intrinsic conformational preferences of KID provide a bias toward the folded state without having to populate bound-like conformations before binding. Furthermore, we argue that these conformational preferences do not represent actual structural constraints which drive binding through a single pathway, which allows for specific interactions with multiple binding partners. Based on this evidence, we propose that the backbone conformational preferences of KID provide a thermodynamic advantage for folding and binding without negatively affecting the kinetics of binding. We further discuss the relation of our results to previous studies to rationalize the functional implications of the conformational preferences of IDPs, such as the optimization of structural disorder in protein-protein interactions. This study illustrates the importance in obtaining atomistic information of intrinsically disordered proteins in real time to reveal functional features arising from their complex conformational space.

Regulatory Phosphorylation of Ikaros by Bruton's Tyrosine Kinase

PubMed Central

Zhang, Jian; Ishkhanian, Rita; Uckun, Fatih M.

2013-01-01

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros. PMID:23977012

Investigating the adduct formation of organic mercury species with carbonic anhydrase and hemoglobin from human red blood cell hemolysate by means of LC/ESI-TOF-MS and LC/ICP-MS.

PubMed

Hogeback, Jens; Schwarzer, Miriam; Wehe, Christoph A; Sperling, Michael; Karst, Uwe

2016-01-01

The interaction of mercury species with human erythrocytes is studied to investigate possible high molecular binding partners for mercury species. Human blood hemolysate was spiked with methylmercury and investigated by means of liquid chromatography (LC) coupled to electrospray ionization time of flight mass spectrometry (ESI-ToF-MS) and inductively coupled plasma mass spectrometry (ICP-MS). Beside adduct formation of mercury species with hemoglobin, the main compound of the erythrocytes, mercury binding to the enzyme carbonic anhydrase was revealed. Due to an enzymatic digest of the protein-mercury adduct, the binding site at the free thiol group of the protein was identified. These results indicate that carbonic anhydrase might play a role in mercury toxicity.

Mathematical simulations for bioanalytical assay development: the (un-)necessity and (im-)possibility of free drug quantification.

PubMed

Staack, Roland F; Jordan, Gregor; Heinrich, Julia

2012-02-01

For every drug development program it needs to be discussed whether discrimination between free and total drug concentrations is required to accurately describe its pharmacokinetic behavior. This perspective describes the application of mathematical simulation approaches to guide this initial decision based on available knowledge about target biology, binding kinetics and expected drug concentrations. We provide generic calculations that can be used to estimate the necessity of free drug quantification for different drug molecules. In addition, mathematical approaches are used to simulate various assay conditions in bioanalytical ligand-binding assays: it is demonstrated that due to the noncovalent interaction between the binding partners and typical assay-related interferences in the equilibrium, a correct quantification of the free drug concentration is highly challenging and requires careful design of different assay procedure steps.

Crystal structure of a catalytically active, non-toxic endopeptidase derivative of Clostridium botulinum toxin A.

PubMed

Masuyer, Geoffrey; Thiyagarajan, Nethaji; James, Peter L; Marks, Philip M H; Chaddock, John A; Acharya, K Ravi

2009-03-27

Botulinum neurotoxins (BoNTs) modulate cholinergic nerve terminals to result in neurotransmitter blockade. BoNTs consists of catalytic (LC), translocation (Hn) and cell-binding domains (Hc). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore, removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic. Thus, a molecule consisting of LC and Hn domains of BoNTs, termed LHn, is a suitable molecule for engineering novel therapeutics. The structure of LHA at 2.6 A reported here provides an understanding of the structural implications and challenges of engineering therapeutic molecules that combine functional properties of LHn of BoNTs with specific ligand partners to target different cell types.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1

PubMed Central

Chu, Wen-Ting; Clarke, Jane; Shammas, Sarah L.; Wang, Jin

2017-01-01

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye—Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process. This model was carefully calibrated with the experimental data on helical content and affinity, and shown to be consistent with previously published experimental data on binding rate changes with respect to ionic strength. We find that intramolecular electrostatic interactions influence the unbound states of PUMA only marginally. Our results further suggest that intermolecular electrostatic interactions, and in particular non-native electrostatic interactions, are involved in formation of the initial encounter complex. We are able to reveal the binding mechanism in more detail than is possible using experimental data alone however, and in particular we uncover the role of non-native electrostatic interactions. We highlight the potential importance of such electrostatic interactions for describing the binding reactions of IDPs. Such approaches could be used to provide predictions for the results of mutational studies. PMID:28369057

Structural characterization of metal binding to a cold-adapted frataxin.

PubMed

Noguera, Martín E; Roman, Ernesto A; Rigal, Juan B; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2015-06-01

Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study

PubMed Central

Rajesh, Durairaj; Muthukumar, Subramanian; Saibaba, Ganesan; Siva, Durairaj; Akbarsha, Mohammad Abdulkader; Gulyás, Balázs; Padmanabhan, Parasuraman; Archunan, Govindaraju

2016-01-01

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management. PMID:27782155

Structural elucidation of estrus urinary lipocalin protein (EULP) and evaluating binding affinity with pheromones using molecular docking and fluorescence study.

PubMed

Rajesh, Durairaj; Muthukumar, Subramanian; Saibaba, Ganesan; Siva, Durairaj; Akbarsha, Mohammad Abdulkader; Gulyás, Balázs; Padmanabhan, Parasuraman; Archunan, Govindaraju

2016-10-26

Transportation of pheromones bound with carrier proteins belonging to lipocalin superfamily is known to prolong chemo-signal communication between individuals belonging to the same species. Members of lipocalin family (MLF) proteins have three structurally conserved motifs for delivery of hydrophobic molecules to the specific recognizer. However, computational analyses are critically required to validate and emphasize the sequence and structural annotation of MLF. This study focused to elucidate the evolution, structural documentation, stability and binding efficiency of estrus urinary lipocalin protein (EULP) with endogenous pheromones adopting in-silico and fluorescence study. The results revealed that: (i) EULP perhaps originated from fatty acid binding protein (FABP) revealed in evolutionary analysis; (ii) Dynamic simulation study shows that EULP is highly stable at below 0.45 Å of root mean square deviation (RMSD); (iii) Docking evaluation shows that EULP has higher binding energy with farnesol and 2-iso-butyl-3-methoxypyrazine (IBMP) than 2-naphthol; and (iv) Competitive binding and quenching assay revealed that purified EULP has good binding interaction with farnesol. Both, In-silico and experimental studies showed that EULP is an efficient binding partner to pheromones. The present study provides impetus to create a point mutation for increasing longevity of EULP to develop pheromone trap for rodent pest management.

Structural Basis of Arc Binding to Synaptic Proteins: Implications for Cognitive Disease

DOE PAGES

Zhang, Wenchi; Wu, Jing; Ward, Matthew D.; ...

2015-04-09

Arc is a cellular immediate-early gene (IEG) that functions at excitatory synapses and is required for learning and memory. Here we report crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was “domesticated” in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPγ2 (Stargazin) and CaMKII peptides and is essential for Arc’s synaptic function. A consensus sequence formore » Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc’s synaptic action may be druggable. Finally, these studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc’s contribution to neural plasticity and disease.« less

Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold

PubMed Central

Huber, Roland G.; Bond, Peter J.

2017-01-01

An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners. PMID:29016650

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

PubMed

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

PubMed

Ivanov, Stefan M; Cawley, Andrew; Huber, Roland G; Bond, Peter J; Warwicker, Jim

2017-01-01

An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

Structural Basis of Arc Binding to Synaptic Proteins: Implications for Cognitive Disease

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

Zhang, Wenchi; Wu, Jing; Ward, Matthew D.

Arc is a cellular immediate-early gene (IEG) that functions at excitatory synapses and is required for learning and memory. Here we report crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was “domesticated” in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPγ2 (Stargazin) and CaMKII peptides and is essential for Arc’s synaptic function. A consensus sequence formore » Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc’s synaptic action may be druggable. Finally, these studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc’s contribution to neural plasticity and disease.« less

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule.

PubMed

Winteringham, Louise N; Endersby, Raelene; Kobelke, Simon; McCulloch, Ross K; Williams, James H; Stillitano, Justin; Cornwall, Scott M; Ingley, Evan; Klinken, S Peter

2006-12-15

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogeneous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

Structural Basis of Arc Binding to Synaptic Proteins: Implications for Cognitive Disease

PubMed Central

Zhang, Wenchi; Wu, Jing; Ward, Matthew D.; Yang, Sunggu; Chuang, Yang-An; Xiao, Meifang; Li, Ruojing; Leahy, Daniel J.; Worley, Paul F.

2015-01-01

SUMMARY Arc is a cellular immediate early gene (IEG) that functions at excitatory synapses and is required for learning and memory. We report crystal structures of Arc subdomains that form a bi-lobar architecture remarkably similar to the capsid domain of human immunodeficiency virus (HIV) gag protein. Analysis indicates Arc originated from the Ty3/Gypsy retrotransposon family and was “domesticated” in higher vertebrates for synaptic functions. The Arc N-terminal lobe evolved a unique hydrophobic pocket that mediates intermolecular binding with synaptic proteins as resolved in complexes with TARPγ2 (Stargazin) and CaMKII peptides, and is essential for Arc’s synaptic function. A consensus sequence for Arc binding identifies several additional partners that include genes implicated in schizophrenia. Arc N-lobe binding is inhibited by small chemicals suggesting Arc’s synaptic action may be druggable. These studies reveal the remarkable evolutionary origin of Arc and provide a structural basis for understanding Arc’s contribution to neural plasticity and disease. PMID:25864631

Mechanism of the calcium-regulation of muscle contraction--in pursuit of its structural basis.

PubMed

Wakabayashi, Takeyuki

2015-01-01

The author reviewed the research that led to establish the structural basis for the mechanism of the calcium-regulation of the contraction of striated muscles. The target of calcium ions is troponin on the thin filaments, of which the main component is the double-stranded helix of actin. A model of thin filament was generated by adding tropomyosin and troponin. During the process to provide the structural evidence for the model, the troponin arm was found to protrude from the calcium-depleted troponin and binds to the carboxyl-terminal region of actin. As a result, the carboxyl-terminal region of tropomyosin shifts and covers the myosin-binding sites of actin to block the binding of myosin. At higher calcium concentrations, the troponin arm changes its partner from actin to the main body of calcium-loaded troponin. Then, tropomyosin shifts back to the position near the grooves of actin double helix, and the myosin-binding sites of actin becomes available to myosin resulting in force generation through actin-myosin interactions.

Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions.

PubMed

Lombardi, Dario; Dittrich, Petra S

2011-01-01

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug-protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.

Using social outcomes to inform decision-making in schizophrenia: Relationships with symptoms and functioning.

PubMed

Campellone, Timothy R; Fisher, Aaron J; Kring, Ann M

2016-02-01

The outcomes of the decisions we make can be used to inform subsequent choices and behavior. We investigated whether and how people with and without schizophrenia use positive and negative social outcomes and emotional displays to inform decisions to place trust in social partners. We also investigated the impact of reversals in social partners' behavior on decisions to trust. Thirty-two people with schizophrenia and 29 control participants completed a task in which they decided how much trust to place in social partners' showing either a dynamic emotional (smiling, scowling) or neutral display. Interactions were predetermined to result in positive (trust reciprocated) or negative (trust abused) outcomes, and we modeled changes in trust decisions over the course of repeated interactions. Compared to controls, people with schizophrenia were less sensitive to positive social outcomes in that they placed less trust in trustworthy social partners during initial interactions. By contrast, people with schizophrenia were more sensitive to negative social outcomes during initial interactions with untrustworthy social partners, placing less trust in these partners compared to controls. People with schizophrenia did not differ from controls in detecting social partner behavior reversals from trustworthy to untrustworthy; however, they had difficulties detecting reversals from untrustworthy to trustworthy. Importantly, decisions to trust were associated with real-world social functioning. We discuss the implications of these findings for understanding social engagement among people with schizophrenia and the development of psychosocial interventions for social functioning. (c) 2016 APA, all rights reserved).

Validating the Outcome of Partnering on Major Capital Projects

DOT National Transportation Integrated Search

2017-01-27

Research on the mechanics of successful collaborative partnering in roadway construction is relatively scarce; however, there is general agreement that successful partnering can result in better budget and schedule control as well as increase safety ...

Multimedia information intervention and its benefits in partners of the head and neck cancer patients.

PubMed

D'Souza, V; Blouin, E; Zeitouni, A; Muller, K; Allison, P J

2017-07-01

We aimed to investigate the levels of anxiety, depression, satisfaction with information provision and cancer-related knowledge in partners of head and neck cancer (HNC) patients receiving a Multimode Comprehensive Tailored Information Package (MCTIP). A non-randomised, controlled trial was conducted with partners of HNC patients recruited at two academic hospitals in Montreal. The Test participants received the MCTIP, while the Control participants received information in an ad hoc manner. All participants were evaluated using the Hospital Anxiety and Depression Scale (HADS), Satisfaction with Cancer Information Profile and a cancer knowledge questionnaire at baseline, and 3 and 6 months later. Data were analysed using descriptive statistics, t-test and chi-square test, and mixed model analysis to test the impact of the intervention. A total of 31 partners of HNC patients participated in this study and completed all the evaluations. The partners in the Test group experienced significantly lower levels of anxiety (P = 0.001) and depression (P = 0.003) symptoms and were more satisfied (P = 0.002) with cancer information provided than partners in the Control group. Providing tailored information seems to have positive outcomes regarding anxiety, depression, and satisfaction in partners of HNC patients. Larger randomised studies are warranted to validate these effects. © 2016 John Wiley & Sons Ltd.

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

PubMed Central

Schuchardt, Brett J.; Mikles, David C.; Hoang, Lawrence M.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

2014-01-01

YAP2 transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well-documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

CD6 and Linker of Activated T Cells are Potential Interaction Partners for T Cell-Specific Adaptor Protein.

PubMed

Hem, C D; Ekornhol, M; Granum, S; Sundvold-Gjerstad, V; Spurkland, A

2017-02-01

The T cell-specific adaptor protein (TSAd) contains several protein interaction domains, and is merging as a modulator of T cell activation. Several interaction partners for the TSAd proline-rich region and phosphotyrosines have been identified, including the Src and Tec family kinases lymphocyte-specific protein tyrosine kinase and interleukin 2-inducible T cell kinase. Via its Src homology 2 (SH2) domain, TSAd may thus function as a link between these enzymes and other signalling molecules. However, few binding partners to the TSAd SH2 domain in T cells are hitherto known. Through the use of in silico ligand prediction, peptide spot arrays, pull-down and immunoprecipitation experiments, we here report novel interactions between the TSAd SH2 domain and CD6 phosphotyrosine (pTyr) 629 and linker of activated T cells (LAT) pTyr 171 , pTyr 191 and pTyr 226 . © 2016 The Foundation for the Scandinavian Journal of Immunology.

Representations of control and psychological symptoms in couples dealing with cancer: a dyadic-regulation approach.

PubMed

Karademas, Evangelos C; Giannousi, Zoe

2013-01-01

The aim of this study was to examine the relation between illness representations of personal and treatment control and psychological symptoms (i.e. symptoms of anxiety and depression) in 72 married couples dealing with a recently diagnosed cancer. Patients were first-diagnosed with early stage (45.83%) or metastatic cancer (54.17%). Dyadic responses were examined with the actor-partner interdependence model. Also, in order to examine whether patients and spouses' representations of control moderate the relation of their partners' corresponding representations to psychological symptoms, we used the relevant bootstrapping framework developed by Hayes and Matthes [(2009). Computational procedures for probing interactions in OLS and logistic regression: SPSS and SAS implementations. Behavior Research Methods, 41, 924-936]. Patients' symptoms of anxiety and depression were associated with both partners' representations of control. Chi-square difference tests indicated that actor and partner effects were equal. Spouses' symptoms of anxiety and depression were related only to their own representations. Moreover, spouses' representations of personal control moderated the relation of patients' corresponding representations to depressive symptoms, whereas patients' representations of treatment control moderated the relation of their spouses' corresponding representations to both anxiety and depression. Findings suggest that both partners' representations of control are important for adaptation to illness. Moreover, they indicate that dyadic regulation may be equally important to self-regulation as far as adaptation to illness is concerned.