Sample records for a2 domain facilitate

  1. LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3

    DOE PAGES

    Golczak, Marcin; Sears, Avery E.; Kiser, Philip D.; ...

    2014-11-10

    Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. In this paper, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompaniedmore » by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. Finally, these findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.« less

  2. Reframing professional boundaries in healthcare: a systematic review of facilitators and barriers to task reallocation from the domain of medicine to the nursing domain.

    PubMed

    Niezen, Maartje G H; Mathijssen, Jolanda J P

    2014-08-01

    To explore the main facilitators and barriers to task reallocation. One of the innovative approaches to dealing with the anticipated shortage of physicians is to reallocate tasks from the professional domain of medicine to the nursing domain. Various (cost-)effectiveness studies demonstrate that nurse practitioners can deliver as high quality care as physicians and can achieve as good outcomes. However, these studies do not examine what factors may facilitate or hinder such task reallocation. A systematic literature review of PubMed and Web of Knowledge supplemented with a snowball research method. The principles of thematic analysis were followed. The 13 identified relevant papers address a broad spectrum of task reallocation (delegation, substitution and complementary care). Thematic analysis revealed four categories of facilitators and barriers: (1) knowledge and capabilities, (2) professional boundaries, (3) organisational environment, and (4) institutional environment. Introducing nurse practitioners in healthcare requires organisational redesign and the reframing of professional boundaries. Especially the facilitators and barriers in the analytical themes of 'professional boundaries' and 'organisational environment' should be considered when reallocating tasks. If not, these factors might hamper the cost-effectiveness of task reallocation in practice. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Barriers and facilitators to preventing pressure ulcers in nursing home residents: A qualitative analysis informed by the Theoretical Domains Framework.

    PubMed

    Lavallée, Jacqueline F; Gray, Trish A; Dumville, Jo; Cullum, Nicky

    2018-06-01

    Pressure ulcers are areas of localised damage to the skin and underlying tissue; and can cause pain, immobility, and delay recovery, impacting on health-related quality of life. The individuals who are most at risk of developing a pressure ulcer are those who are seriously ill, elderly, have impaired mobility and/or poor nutrition; thus, many nursing home residents are at risk. To understand the context of pressure ulcer prevention in nursing homes and to explore the potential barriers and facilitators to evidence-informed practices. Semi-structured interviews were conducted with nursing home nurses, healthcare assistants and managers, National Health Service community-based wound specialist nurses (known in the UK as tissue viability nurses) and a nurse manager in the North West of England. The interview guide was developed using the Theoretical Domains Framework to explore the barriers and facilitators to pressure ulcer prevention in nursing home residents. Data were analysed using a framework analysis and domains were identified as salient based on their frequency and the potential strength of their impact. 25 participants (nursing home: 2 managers, 7 healthcare assistants, 11 qualified nurses; National Health Service community services: 4 tissue viability nurses, 1 manager) were interviewed. Depending upon the behaviours reported and the context, the same domain could be classified as both a barrier and a facilitator. We identified seven domains as relevant in the prevention of pressure ulcers in nursing home residents mapping to four "barrier" domains and six "facilitator" domains. The four "barrier" domains were knowledge, physical skills, social influences and environmental context and resources and the six "facilitator" domains were interpersonal skills, environmental context and resources, social influences, beliefs about capabilities, beliefs about consequences and social/professional role and identity). Knowledge and insight into these barriers and

  4. Structures of Ca(V) Ca**2+/CaM-IQ Domain Complexes Reveal Binding Modes That Underlie Calcium-Dependent Inactivation And Facilitation

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

    Kim, E.Y.; Rumpf, C.H.; Fujiwara, Y.

    2009-05-20

    Calcium influx drives two opposing voltage-activated calcium channel (Ca{sub V}) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca{sup 2+}/calmodulin (Ca{sup 2+}/CaM) lobes produce CDI and CDF through interactions with the Ca{sub V}{alpha}{sub 1} subunit IQ domain. Curiously, Ca{sup 2+}/CaM lobe modulation polarity appears inverted between Ca{sub V}1s and Ca{sub V}2s. Here, we present crystal structures of Ca{sub V}2.1, Ca{sub V}2.2, and Ca{sub V}2.3 Ca{sup 2+}/CaM-IQ domain complexes. All display binding orientations opposite to Ca{sub V}1.2 with a physical reversal of the CaM lobe positions relative to the IQ {alpha}-helix. Titration calorimetry reveals lobe competition for a high-affinitymore » site common to Ca{sub V}1 and Ca{sub V}2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca{sub V}2 Ca{sup 2+}/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca{sub V} feedback modulation and indicate that Ca{sub V}1 and Ca{sub V}2 IQ domains bear a dedicated CDF site that exchanges Ca{sup 2+}/CaM lobe occupants.« less

  5. Expertise facilitates the transfer of anticipation skill across domains.

    PubMed

    Rosalie, Simon M; Müller, Sean

    2014-02-01

    It is unclear whether perceptual-motor skill transfer is based upon similarity between the learning and transfer domains per identical elements theory, or facilitated by an understanding of underlying principles in accordance with general principle theory. Here, the predictions of identical elements theory, general principle theory, and aspects of a recently proposed model for the transfer of perceptual-motor skill with respect to expertise in the learning and transfer domains are examined. The capabilities of expert karate athletes, near-expert karate athletes, and novices to anticipate and respond to stimulus skills derived from taekwondo and Australian football were investigated in ecologically valid contexts using an in situ temporal occlusion paradigm and complex whole-body perceptual-motor skills. Results indicated that the karate experts and near-experts are as capable of using visual information to anticipate and guide motor skill responses as domain experts and near-experts in the taekwondo transfer domain, but only karate experts could perform like domain experts in the Australian football transfer domain. Findings suggest that transfer of anticipation skill is based upon expertise and an understanding of principles but may be supplemented by similarities that exist between the stimulus and response elements of the learning and transfer domains.

  6. Facilitation of Ferroelectric Switching via Mechanical Manipulation of Hierarchical Nanoscale Domain Structures.

    PubMed

    Chen, Zibin; Hong, Liang; Wang, Feifei; Ringer, Simon P; Chen, Long-Qing; Luo, Haosu; Liao, Xiaozhou

    2017-01-06

    Heterogeneous ferroelastic transition that produces hierarchical 90° tetragonal nanodomains via mechanical loading and its effect on facilitating ferroelectric domain switching in relaxor-based ferroelectrics were explored. Combining in situ electron microscopy characterization and phase-field modeling, we reveal the nature of the transition process and discover that the transition lowers by 40% the electrical loading threshold needed for ferroelectric domain switching. Our results advance the fundamental understanding of ferroelectric domain switching behavior.

  7. Evolution of Src Homology 2 (SH2) Domain to Recognize Sulfotyrosine.

    PubMed

    Ju, Tong; Niu, Wei; Guo, Jiantao

    2016-09-16

    Protein tyrosine O-sulfation is considered as the most common type of post-translational tyrosine modification in nature and plays important roles in extracellular biomolecular interactions. To facilitate the mapping, biological study, and medicinal application of this type of post-translational modification, we seek to evolve a small protein scaffold that recognizes sulfotyrosine with high affinity. We focused our efforts on the engineering of the Src Homology 2 (SH2) domain, which represents the largest class of known phosphotyrosine-recognition domain in nature and has a highly evolvable binding pocket. By using phage display, we successfully engineered the SH2 domain to recognize sulfotyrosine with high affinity. The best mutant, SH2-60.1, displayed more than 1700 fold higher sulfotyrosine-binding affinity than that of the wild-type SH2 domain. We also demonstrated that the evolved SH2 domain mutants could be used to detect sulfoprotein levels on the cell surface. These evolved SH2 domain mutants can be potentially applied to the study of protein tyrosine O-sulfation with proper experimental designs.

  8. The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.

    PubMed

    Sánchez-Bautista, Sonia; Marín-Vicente, Consuelo; Gómez-Fernández, Juan C; Corbalán-García, Senena

    2006-10-06

    The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+ -binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCalpha when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCalpha. These findings entail revision of the currently accepted model of PKCalpha recruitment to the membrane and its activation.

  9. Use of Heuristics to Facilitate Scientific Discovery Learning in a Simulation Learning Environment in a Physics Domain

    ERIC Educational Resources Information Center

    Veermans, Koen; van Joolingen, Wouter; de Jong, Ton

    2006-01-01

    This article describes a study into the role of heuristic support in facilitating discovery learning through simulation-based learning. The study compares the use of two such learning environments in the physics domain of collisions. In one learning environment (implicit heuristics) heuristics are only used to provide the learner with guidance…

  10. Barriers and facilitators to the implementation of a school-based physical activity policy in Canada: application of the theoretical domains framework.

    PubMed

    Weatherson, Katie A; McKay, Rhyann; Gainforth, Heather L; Jung, Mary E

    2017-10-23

    In British Columbia Canada, a Daily Physical Activity (DPA) policy was mandated that requires elementary school teachers to provide students with opportunities to achieve 30 min of physical activity during the school day. However, the implementation of school-based physical activity policies is influenced by many factors. A theoretical examination of the factors that impede and enhance teachers' implementation of physical activity policies is necessary in order to develop strategies to improve policy practice and achieve desired outcomes. This study used the Theoretical Domains Framework (TDF) to understand teachers' barriers and facilitators to the implementation of the DPA policy in one school district. Additionally, barriers and facilitators were examined and compared according to how the teacher implemented the DPA policy during the instructional school day. Interviews were conducted with thirteen teachers and transcribed verbatim. One researcher performed barrier and facilitator extraction, with double extraction occurring across a third of the interview transcripts by a second researcher. A deductive and inductive analytical approach in a two-stage process was employed whereby barriers and facilitators were deductively coded using TDF domains (content analysis) and analyzed for sub-themes within each domain. Two researchers performed coding. A total of 832 items were extracted from the interview transcripts. Some items were coded into multiple TDF domains, resulting in a total of 1422 observations. The most commonly coded TDF domains accounting for 75% of the total were Environmental context and resources (ECR; n = 250), Beliefs about consequences (n = 225), Social influences (n = 193), Knowledge (n = 100), and Intentions (n = 88). Teachers who implemented DPA during instructional time differed from those who relied on non-instructional time in relation to Goals, Behavioural regulation, Social/professional role and identity, Beliefs about

  11. Classification and Lineage Tracing of SH2 Domains Throughout Eukaryotes.

    PubMed

    Liu, Bernard A

    2017-01-01

    Today there exists a rapidly expanding number of sequenced genomes. Cataloging protein interaction domains such as the Src Homology 2 (SH2) domain across these various genomes can be accomplished with ease due to existing algorithms and predictions models. An evolutionary analysis of SH2 domains provides a step towards understanding how SH2 proteins integrated with existing signaling networks to position phosphotyrosine signaling as a crucial driver of robust cellular communication networks in metazoans. However organizing and tracing SH2 domain across organisms and understanding their evolutionary trajectory remains a challenge. This chapter describes several methodologies towards analyzing the evolutionary trajectory of SH2 domains including a global SH2 domain classification system, which facilitates annotation of new SH2 sequences essential for tracing the lineage of SH2 domains throughout eukaryote evolution. This classification utilizes a combination of sequence homology, protein domain architecture and the boundary positions between introns and exons within the SH2 domain or genes encoding these domains. Discrete SH2 families can then be traced across various genomes to provide insight into its origins. Furthermore, additional methods for examining potential mechanisms for divergence of SH2 domains from structural changes to alterations in the protein domain content and genome duplication will be discussed. Therefore a better understanding of SH2 domain evolution may enhance our insight into the emergence of phosphotyrosine signaling and the expansion of protein interaction domains.

  12. Facilitation of learning: part 2.

    PubMed

    Warburton, Tyler; Houghton, Trish; Barry, Debbie

    2016-04-27

    The previous article in this series of 11, Facilitation of learning: part 1, reviewed learning theories and how they relate to clinical practice. Developing an understanding of these theories is essential for mentors and practice teachers to enable them to deliver evidence-based learning support. This is important given that effective learning support is dependent on an educator who possesses knowledge of their specialist area as well as the relevent tools and methods to support learning. The second domain of the Nursing and Midwifery Council's Standards to Support Learning and Assessment in Practice relates to the facilitation of learning. To fulfil this domain, mentors and practice teachers are required to demonstrate their ability to recognise the needs of learners and provide appropriate support to meet those needs. This article expands on some of the discussions from part 1 of this article and considers these from a practical perspective, in addition to introducing some of the tools that can be used to support learning.

  13. Barriers and facilitators for implementing a new screening tool in an emergency department: A qualitative study applying the Theoretical Domains Framework.

    PubMed

    Kirk, Jeanette W; Sivertsen, Ditte M; Petersen, Janne; Nilsen, Per; Petersen, Helle V

    2016-10-01

    The aim was to identify the factors that were perceived as most important as facilitators or barriers to the introduction and intended use of a new tool in the emergency department among nurses and a geriatric team. A high incidence of functional decline after hospitalisation for acute medical illness has been shown in the oldest patients and those who are physically frail. In Denmark, more than 35% of older medical patients acutely admitted to the emergency department are readmitted within 90 days after discharge. A new screening tool for use in the emergency department aiming to identify patients at particularly high risk of functional decline and readmission was developed. Qualitative study based on semistructured interviews with nurses and a geriatric team in the emergency department and semistructured single interviews with their managers. The Theoretical Domains Framework guided data collection and analysis. Content analysis was performed whereby new themes and themes already existing within each domain were described. Six predominant domains were identified: (1) professional role and identity; (2) beliefs about consequences; (3) goals; (4) knowledge; (5) optimism and (6) environmental context and resources. The content analysis identified three themes, each containing two subthemes. The themes were professional role and identity, beliefs about consequences and preconditions for a successful implementation. Two different cultures were identified in the emergency department. These cultures applied to different professional roles and identity, different actions and sense making and identified how barriers and facilitators linked to the new screening tool were perceived. The results show that different cultures exist in the same local context and influence the perception of barriers and facilitators differently. These cultures must be identified and addressed when implementation is planned. © 2016 The Authors. Journal of Clinical Nursing Published by John

  14. The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

    PubMed Central

    Yang, Yinshan; Labesse, Gilles; Carrère-Kremer, Séverine; Esteves, Kevin; Kremer, Laurent

    2012-01-01

    MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg2+ deprivation, but previous work suggested that MgtC is not a Mg2+ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg2+ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg2+ concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg2+ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain. PMID:22984256

  15. Identification of a second binding site on the TRIM25 B30.2 domain.

    PubMed

    D'Cruz, Akshay A; Kershaw, Nadia J; Hayman, Thomas J; Linossi, Edmond M; Chiang, Jessica J; Wang, May K; Dagley, Laura F; Kolesnik, Tatiana B; Zhang, Jian-Guo; Masters, Seth L; Griffin, Michael D W; Gack, Michaela U; Murphy, James M; Nicola, Nicos A; Babon, Jeffrey J; Nicholson, Sandra E

    2018-01-23

    The r etinoic acid- i nducible g ene- I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tri partite m otif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 ( c aspase a ctivation and r ecruitment d omains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  16. Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

    PubMed

    Yan, Jin; Leal, Karina; Magupalli, Venkat G; Nanou, Evanthia; Martinez, Gilbert Q; Scheuer, Todd; Catterall, William A

    2014-11-01

    Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

  17. The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

    PubMed

    Su'etsugu, Masayuki; Harada, Yuji; Keyamura, Kenji; Matsunaga, Chika; Kasho, Kazutoshi; Abe, Yoshito; Ueda, Tadashi; Katayama, Tsutomu

    2013-12-01

    DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  18. MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

    PubMed

    Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

    2013-05-01

    The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

  19. Barriers and facilitators to hospital pharmacists' engagement in medication safety activities: a qualitative study using the theoretical domains framework.

    PubMed

    Mekonnen, Alemayehu B; McLachlan, Andrew J; Brien, Jo-Anne E; Mekonnen, Desalew; Abay, Zenahebezu

    2018-01-01

    Hospital pharmacists play a central role in medication safety activities. However, in Ethiopia, this role has been launched recently and little is known regarding the current status of this extended service. Using the Theoretical Domains Framework (TDF), we aimed to identify the barriers and facilitators to hospital pharmacists' engagement in medication safety activities across various public hospitals in the Amhara region of Ethiopia. Eight focus group discussions, using an interview guide that was drawn upon the TDF, were conducted with 44 hospital pharmacists to explore their beliefs regarding their involvement in clinical services. Group discussions were audio-recorded, transcribed verbatim, and analysed using directed content analysis based on the TDF. Relevant domains were identified by applying relevance criteria to each of the domains in the TDF. Content analysis revealed six domains that influence hospital pharmacists' engagement in medication safety activities. These domains included 'Knowledge', 'Skills', 'Environmental context and resources', 'Motivations and goals', 'Social influences' and 'Social/professional role'. Most hospital pharmacists believed knowledge gap was an issue, as was the lack of training and supportive skills although some expressed as they were competent enough for their skills in identifying medication related problems. Most participants were very much enthusiastic for their extended roles and were positive towards the future of the profession; however, competing priorities along with the lack of remuneration and awareness (of other health care professionals) regarding the profession's role were barriers to service delivery. There were also a number of resource constraints, such as staffing, infrastructure and government funding, and acceptance rate of pharmacist's recommendation that were likely to influence the clinical practice of pharmacists. Using the TDF , this study identified a wide range of barriers and facilitators to

  20. PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

    PubMed

    Gunawardana, Dilantha

    2016-01-01

    Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes.

  1. An Efficient Semi-supervised Learning Approach to Predict SH2 Domain Mediated Interactions.

    PubMed

    Kundu, Kousik; Backofen, Rolf

    2017-01-01

    Src homology 2 (SH2) domain is an important subclass of modular protein domains that plays an indispensable role in several biological processes in eukaryotes. SH2 domains specifically bind to the phosphotyrosine residue of their binding peptides to facilitate various molecular functions. For determining the subtle binding specificities of SH2 domains, it is very important to understand the intriguing mechanisms by which these domains recognize their target peptides in a complex cellular environment. There are several attempts have been made to predict SH2-peptide interactions using high-throughput data. However, these high-throughput data are often affected by a low signal to noise ratio. Furthermore, the prediction methods have several additional shortcomings, such as linearity problem, high computational complexity, etc. Thus, computational identification of SH2-peptide interactions using high-throughput data remains challenging. Here, we propose a machine learning approach based on an efficient semi-supervised learning technique for the prediction of 51 SH2 domain mediated interactions in the human proteome. In our study, we have successfully employed several strategies to tackle the major problems in computational identification of SH2-peptide interactions.

  2. Domain Architecture of the Catalytic Subunit in the ISW2-Nucleosome Complex▿

    PubMed Central

    Dang, Weiwei; Bartholomew, Blaine

    2007-01-01

    ATP-dependent chromatin remodeling has an important role in the regulation of cellular differentiation and development. For the first time, a topological view of one of these complexes has been revealed, by mapping the interactions of the catalytic subunit Isw2 with nucleosomal and extranucleosomal DNA in the complex with all four subunits of ISW2 bound to nucleosomes. Different domains of Isw2 were shown to interact with the nucleosome near the dyad axis, another near the entry site of the nucleosome, and another with extranucleosomal DNA. The conserved DEXD or ATPase domain was found to contact the superhelical location 2 (SHL2) of the nucleosome, providing a direct physical connection of ATP hydrolysis with this region of nucleosomes. The C terminus of Isw2, comprising the SLIDE (SANT-like domain) and HAND domains, was found to be associated with extranucleosomal DNA and the entry site of nucleosomes. It is thus proposed that the C-terminal domains of Isw2 are involved in anchoring the complex to nucleosomes through their interactions with linker DNA and that they facilitate the movement of DNA along the surface of nucleosomes. PMID:17908792

  3. The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

    PubMed

    Ferrao, Ryan; Lupardus, Patrick J

    2017-01-01

    The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

  4. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9)

    PubMed Central

    Fu, Qinqin; Yuan, Y. Adam

    2013-01-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA. PMID:23361462

  5. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    PubMed

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  6. Systematic characterization of the specificity of the SH2 domains of cytoplasmic tyrosine kinases.

    PubMed

    Zhao, Bing; Tan, Pauline H; Li, Shawn S C; Pei, Dehua

    2013-04-09

    Cytoplasmic tyrosine kinases (CTK) generally contain a Src-homology 2 (SH2) domain, whose role in the CTK family is not fully understood. Here we report the determination of the specificity of 25 CTK SH2 domains by screening one-bead-one-compound (OBOC) peptide libraries. Based on the peptide sequences selected by the SH2 domains, we built Support Vector Machine (SVM) models for the prediction of binding ligands for the SH2 domains. These models yielded support for the progressive phosphorylation model for CTKs in which the overlapping specificity of the CTK SH2 and kinase domains has been proposed to facilitate targeting of the CTK substrates with at least two potential phosphotyrosine (pTyr) sites. We curated 93 CTK substrates with at least two pTyr sites catalyzed by the same CTK, and showed that 71% of these substrates had at least two pTyr sites predicted to bind a common CTK SH2 domain. More importantly, we found 34 instances where there was at least one pTyr site predicted to be recognized by the SH2 domain of the same CTK, suggesting that the SH2 and kinase domains of the CTKs may cooperate to achieve progressive phosphorylation of a protein substrate. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Voltage-sensing phosphatase modulation by a C2 domain.

    PubMed

    Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

    2015-01-01

    The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  8. Nucleosome mobilization by ISW2 requires the concerted action of the ATPase and SLIDE domains

    PubMed Central

    Hota, Swetansu K.; Bhardwaj, Saurabh K.; Deindl, Sebastian; Lin, Yuan-chi; Zhuang, Xiaowei; Bartholomew, Blaine

    2013-01-01

    The ISWI family of ATP-dependent chromatin remodelers represses transcription by changing nucleosome positioning. The interactions with extranucleosomal DNA and the requirement of a minimal length of extranucleosomal DNA by ISWI mediate the spacing of nucleosomes. ISW2 from Saccharomyces cerevisiae, a member of the ISWI family, has a conserved domain called SLIDE (SANT-like ISWI domain), whose binding to extranucleosomal DNA ~19 bp from the edge of nucleosomes is required for efficiently pushing DNA into nucleosomes and maintaining the unidirectional movement of nucleosomes, as reported here. Loss of SLIDE binding does not perturb ATPase domain binding to the SHL2 site of nucleosomes or its initial movement of DNA inside of nucleosomes. ISW2 has therefore two distinct roles in mobilizing nucleosomes, with the ATPase domain translocating and moving DNA inside nucleosomes, and the SLIDE domain facilitating the entry of linker DNA into nucleosomes. PMID:23334290

  9. Voltage-sensing phosphatase modulation by a C2 domain

    PubMed Central

    Castle, Paul M.; Zolman, Kevin D.; Kohout, Susy C.

    2015-01-01

    The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

  10. The Robustness of a Signaling Complex to Domain Rearrangements Facilitates Network Evolution

    PubMed Central

    Sato, Paloma M.; Yoganathan, Kogulan; Jung, Jae H.; Peisajovich, Sergio G.

    2014-01-01

    The rearrangement of protein domains is known to have key roles in the evolution of signaling networks and, consequently, is a major tool used to synthetically rewire networks. However, natural mutational events leading to the creation of proteins with novel domain combinations, such as in frame fusions followed by domain loss, retrotranspositions, or translocations, to name a few, often simultaneously replace pre-existing genes. Thus, while proteins with new domain combinations may establish novel network connections, it is not clear how the concomitant deletions are tolerated. We investigated the mechanisms that enable signaling networks to tolerate domain rearrangement-mediated gene replacements. Using as a model system the yeast mitogen activated protein kinase (MAPK)-mediated mating pathway, we analyzed 92 domain-rearrangement events affecting 11 genes. Our results indicate that, while domain rearrangement events that result in the loss of catalytic activities within the signaling complex are not tolerated, domain rearrangements can drastically alter protein interactions without impairing function. This suggests that signaling complexes can maintain function even when some components are recruited to alternative sites within the complex. Furthermore, we also found that the ability of the complex to tolerate changes in interaction partners does not depend on long disordered linkers that often connect domains. Taken together, our results suggest that some signaling complexes are dynamic ensembles with loose spatial constraints that could be easily re-shaped by evolution and, therefore, are ideal targets for cellular engineering. PMID:25490747

  11. The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

    PubMed

    Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

    2014-10-01

    JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain.

    PubMed

    Curto, M-Ángeles; Moro, Sandra; Yanguas, Francisco; Gutiérrez-González, Carmen; Valdivieso, M-Henar

    2018-05-01

    Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8-10 aa)C signature motif. Structure-function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

  13. RHOMBOID DOMAIN CONTAINING 2 (RHBDD2)

    PubMed Central

    Abba, MC; Lacunza, E; Nunez, MI; Colussi, A; Isla-Larrain, M; Segal-Eiras, A; Croce, MV; Aldaz, CM

    2009-01-01

    In the course of breast cancer global gene expression studies, we identified an uncharacterized gene known as RHBDD2 (Rhomboid domain containing 2) to be markedly over-expressed in primary tumors from patients with recurrent disease. In this study, we identified RHBDD2 mRNA and protein expression significantly elevated in breast carcinomas compared with normal breast samples as analyzed by SAGE (n=46) and immunohistochemistry (n=213). Interestingly, specimens displaying RHBDD2 over-expression were predominantly advanced stage III breast carcinomas (p=0.001). Western-blot, RT-PCR and cDNA sequencing analyses allowed us to identify two RHBDD2 alternatively spliced mRNA isoforms expressed in breast cancer cell lines. We further investigated the occurrence and frequency of gene amplification and over-expression affecting RHBDD2 in 131 breast samples. RHBDD2 gene amplification was detected in 21% of 98 invasive breast carcinomas analyzed. However, no RHBDD2 amplification was detected in normal breast tissues (n=17) or breast benign lesions (n=16) (p=0.014). Interestingly, siRNA mediated silencing of RHBDD2 expression results in a decrease of MCF7 breast cancer cells proliferation compared with the corresponding controls (p=0.001). In addition, analysis of publicly available gene expression data showed a strong association between high RHBDD2 expression and decreased overall survival (p=0.0023), relapse-free survival (p= 0.0013), and metastasis-free interval (p=0.006) in patients with primary ER-negative breast carcinomas. In conclusion, our findings suggest that RHBDD2 over-expression behaves as an indicator of poor prognosis and may play a role facilitating breast cancer progression. PMID:19616622

  14. The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

    PubMed Central

    Budhidarmo, Rhesa; Day, Catherine L.

    2014-01-01

    The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

  15. A role of the SAM domain in EphA2 receptor activation.

    PubMed

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W

    2017-03-24

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

  16. A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT

    PubMed Central

    Mao, Peng; Kyriss, McKenna N. M.; Hodges, Amelia J.; Duan, Mingrui; Morris, Robert T.; Lavine, Mark D.; Topping, Traci B.; Gloss, Lisa M.; Wyrick, John J.

    2016-01-01

    Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A–H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A–H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A–H2B deposition by FACT. PMID:27369377

  17. Using theory to explore facilitators and barriers to delayed prescribing in Australia: a qualitative study using the Theoretical Domains Framework and the Behaviour Change Wheel.

    PubMed

    Sargent, Lucy; McCullough, Amanda; Del Mar, Chris; Lowe, John

    2017-02-13

    Delayed antibiotic prescribing reduces antibiotic use for acute respiratory infections in trials in general practice, but the uptake in clinical practice is low. The aim of the study was to identify facilitators and barriers to general practitioners' (GPs') use of delayed prescribing and to gain pharmacists' and the public's views about delayed prescribing in Australia. This study used the Theoretical Domains Framework and the Behaviour Change Wheel to explore facilitators and barriers to delayed prescribing in Australia. Forty-three semi-structured, face-to-face interviews with general practitioners, pharmacists and patients were conducted. Responses were coded into domains of the Theoretical Domains Framework, and specific criteria from the Behaviour Change Wheel were used to identify which domains were relevant to increasing the use of delayed prescribing by GPs. The interviews revealed nine key domains that influence GPs' use of delayed prescribing: knowledge; cognitive and interpersonal skills; memory, attention and decision-making processes; optimism; beliefs about consequences; intentions; goals; emotion; and social influences: GPs knew about delayed prescribing; however, they did not use it consistently, preferring to bring patients back for review and only using it with patients in a highly selective way. Pharmacists would support GPs and the public in delayed prescribing but would fill the prescription if people insisted. The public said they would delay taking their antibiotics if asked by their GP and given the right information on managing symptoms and when to take antibiotics. Using a theory-driven approach, we identified nine key domains that influence GPs' willingness to provide a delayed prescription to patients with an acute respiratory infection presenting to general practice. These data can be used to develop a structured intervention to change this behaviour and thus reduce antibiotic use for acute respiratory infections in general practice.

  18. An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry

    PubMed Central

    Hochrein, James M.; Lerner, Edwina C.; Schiavone, Anthony P.; Smithgall, Thomas E.; Engen, John R.

    2006-01-01

    The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains. PMID:16322569

  19. Ubiquitin Interacts with the Tollip C2 and CUE Domains and Inhibits Binding of Tollip to Phosphoinositides*

    PubMed Central

    Mitra, Sharmistha; Traughber, C. Alicia; Brannon, Mary K.; Gomez, Stephanie; Capelluto, Daniel G. S.

    2013-01-01

    A large number of cellular signaling processes are directed through internalization, via endocytosis, of polyubiquitinated cargo proteins. Tollip is an adaptor protein that facilitates endosomal cargo sorting for lysosomal degradation. Tollip preferentially binds phosphatidylinositol 3-phosphate (PtdIns(3)P) via its C2 domain, an association that may be required for endosomal membrane targeting. Here, we show that Tollip binds ubiquitin through its C2 and CUE domains and that its association with the C2 domain inhibits PtdIns(3)P binding. NMR analysis demonstrates that the C2 and CUE domains bind to overlapping sites on ubiquitin, suggesting that two ubiquitin molecules associate with Tollip simultaneously. Hydrodynamic studies reveal that ubiquitin forms heterodimers with the CUE domain, indicating that the association disrupts the dimeric state of the CUE domain. We propose that, in the absence of polyubiquitinated cargo, the dual binding of ubiquitin partitions Tollip into membrane-bound and membrane-free states, a function that contributes to the engagement of Tollip in both membrane trafficking and cytosolic pathways. PMID:23880770

  20. Modeling of leachate generation from MSW landfills by a 2-dimensional 2-domain approach

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

    Fellner, Johann, E-mail: j.fellner@tuwien.ac.a; Brunner, Paul H., E-mail: paul.h.brunner@tuwien.ac.a

    2010-11-15

    The flow of water through Municipal Solid Waste (MSW) landfills is highly non-uniform and dominated by preferential pathways. Thus, concepts to simulate landfill behavior require that a heterogeneous flow regime is considered. Recent models are based on a 2-domain approach, differentiating between channel domain with high hydraulic conductivity, and matrix domain of slow water movement with high water retention capacity. These models focus on the mathematical description of rapid water flow in channel domain. The present paper highlights the importance of water exchange between the two domains, and expands the 1-dimensional, 2-domain flow model by taking into account water flowsmore » in two dimensions. A flow field consisting of a vertical path (channel domain) surrounded by the waste mass (matrix domain) is defined using the software HYDRUS-2D. When the new model is calibrated using data sets from a MSW-landfill site the predicted leachate generation corresponds well with the observed leachate discharge. An overall model efficiency in terms of r{sup 2} of 0.76 was determined for a simulation period of almost 4 years. The results confirm that water in landfills follows a preferential path way characterized by high permeability (K{sub s} = 300 m/d) and zero retention capacity, while the bulk of the landfill (matrix domain) is characterized by low permeability (K{sub s} = 0.1 m/d) and high retention capacity. The most sensitive parameters of the model are the hydraulic conductivities of the channel domain and the matrix domain, and the anisotropy of the matrix domain.« less

  1. The Habc Domain of the SNARE Vam3 Interacts with the HOPS Tethering Complex to Facilitate Vacuole Fusion*

    PubMed Central

    Lürick, Anna; Kuhlee, Anne; Bröcker, Cornelia; Kümmel, Daniel; Raunser, Stefan; Ungermann, Christian

    2015-01-01

    Membrane fusion at vacuoles requires a consecutive action of the HOPS tethering complex, which is recruited by the Rab GTPase Ypt7, and vacuolar SNAREs to drive membrane fusion. It is assumed that the Sec1/Munc18-like Vps33 within the HOPS complex is largely responsible for SNARE chaperoning. Here, we present direct evidence for HOPS binding to SNAREs and the Habc domain of the Vam3 SNARE protein, which may explain its function during fusion. We show that HOPS interacts strongly with the Vam3 Habc domain, assembled Q-SNAREs, and the R-SNARE Ykt6, but not the Q-SNARE Vti1 or the Vam3 SNARE domain. Electron microscopy combined with Nanogold labeling reveals that the binding sites for vacuolar SNAREs and the Habc domain are located in the large head of the HOPS complex, where Vps16 and Vps33 have been identified before. Competition experiments suggest that HOPS bound to the Habc domain can still interact with assembled Q-SNAREs, whereas Q-SNARE binding prevents recognition of the Habc domain. In agreement, membranes carrying Vam3ΔHabc fuse poorly unless an excess of HOPS is provided. These data suggest that the Habc domain of Vam3 facilitates the assembly of the HOPS/SNARE machinery at fusion sites and thus supports efficient membrane fusion. PMID:25564619

  2. BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

    PubMed

    Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2017-10-03

    DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM -knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

  3. BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis

    PubMed Central

    Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2017-01-01

    DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors. PMID:28923951

  4. SH2 Domain Histochemistry.

    PubMed

    Buhs, Sophia; Nollau, Peter

    2017-01-01

    Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.

  5. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from particular...

  6. Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance

    PubMed Central

    Barber-Zucker, Shiran; Uebe, René; Davidov, Geula; Navon, Yotam; Sherf, Dror; Chill, Jordan H.; Kass, Itamar; Bitton, Ronit; Schüler, Dirk; Zarivach, Raz

    2016-01-01

    Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins’ proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human. PMID:27550551

  7. The SH2 domain interaction landscape.

    PubMed

    Tinti, Michele; Kiemer, Lars; Costa, Stefano; Miller, Martin L; Sacco, Francesca; Olsen, Jesper V; Carducci, Martina; Paoluzi, Serena; Langone, Francesca; Workman, Christopher T; Blom, Nikolaj; Machida, Kazuya; Thompson, Christopher M; Schutkowski, Mike; Brunak, Søren; Mann, Matthias; Mayer, Bruce J; Castagnoli, Luisa; Cesareni, Gianni

    2013-04-25

    Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  8. CTLs, a new class of RING-H2 ubiquitin ligases uncovered by YEELL, a motif close to the RING domain that is present across eukaryotes.

    PubMed

    Jiménez-López, Domingo; Aguilar-Henonin, Laura; González-Prieto, Juan Manuel; Aguilar-Hernández, Victor; Guzmán, Plinio

    2018-01-01

    RING ubiquitin E3 ligases enclose a RING domain for ubiquitin ligase activity and associated domains and/or conserved motifs outside the RING domain that collectively facilitate their classification and usually reveal some of key information related to mechanism of action. Here we describe a new family of E3 ligases that encodes a RING-H2 domain related in sequence to the ATL and BTL RING-H2 domains. This family, named CTL, encodes a motif designed as YEELL that expands 21 amino acids next to the RING-H2 domain that is present across most eukaryotic lineages. E3 ubiquitin ligase BIG BROTHER is a plant CTL that regulates organ size, and SUMO-targeted ubiquitin E3 ligase RNF111/ARKADIA is a vertebrate CTL. Basal animal and vertebrate, as well as fungi species, encode a single CTL gene that constraints the number of paralogs observed in vertebrates. Conversely, as previously described in ATL and BTL families in plants, CTL genes range from a single copy in green algae and 3 to 5 copies in basal species to 9 to 35 copies in angiosperms. Our analysis describes key structural features of a novel family of E3 ubiquitin ligases as an integral component of the set of core eukaryotic genes.

  9. CTLs, a new class of RING-H2 ubiquitin ligases uncovered by YEELL, a motif close to the RING domain that is present across eukaryotes

    PubMed Central

    Jiménez-López, Domingo; Aguilar-Henonin, Laura; González-Prieto, Juan Manuel; Aguilar-Hernández, Victor

    2018-01-01

    RING ubiquitin E3 ligases enclose a RING domain for ubiquitin ligase activity and associated domains and/or conserved motifs outside the RING domain that collectively facilitate their classification and usually reveal some of key information related to mechanism of action. Here we describe a new family of E3 ligases that encodes a RING-H2 domain related in sequence to the ATL and BTL RING-H2 domains. This family, named CTL, encodes a motif designed as YEELL that expands 21 amino acids next to the RING-H2 domain that is present across most eukaryotic lineages. E3 ubiquitin ligase BIG BROTHER is a plant CTL that regulates organ size, and SUMO-targeted ubiquitin E3 ligase RNF111/ARKADIA is a vertebrate CTL. Basal animal and vertebrate, as well as fungi species, encode a single CTL gene that constraints the number of paralogs observed in vertebrates. Conversely, as previously described in ATL and BTL families in plants, CTL genes range from a single copy in green algae and 3 to 5 copies in basal species to 9 to 35 copies in angiosperms. Our analysis describes key structural features of a novel family of E3 ubiquitin ligases as an integral component of the set of core eukaryotic genes. PMID:29324855

  10. Functional analysis of the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1

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

    Yoon, Jung Hwan; Choi, Yoo Jin; Choi, Won Suk

    2013-11-01

    Highlights: •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited tumor cell growth. •NH{sub 2}-terminal and BRICHOS domain of GKN1 regulated cell cycle. •NH{sub 2}-terminal and BRICHOS domain of GKN1 inhibited epigenetic regulators. -- Abstract: Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1{sup D13N}, pGKN1{sup Δ68–199}, and pGKN1{sup Δ1–67,165–199} weremore » shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH{sub 2}-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities.« less

  11. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold*

    PubMed Central

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn; Agacan, Mark; Tempel, Wolfram; Tong, Yufeng; Nedyalkova, Lyudmila; Park, Hee-Won; Leslie, Nicholas R.; van Aalten, Daan M. F.; Downes, C. Peter; Batty, Ian H.

    2012-01-01

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105–107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules. PMID:22493426

  12. IQGAP Proteins Reveal an Atypical Phosphoinositide (aPI) Binding Domain with a Pseudo C2 Domain Fold

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

    Dixon, Miles J.; Gray, Alexander; Schenning, Martijn

    2012-10-16

    Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3). The binding affinity for PtdInsP3, together with other, secondary target-recognition characteristics, are comparable with those ofmore » the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP3 effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.« less

  13. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity of the C2 Domains of Factor V and Factor VIII

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

    Lin,L.; Huai, Q.; Huang, M.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 Angstroms. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C? atoms of 0.9 Angstroms and 1.2 Angstroms, and sequence identities of 43% and 38%, respectively). The lactadherin C2more » domain has a discoidin-like fold containing two ?-sheets of five and three antiparallel ?-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One ?-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain ?-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.« less

  14. Crystal Structure of the Bovine lactadherin C2 Domain, a Membrane Binding Motif, Shows Similarity to the C2 Domains of Factor V and Factor VIII

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

    Lin,L.

    2007-01-01

    Lactadherin, a glycoprotein secreted by a variety of cell types, contains two EGF domains and two C domains with sequence homology to the C domains of blood coagulation proteins factor V and factor VIII. Like these proteins, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity. We determined the crystal structure of the bovine lactadherin C2 domain (residues 1 to 158) at 2.4 {angstrom}. The lactadherin C2 structure is similar to the C2 domains of factors V and VIII (rmsd of C{sub {alpha}} atoms of 0.9 {angstrom} and 1.2 {angstrom}, and sequence identities of 43% and 38%, respectively). The lactadherinmore » C2 domain has a discoidin-like fold containing two {beta}-sheets of five and three antiparallel {beta}-strands packed against one another. The N and C termini are linked by a disulfide bridge between Cys1 and Cys158. One {beta}-turn and two loops containing solvent-exposed hydrophobic residues extend from the C2 domain {beta}-sandwich core. In analogy with the C2 domains of factors V and VIII, some or all of these solvent-exposed hydrophobic residues, Trp26, Leu28, Phe31, and Phe81, likely participate in membrane binding. The C2 domain of lactadherin may serve as a marker of cell surface phosphatidylserine exposure and may have potential as a unique anti-thrombotic agent.« less

  15. What factors influence uptake of retinal screening among young adults with type 2 diabetes? A qualitative study informed by the theoretical domains framework.

    PubMed

    Lake, Amelia J; Browne, Jessica L; Rees, Gwyneth; Speight, Jane

    2017-06-01

    Young adults with type 2 diabetes (T2D, 18-39years) face increased risk of vision loss from diabetic retinopathy (DR). Retinal screening is essential to detect DR, yet screening rates for this group are low and little is known about the underlying factors influencing this important behavior. Using the theoretical domains framework (TDF) to guide data collection and analysis, we explored screening barriers and facilitator, contrasting them with a comparator group of older adults with T2D (40+ years). Thirty semi-structured telephone interviews (10 younger, 20 older adults) were conducted. Data were coded into TDF domains with salience identified by "frequency" of reference. Screening facilitators and barriers were systematically compared between groups. Although many screening facilitators and barriers were shared by younger and older adults, additional factors highly relevant to the former included: social comparison with others ('social influences'); concern for the impact on the family unit, unrealistic optimism and perceived invulnerability ('beliefs about consequences'); lack of time and financial resources ('environmental context and resources'), and DR misconceptions ('knowledge'). This study demonstrated that young adult retinal screening behavior was influenced by additional social cognitive factors compared to older adults, providing a first-step evidence base for clinicians and other health professionals, and potential targets for future eye health and retinal screening interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.

    PubMed

    Seo, Min-Duk; Park, Sung Jean; Kim, Hyun-Jung; Lee, Bong Jin

    2007-01-09

    Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.

  17. RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

    PubMed

    Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

    2011-06-24

    DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

  18. The SAM domain inhibits EphA2 interactions in the plasma membrane.

    PubMed

    Singh, Deo R; Ahmed, Fozia; Paul, Michael D; Gedam, Manasee; Pasquale, Elena B; Hristova, Kalina

    2017-01-01

    All members of the Eph receptor family of tyrosine kinases contain a SAM domain near the C terminus, which has been proposed to play a role in receptor homotypic interactions and/or interactions with binding partners. The SAM domain of EphA2 is known to be important for receptor function, but its contribution to EphA2 lateral interactions in the plasma membrane has not been determined. Here we use a FRET-based approach to directly measure the effect of the SAM domain on the stability of EphA2 dimers on the cell surface in the absence of ligand binding. We also investigate the functional consequences of EphA2 SAM domain deletion. Surprisingly, we find that the EphA2 SAM domain inhibits receptor dimerization and decreases EphA2 tyrosine phosphorylation. This role is dramatically different from the role of the SAM domain of the related EphA3 receptor, which we previously found to stabilize EphA3 dimers and increase EphA3 tyrosine phosphorylation in cells in the absence of ligand. Thus, the EphA2 SAM domain likely contributes to a unique mode of EphA2 interaction that leads to distinct signaling outputs. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

    PubMed

    Garcia, P; Shoelson, S E; Drew, J S; Miller, W T

    1994-12-02

    Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

  20. Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein.

    PubMed

    Wang, Hongpeng; Zhang, Yan; Zhang, Zhenyi; Jin, Wei Lin; Wu, Geng

    2014-01-01

    Bin-Amphiphysin-Rvs (BAR) domain proteins play essential roles in diverse cellular processes by inducing membrane invaginations or membrane protrusions. Among the BAR superfamily, the `classical' BAR and Fes/CIP4 homology BAR (F-BAR) subfamilies of proteins usually promote membrane invaginations, whereas the inverse BAR (I-BAR) subfamily generally incur membrane protrusions. Despite possessing an N-terminal F-BAR domain, the srGAP2 protein regulates neurite outgrowth and neuronal migration by causing membrane protrusions reminiscent of the activity of I-BAR domain proteins. In this study, the inverse F-BAR (IF-BAR) domain of human srGAP2 was overexpressed, purified and crystallized. The crystals of the srGAP2 IF-BAR domain protein diffracted to 3.50 Å resolution and belonged to space group P2(1). These results will facilitate further structural determination of the srGAP2 IF-BAR domain and the ultimate elucidation of its peculiar behaviour of inducing membrane protrusions rather than membrane invaginations.

  1. An assembly of proteins and lipid domains regulates transport of phosphatidylserine to phosphatidylserine decarboxylase 2 in Saccharomyces cerevisiae.

    PubMed

    Riekhof, Wayne R; Wu, Wen-I; Jones, Jennifer L; Nikrad, Mrinalini; Chan, Mallory M; Loewen, Christopher J R; Voelker, Dennis R

    2014-02-28

    Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.

  2. SH2 domains: modulators of nonreceptor tyrosine kinase activity.

    PubMed

    Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

    2009-12-01

    The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

  3. Expression and Production of SH2 Domain Proteins.

    PubMed

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  4. Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1.

    PubMed

    Oide, Mao; Okajima, Koji; Kashojiya, Sachiko; Takayama, Yuki; Oroguchi, Tomotaka; Hikima, Takaaki; Yamamoto, Masaki; Nakasako, Masayoshi

    2016-09-16

    Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Simultaneous Binding of Two Peptidyl Ligands by a Src Homology 2 Domain

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

    Zhang, Yanyan; Zhang, Jinjin; Yuan, Chunhua

    Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing phosphotyrosine (pY)-containing sequences of target proteins. In all of the SH2 domain-pY peptide interactions described to date, the SH2 domain binds to a single pY peptide. Here, determination of the cocrystal structure of the N-terminal SH2 domain of phosphatase SHP-2 bound to a class IV peptide (VIpYFVP) revealed a noncanonical 1:2 (protein-peptide) complex. The first peptide binds in a canonical manner with its pY side chain inserted in the usual binding pocket, while the second pairs up with the first to form two antiparallel {beta}-strands that extend the central {beta}-sheetmore » of the SH2 domain. This unprecedented binding mode was confirmed in the solution phase by NMR experiments and shown to be adopted by pY peptides derived from cellular proteins. Site-directed mutagenesis and surface plasmon resonance studies revealed that the binding of the first peptide is pY-dependent, but phosphorylation is not required for the second peptide. Our findings suggest a potential new function for the SH2 domain as a molecular clamp to promote dimerization of signaling proteins.« less

  6. Domain engineering of the metastable domains in the 4f-uniaxial-ferromagnet CeRu2Ga2B

    NASA Astrophysics Data System (ADS)

    Wulferding, D.; Kim, H.; Yang, I.; Jeong, J.; Barros, K.; Kato, Y.; Martin, I.; Ayala-Valenzuela, O. E.; Lee, M.; Choi, H. C.; Ronning, F.; Civale, L.; Baumbach, R. E.; Bauer, E. D.; Thompson, J. D.; Movshovich, R.; Kim, Jeehoon

    2017-04-01

    In search of novel, improved materials for magnetic data storage and spintronic devices, compounds that allow a tailoring of magnetic domain shapes and sizes are essential. Good candidates are materials with intrinsic anisotropies or competing interactions, as they are prone to host various domain phases that can be easily and precisely selected by external tuning parameters such as temperature and magnetic field. Here, we utilize vector magnetic fields to visualize directly the magnetic anisotropy in the uniaxial ferromagnet CeRu2Ga2B. We demonstrate a feasible control both globally and locally of domain shapes and sizes by the external field as well as a smooth transition from single stripe to bubble domains, which opens the door to future applications based on magnetic domain tailoring.

  7. Domain engineering of the metastable domains in the 4f-uniaxial-ferromagnet CeRu 2Ga 2B

    DOE PAGES

    Wulferding, Dirk; Kim, Hoon; Yang, Ilkyu; ...

    2017-04-10

    In search of novel, improved materials for magnetic data storage and spintronic devices, compounds that allow a tailoring of magnetic domain shapes and sizes are essential. Good candidates are materials with intrinsic anisotropies or competing interactions, as they are prone to host various domain phases that can be easily and precisely selected by external tuning parameters such as temperature and magnetic field. Here, we utilize vector magnetic fields to visualize directly the magnetic anisotropy in the uniaxial ferromagnet CeRu 2Ga 2B. We demonstrate a feasible control both globally and locally of domain shapes and sizes by the external field asmore » well as a smooth transition from single stripe to bubble domains, which opens the door to future applications based on magnetic domain tailoring.« less

  8. The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization*

    PubMed Central

    Leslie, Patrick L.; Ke, Hengming; Zhang, Yanping

    2015-01-01

    The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization. PMID:25809483

  9. The MDM2 RING domain and central acidic domain play distinct roles in MDM2 protein homodimerization and MDM2-MDMX protein heterodimerization.

    PubMed

    Leslie, Patrick L; Ke, Hengming; Zhang, Yanping

    2015-05-15

    The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

    PubMed

    Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

    1998-11-01

    The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

  11. Progress towards the development of SH2 domain inhibitors.

    PubMed

    Kraskouskaya, Dziyana; Duodu, Eugenia; Arpin, Carolynn C; Gunning, Patrick T

    2013-04-21

    Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

  12. Barriers and facilitators towards implementing the Sepsis Six care bundle (BLISS-1): a mixed methods investigation using the theoretical domains framework.

    PubMed

    Roberts, Neil; Hooper, Guy; Lorencatto, Fabiana; Storr, Wendell; Spivey, Michael

    2017-09-19

    The 'Sepsis 6', a care bundle of basic, but vital, measures (e.g. intravenous fluid, antibiotics) has been implemented to improve sepsis treatment. However, uptake has been variable. Tools from behavioral sciences, such as the Theoretical Domains Framework (TDF) may be used to understand and address such implementation issues. This study used a behavioral science approach to identify barriers and facilitators towards Sepsis Six implementation at a case study hospital. Semi-structured interviews based on the TDF were conducted with a sample group of consultants, junior doctors and nurses from Emergency Department, Medical and Surgical Admissions, to explore barriers/facilitators to Sepsis Six performance. Transcripts were analyzed following the combined principles of content and framework analysis. Emerging themes informed a questionnaire to explore generalizability and importance across a sample of 261 stakeholders. Median importance and agreement ratings for each theme were calculated overall and for each role and clinical area. These were used to identify important barriers and important facilitators as targets for performance improvement. No new belief statements were discovered and data saturation was deemed achieved after 10 interviews. 1699 utterances were coded into 64 belief statements, then collated into a 51-item questionnaire. 113 questionnaire responses were obtained (44.3% response rate). Important barriers included insufficient audit and feedback, poor teamwork and communication, concerns about using the Sepsis Six in certain patients, insufficient training, and resource concerns. Facilitators included confidence in knowledge and skills, beliefs in overall benefits of the bundle, beliefs that identification and management of septic patients fell within everyone's role, and that regular use of the bundle made it easier to remember. Some beliefs were applicable for the entire group, others were specific to particular staff groups. A range of barriers

  13. Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

    PubMed Central

    Yoakim, M; Hou, W; Liu, Y; Carpenter, C L; Kapeller, R; Schaffhausen, B S

    1992-01-01

    The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins. Images PMID:1380095

  14. Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer

    PubMed Central

    1976-01-01

    We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold. PMID:6619

  15. Introduction: History of SH2 Domains and Their Applications.

    PubMed

    Liu, Bernard A; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain is the prototypical protein interaction module that lies at the heart of phosphotyrosine signaling. Since its serendipitous discovery, there has been a tremendous advancement in technologies and an array of techniques available for studying SH2 domains and phosphotyrosine signaling. In this chapter, we provide a glimpse of the history of SH2 domains and describe many of the tools and techniques that have been developed along the way and discuss future directions for SH2 domain studies. We highlight the gist of each chapter in this volume in the context of: the structural biology and phosphotyrosine binding; characterizing SH2 specificity and generating prediction models; systems biology and proteomics; SH2 domains in signal transduction; and SH2 domains in disease, diagnostics, and therapeutics. Many of the individual chapters provide an in-depth approach that will allow scientists to interrogate the function and role of SH2 domains.

  16. Lipid-Coated Gold Nanoparticles and FRET Allow Sensitive Monitoring of Liposome Clustering Mediated by the Synaptotagmin-7 C2A Domain.

    PubMed

    Hamilton, Desmond J; Coffman, Matthew D; Knight, Jefferson D; Reed, Scott M

    2017-09-12

    Synaptotagmin (Syt) family proteins contain tandem C2 domains, C2A and C2B, which insert into anionic membranes in response to increased cytosolic Ca 2+ concentration and facilitate exocytosis in neuronal and endocrine cells. The C2A domain from Syt7 binds lipid membranes much more tightly than the corresponding domain from Syt1, but the implications of this difference for protein function are not yet clear. In particular, the ability of the isolated Syt7 C2A domain to initiate membrane apposition and/or aggregation has been previously unexplored. Here, we demonstrate that Syt7 C2A induces apposition and aggregation of liposomes using Förster resonance energy transfer (FRET) assays, dynamic light scattering, and spectroscopic techniques involving lipid-coated gold nanoparticles (LCAuNPs). Protein-membrane binding, membrane apposition, and macroscopic aggregation are three separate phenomena with distinct Ca 2+ requirements: the threshold Ca 2+ concentration for membrane binding is lowest, followed by apposition and aggregation. However, aggregation is highly sensitive to protein concentration and can occur even at submicromolar Syt7 C2A; thus, highly sensitive assays are needed for measuring apposition without complications arising from aggregation. Notably, the localized surface plasmon resonance of the LCAuNP is sensitive to ≤10 nM Syt7 C2A concentrations. Furthermore, when the LCAuNPs were added into a FRET-based liposome apposition assay, the resultant energy transfer increased; possible explanations are discussed. Overall, LCAuNP-based methods allow for highly sensitive detection of protein-induced membrane apposition under conditions that miminize large-scale aggregation.

  17. The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function

    PubMed Central

    Warren, David; Heilpern, Andrew J.; Berg, Kent; Rosenberg, Naomi

    2000-01-01

    Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway. PMID:10775585

  18. Cloning and characterization of a novel human STAR domain containing cDNA KHDRBS2.

    PubMed

    Wang, Liu; Xu, Jian; Zeng, Li; Ye, Xin; Wu, Qihan; Dai, Jianfeng; Ji, Chaoneng; Gu, Shaohua; Zhao, Chunhua; Xie, Yi; Mao, Yumin

    2002-12-01

    KHDRBS2, KH domain containing, RNA binding, signal transduction associated 2, is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. It contains a KH domain,which is embedded in a larger conserved domain called the STAR domain. This protein has a 99% sequence identity with rat SLM-1 (the Sam68-like mammalian protein 1) and 98% sequence identity with mouse SLM-1 in its STAR domain. KHDRBS2 has the characteristic Sam68 SH2 and SH3 domain binding sites. RT-PCR analysis showed its transcript is ubiquitously expressed. The characterization of KHDRBS2 indicates it may link tyrosine kinase signaling cascades with some aspect of RNA metabolism.

  19. Dynamically Coupled Residues within the SH2 Domain of FYN Are Key to Unlocking Its Activity.

    PubMed

    Huculeci, Radu; Cilia, Elisa; Lyczek, Agatha; Buts, Lieven; Houben, Klaartje; Seeliger, Markus A; van Nuland, Nico; Lenaerts, Tom

    2016-11-01

    Src kinase activity is controlled by various mechanisms involving a coordinated movement of kinase and regulatory domains. Notwithstanding the extensive knowledge related to the backbone dynamics, little is known about the more subtle side-chain dynamics within the regulatory domains and their role in the activation process. Here, we show through experimental methyl dynamic results and predicted changes in side-chain conformational couplings that the SH2 structure of Fyn contains a dynamic network capable of propagating binding information. We reveal that binding the phosphorylated tail of Fyn perturbs a residue cluster near the linker connecting the SH2 and SH3 domains of Fyn, which is known to be relevant in the regulation of the activity of Fyn. Biochemical perturbation experiments validate that those residues are essential for inhibition of Fyn, leading to a gain of function upon mutation. These findings reveal how side-chain dynamics may facilitate the allosteric regulation of the different members of the Src kinase family. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    DOEpatents

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  1. Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

    PubMed Central

    Poon, Betty; Chen, Irvin S. Y.

    1998-01-01

    In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication. PMID:9499049

  2. μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

    PubMed

    Chen, Yi-Ting; Tai, Chin-Yin

    2017-05-01

    Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

    PubMed

    Pang, Xiaodong; Zhou, Huan-Xiang

    2014-05-01

    Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.

  4. BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

    PubMed

    Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

    2016-06-21

    The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Domain 2: Sport Safety and Injury Prevention

    ERIC Educational Resources Information Center

    Gurchiek, Larry; Mokha, Monique Butcher

    2004-01-01

    Most coaches recognize the importance of creating a safe environment and preventing injuries of their athletes. Domain 2 is dedicated to this important aspect of coaching, and outlines specific areas within safety and injury prevention that coaches should address. Domain 2 sets the standards for facility, equipment, and environmental safety…

  6. High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

    PubMed

    Higo, Kunitake; Ikura, Teikichi; Oda, Masayuki; Morii, Hisayuki; Takahashi, Jun; Abe, Ryo; Ito, Nobutoshi

    2013-01-01

    Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.

  7. The quaternary architecture of RARβ–RXRα heterodimer facilitates domain–domain signal transmission

    DOE PAGES

    Chandra, Vikas; Wu, Dalei; Li, Sheng; ...

    2017-10-11

    Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor beta-retinoic X receptor alpha (RAR beta-RXR alpha) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen-deuterium exchange mass spectrometry, mutagenesis and functional studies. The RAR beta ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within theirmore » quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its hetero-dimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.« less

  8. The quaternary architecture of RARβ–RXRα heterodimer facilitates domain–domain signal transmission

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

    Chandra, Vikas; Wu, Dalei; Li, Sheng

    Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor beta-retinoic X receptor alpha (RAR beta-RXR alpha) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen-deuterium exchange mass spectrometry, mutagenesis and functional studies. The RAR beta ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within theirmore » quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its hetero-dimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.« less

  9. Superbinder SH2 domains act as antagonists of cell signaling.

    PubMed

    Kaneko, Tomonori; Huang, Haiming; Cao, Xuan; Li, Xing; Li, Chengjun; Voss, Courtney; Sidhu, Sachdev S; Li, Shawn S C

    2012-09-25

    Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

  10. Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

    PubMed Central

    Ravichandran, K S; Igras, V; Shoelson, S E; Fesik, S W; Burakoff, S J

    1996-01-01

    Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643566

  11. The facilitative effects of glucose ingestion on memory retrieval in younger and older adults: is task difficulty or task domain critical?

    PubMed

    Riby, Leigh M; McMurtrie, Hazel; Smallwood, Jonathan; Ballantyne, Carrie; Meikle, Andrew; Smith, Emily

    2006-02-01

    The ingestion of a glucose-containing drink has been shown to improve cognitive performance, particularly memory functioning. However, it remains unclear as to the extent to which task domain and task difficulty moderate the glucose enhancement effect. The aim of this research was to determine whether boosts in performance are restricted to particular classes of memory (episodic v. semantic) or to tasks of considerable cognitive load. A repeated measures (25 g glucose v. saccharin), counterbalanced, double-blind design was used with younger and older adults. Participants performed a battery of episodic (e.g. paired associate learning) and semantic memory (e.g. category verification) tasks under low and high cognitive load. Electrophysiological measures (heart rate and galvanic skin response) of arousal and mental effort were also gathered. The results indicated that whilst glucose appeared to aid episodic remembering, cognitive load did not exaggerate the facilitative effect. For semantic memory, there was little evidence to suggest that glucose can boost semantic memory retrieval even when the load was manipulated. One exception was that glucose facilitated performance during the difficult category fluency task. Regardless, the present findings are consistent with the domain-specific account in which glucose acts primarily on the hippocampal region, which is known to support episodic memory. The possible contribution of the hippocampus in semantic memory processing is also discussed.

  12. Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

    PubMed

    Mikkola, Esa T; Gahmberg, Carl G

    2010-06-18

    The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  13. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

    PubMed Central

    Crisci, Angela; Raleff, Flore; Bagdiul, Ivona; Raabe, Monika; Urlaub, Henning; Rain, Jean-Christophe; Krämer, Angela

    2015-01-01

    Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions. PMID:26420826

  14. Domain analysis of Ras-association domain family member 6 upon interaction with MDM2.

    PubMed

    Sarkar, Aradhan; Iwasa, Hiroaki; Hossain, Shakhawoat; Xu, Xiaoyin; Sawada, Takeru; Shimizu, Takanobu; Maruyama, Junichi; Arimoto-Matsuzaki, Kyoko; Hata, Yutaka

    2017-01-01

    The tumor suppressor Ras-association domain family member 6 (RASSF6) has Ras-association domain (RA) and Salvador/RASSF/Hippo domain (SARAH). RASSF6 antagonizes MDM2, stabilizes p53, and induces apoptosis and cell cycle arrest. We previously demonstrated the interaction between RASSF6 and MDM2, but did not determine how both proteins interact with each other. We have shown here that N-terminal, RA, and SARAH domains of RASSF6 interact with MDM2 at distinct regions. RA binds to the RING-finger region of MDM2 and stabilizes p53. SARAH binds RA and blocks the interaction between RA and MDM2. RA overexpression induces p53-dependent apoptosis and senescence. In the presence of active KRas, the interaction between RA and MDM2 is recovered. In this way, RA and SARAH play an important role in Ras-mediated regulation of p53. © 2017 Federation of European Biochemical Societies.

  15. Structure of the SH3 Domain of Rat Endophilin A2

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

    Loll,P.; Swain, E.; Chen, Y.

    2008-01-01

    The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 Angstroms to R and Rfree values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition ofmore » a basic residue by a cluster of acidic side chains on the RT loop.« less

  16. A mutation-led search for novel functional domains in MeCP2.

    PubMed

    Guy, Jacky; Alexander-Howden, Beatrice; FitzPatrick, Laura; DeSousa, Dina; Koerner, Martha V; Selfridge, Jim; Bird, Adrian

    2018-04-27

    Most missense mutations causing Rett syndrome affect domains of MeCP2 that have been shown to either bind methylated DNA or interact with a transcriptional co-repressor complex. Several mutations, however, including the C-terminal truncations that account for ∼10% of cases, fall outside these characterised domains. We studied the molecular consequences of four of these "non-canonical" mutations in cultured neurons and mice to see if they reveal additional essential domains without affecting known properties of MeCP2. The results show that the mutations partially or strongly deplete the protein and also in some cases interfere with co-repressor recruitment. These mutations therefore impact the activity of known functional domains and do not invoke new molecular causes of Rett syndrome. The finding that a stable C-terminal truncation does not compromise MeCP2 function raises the possibility that small molecules which stabilise these mutant proteins may be of therapeutic value.

  17. Functional diversity of Csk, Chk, and Src SH2 domains due to a single residue variation.

    PubMed

    Ayrapetov, Marina K; Nam, Nguyen Hai; Ye, Guofeng; Kumar, Anil; Parang, Keykavous; Sun, Gongqin

    2005-07-08

    The C-terminal Src kinase (Csk) family of protein tyrosine kinases contains two members: Csk and Csk homologous kinase (Chk). Both phosphorylate and inactivate Src family kinases. Recent reports suggest that the Src homology (SH) 2 domains of Csk and Chk may bind to different phosphoproteins, which provides a basis for different cellular functions for Csk and Chk. To verify and characterize such a functional divergence, we compared the binding properties of the Csk, Chk, and Src SH2 domains and investigated the structural basis for the functional divergence. First, the study demonstrated striking functional differences between the Csk and Chk SH2 domains and revealed functional similarities between the Chk and Src SH2 domains. Second, structural analysis and mutagenic studies revealed that the functional differences among the three SH2 domains were largely controlled by one residue, Glu127 in Csk, Ile167 in Chk, and Lys200 in Src. Mutating these residues in the Csk or Chk SH2 domain to the Src counterpart resulted in dramatic gain of function similar to Src SH2 domain, whereas mutating Lys200 in Src SH2 domain to Glu (the Csk counterpart) resulted in loss of Src SH2 function. Third, a single point mutation of E127K rendered Csk responsive to activation by a Src SH2 domain ligand. Finally, the optimal phosphopeptide sequence for the Chk SH2 domain was determined. These results provide a compelling explanation for the functional differences between two homologous protein tyrosine kinases and reveal a new structure-function relationship for the SH2 domains.

  18. Domain-General and Domain-Specific Strategies for the Assessment of Distress Intolerance

    PubMed Central

    McHugh, R. Kathryn; Otto, Michael W.

    2011-01-01

    Recent research has provided evidence that distress intolerance—the perceived inability to tolerate distressing states—varies based on the domain of distress (e.g., pain, anxiety). Although domain-specific assessment strategies may provide information targeted to specific disorders or maladaptive behaviors, domain-general measures have the potential to facilitate comparisons across studies, disorders, and populations. The current study evaluated the utilization of self-report measures of distress intolerance as domain-general measures by examining their association with indices of behavioral avoidance and substance craving. Two groups of participants (N = 55) were recruited including a substance-dependent group and a comparison group equated based on the presence of an affective disorder. Results provided support for the validity of domain-general measures for assessing distress intolerance across varied domains. The importance of both domain-general and domain-specific measurement of distress intolerance is discussed. PMID:21823763

  19. Barriers and facilitators to the implementation of physical activity policies in schools: A systematic review.

    PubMed

    Nathan, Nicole; Elton, Ben; Babic, Mark; McCarthy, Nicole; Sutherland, Rachel; Presseau, Justin; Seward, Kirsty; Hodder, Rebecca; Booth, Debbie; Yoong, Sze Lin; Wolfenden, Luke

    2018-02-01

    Research consistently indicates that schools fail to implement mandatory physical activity policies. This review aimed to describe factors (barriers and facilitators) that may influence the implementation of school physical activity policies which specify the time or intensity that physical activity should be implemented and to map these factors to a theoretical framework. A systematic search was undertaken in six databases for quantitative or qualitative studies published between 1995-March 2016 that examined teachers', principals' or school administrators' reported barriers and/or facilitators to implementing mandated school physical activity policies. Two independent reviewers screened texts, extracted and coded data from identified articles using the Theoretical Domains Framework (TDF). Of the 10,346 articles identified, 17 studies met the inclusion criteria (8 quantitative, 9 qualitative). Barriers and facilitators identified in qualitative studies covered 9 and 10 TDF domains respectively. Barriers and facilitators reported in quantitative studies covered 8 TDF domains each. The most common domains identified were: 'environmental context and resources' (e.g., availability of equipment, time or staff), 'goals' (e.g., the perceived priority of the policy in the school), 'social influences' (e.g., support from school boards), and 'skills' (e.g., teachers' ability to implement the policy). Implementation support strategies that target these factors may represent promising means to improve implementation of physical activity policies and increase physical activity among school-aged children. Future studies assessing factors that influence school implementation of physical activity policies would benefit from using a comprehensive framework to help identify if any domains have been overlooked in the current literature. This review was prospectively registered with PROSPERO (CRD42016051649) on the 8th December 2016. Copyright © 2017 Elsevier Inc. All rights

  20. NMR solution structure of the activation domain of human procarboxypeptidase A2

    PubMed Central

    Jiménez, M. Angeles; Villegas, Virtudes; Santoro, Jorge; Serrano, Luis; Vendrell, Josep; Avilés, Francesc X.; Rico, Manuel

    2003-01-01

    The activation domain of human procarboxypeptidase A2, ADA2h, is an 81-residue globular domain released during the proteolytic activation of the proenzyme. The role of this and other similar domains as assistants of the correct folding of the enzyme is not fully understood. The folding pathway of ADA2h was characterized previously, and it was also observed that under certain conditions it may convert into amyloid fibrils in vitro. To gain insight into these processes, a detailed description of its three-dimensional structure in aqueous solution is required so that eventual changes could be properly monitored. A complete assignment of the 1H and 15N resonances of ADA2h was performed, and the solution structure, as derived from a set of 1688 nonredundant constraints, is very well defined (pairwise backbone RMSD = 0.67 ± 0.17 Å for residues 10–80). The structure is composed of two antiparallel α-helices comprising residues 19–32 and 58–69 packed on the same side of a three-stranded β-sheet spanning residues 10–15, 50–55, and 73–75. The global fold for the isolated human A2 activation domain is very similar to that of porcine carboxypeptidase B, as well as to the structure of the domain in the crystal of the intact human proenzyme. The observed structural differences relative to the intact human proenzyme are located at the interface between the activation domain and the enzyme and can be related with the activation mechanism. The backbone amide proton exchange behavior of ADA2h was also examined. The global free energy of unfolding obtained from exchange data of the most protected amide protons at pH 7.0 and 298K is 4.9 ± 0.3 kcal.mole−1, in good agreement with the values determined by thermal or denaturant unfolding studies. PMID:12538893

  1. The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

    PubMed

    Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

    2011-06-01

    Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

  2. Solution structure of the Big domain from Streptococcus pneumoniae reveals a novel Ca2+-binding module

    PubMed Central

    Wang, Tao; Zhang, Jiahai; Zhang, Xuecheng; Xu, Chao; Tu, Xiaoming

    2013-01-01

    Streptococcus pneumoniae is a pathogen causing acute respiratory infection, otitis media and some other severe diseases in human. In this study, the solution structure of a bacterial immunoglobulin-like (Big) domain from a putative S. pneumoniae surface protein SP0498 was determined by NMR spectroscopy. SP0498 Big domain adopts an eight-β-strand barrel-like fold, which is different in some aspects from the two-sheet sandwich-like fold of the canonical Ig-like domains. Intriguingly, we identified that the SP0498 Big domain was a Ca2+ binding domain. The structure of the Big domain is different from those of the well known Ca2+ binding domains, therefore revealing a novel Ca2+-binding module. Furthermore, we identified the critical residues responsible for the binding to Ca2+. We are the first to report the interactions between the Big domain and Ca2+ in terms of structure, suggesting an important role of the Big domain in many essential calcium-dependent cellular processes such as pathogenesis. PMID:23326635

  3. A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

    1998-01-01

    To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

  4. The HhH(2)/NDD Domain of the Drosophila Nod Chromokinesin-like Protein Is Required for Binding to Chromosomes in the Oocyte Nucleus

    PubMed Central

    Cui, Wei; Hawley, R. Scott

    2005-01-01

    Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9–10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes. PMID:16143607

  5. Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

    PubMed Central

    Gaffaney, Jon D.; Dunning, F. Mark; Wang, Zhao; Hui, Enfu; Chapman, Edwin R.

    2008-01-01

    Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B. PMID:18784080

  6. Big Domains Are Novel Ca2+-Binding Modules: Evidences from Big Domains of Leptospira Immunoglobulin-Like (Lig) Proteins

    PubMed Central

    Palaniappan, Raghavan U. M.; Lin, Yi-Pin; He, Hongxuan; McDonough, Sean P.; Sharma, Yogendra; Chang, Yung-Fu

    2010-01-01

    Background Many bacterial surface exposed proteins mediate the host-pathogen interaction more effectively in the presence of Ca2+. Leptospiral immunoglobulin-like (Lig) proteins, LigA and LigB, are surface exposed proteins containing Bacterial immunoglobulin like (Big) domains. The function of proteins which contain Big fold is not known. Based on the possible similarities of immunoglobulin and βγ-crystallin folds, we here explore the important question whether Ca2+ binds to a Big domains, which would provide a novel functional role of the proteins containing Big fold. Principal Findings We selected six individual Big domains for this study (three from the conserved part of LigA and LigB, denoted as Lig A3, Lig A4, and LigBCon5; two from the variable region of LigA, i.e., 9th (Lig A9) and 10th repeats (Lig A10); and one from the variable region of LigB, i.e., LigBCen2. We have also studied the conserved region covering the three and six repeats (LigBCon1-3 and LigCon). All these proteins bind the calcium-mimic dye Stains-all. All the selected four domains bind Ca2+ with dissociation constants of 2–4 µM. Lig A9 and Lig A10 domains fold well with moderate thermal stability, have β-sheet conformation and form homodimers. Fluorescence spectra of Big domains show a specific doublet (at 317 and 330 nm), probably due to Trp interaction with a Phe residue. Equilibrium unfolding of selected Big domains is similar and follows a two-state model, suggesting the similarity in their fold. Conclusions We demonstrate that the Lig are Ca2+-binding proteins, with Big domains harbouring the binding motif. We conclude that despite differences in sequence, a Big motif binds Ca2+. This work thus sets up a strong possibility for classifying the proteins containing Big domains as a novel family of Ca2+-binding proteins. Since Big domain is a part of many proteins in bacterial kingdom, we suggest a possible function these proteins via Ca2+ binding. PMID:21206924

  7. Fusion protein based on Grb2-SH2 domain for cancer therapy

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

    Saito, Yuriko; Graduate School of Pharmaceutical Sciences, Chiba University; Furukawa, Takako, E-mail: tfuru@nirs.go.jp

    2010-08-20

    Research highlights: {yields} Grb2 mediates EGFR signaling through binding to phosphorylate EGFR with SH2 domain. {yields} We generated fusion proteins containing 1 or 2 SH2 domains of Grb2 added with TAT. {yields} The one with 2 SH2 domains (TSSF) interfered ERK phosphorylation. {yields} TSSF significantly delayed the growth of EGFR overexpressing tumor in a mouse model. -- Abstract: Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylatedmore » EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR-Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR-Grb2 signaling and could have a potential to treat EGFR-activated cancer.« less

  8. Implementation of pregnancy weight management and obesity guidelines: a meta-synthesis of healthcare professionals' barriers and facilitators using the Theoretical Domains Framework.

    PubMed

    Heslehurst, N; Newham, J; Maniatopoulos, G; Fleetwood, C; Robalino, S; Rankin, J

    2014-06-01

    Obesity in pregnancy is rising and is associated with severe health consequences for both the mother and the child. There is an increasing international focus on guidelines to manage the clinical risks of maternal obesity, and for pregnancy weight management. However, passive dissemination of guidelines is not effective and more active strategies are required for effective guideline implementation into practice. Implementation of guidelines is a form of healthcare professional behaviour change, and therefore implementation strategies should be based on appropriate behaviour change theory. This systematic review aimed to identify the determinants of healthcare professionals' behaviours in relation to maternal obesity and weight management. Twenty-five studies were included. Data synthesis of the existing international qualitative and quantitative evidence base used the Theoretical Domains Framework to identify the barriers and facilitators to healthcare professionals' maternal obesity and weight management practice. The domains most frequently identified included 'knowledge', 'beliefs about consequences' and 'environmental context and resources'. Healthcare professionals' weight management practice had the most barriers compared with any other area of maternal obesity practice. The results of this review will be used to inform the development of an intervention to support healthcare professional behaviour change. © 2014 The Authors. obesity reviews © 2014 International Association for the Study of Obesity.

  9. Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

    PubMed

    Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

    2009-05-27

    The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

  10. Facilitators and barriers to quality of care in maternal, newborn and child health: a global situational analysis through metareview

    PubMed Central

    Nair, Manisha; Yoshida, Sachiyo; Lambrechts, Thierry; Boschi-Pinto, Cynthia; Bose, Krishna; Mason, Elizabeth Mary; Mathai, Matthews

    2014-01-01

    Objective Conduct a global situational analysis to identify the current facilitators and barriers to improving quality of care (QoC) for pregnant women, newborns and children. Study design Metareview of published and unpublished systematic reviews and meta-analyses conducted between January 2000 and March 2013 in any language. Assessment of Multiple Systematic Reviews (AMSTAR) is used to assess the methodological quality of systematic reviews. Settings Health systems of all countries. Study outcome: QoC measured using surrogate indicators––effective, efficient, accessible, acceptable/patient centred, equitable and safe. Analysis Conducted in two phases (1) qualitative synthesis of extracted data to identify and group the facilitators and barriers to improving QoC, for each of the three population groups, into the six domains of WHO's framework and explore new domains and (2) an analysis grid to map the common facilitators and barriers. Results We included 98 systematic reviews with 110 interventions to improve QoC from countries globally. The facilitators and barriers identified fitted the six domains of WHO's framework––information, patient–population engagement, leadership, regulations and standards, organisational capacity and models of care. Two new domains, ‘communication’ and ‘satisfaction’, were generated. Facilitators included active and regular interpersonal communication between users and providers; respect, confidentiality, comfort and support during care provision; engaging users in decision-making; continuity of care and effective audit and feedback mechanisms. Key barriers identified were language barriers in information and communication; power difference between users and providers; health systems not accounting for user satisfaction; variable standards of implementation of standard guidelines; shortage of resources in health facilities and lack of studies assessing the role of leadership in improving QoC. These were common across

  11. A Hybrid Approach to Finding Relevant Social Media Content for Complex Domain Specific Information Needs.

    PubMed

    Cameron, Delroy; Sheth, Amit P; Jaykumar, Nishita; Thirunarayan, Krishnaprasad; Anand, Gaurish; Smith, Gary A

    2014-12-01

    While contemporary semantic search systems offer to improve classical keyword-based search, they are not always adequate for complex domain specific information needs. The domain of prescription drug abuse, for example, requires knowledge of both ontological concepts and "intelligible constructs" not typically modeled in ontologies. These intelligible constructs convey essential information that include notions of intensity, frequency, interval, dosage and sentiments, which could be important to the holistic needs of the information seeker. In this paper, we present a hybrid approach to domain specific information retrieval that integrates ontology-driven query interpretation with synonym-based query expansion and domain specific rules, to facilitate search in social media on prescription drug abuse. Our framework is based on a context-free grammar (CFG) that defines the query language of constructs interpretable by the search system. The grammar provides two levels of semantic interpretation: 1) a top-level CFG that facilitates retrieval of diverse textual patterns, which belong to broad templates and 2) a low-level CFG that enables interpretation of specific expressions belonging to such textual patterns. These low-level expressions occur as concepts from four different categories of data: 1) ontological concepts, 2) concepts in lexicons (such as emotions and sentiments), 3) concepts in lexicons with only partial ontology representation, called lexico-ontology concepts (such as side effects and routes of administration (ROA)), and 4) domain specific expressions (such as date, time, interval, frequency and dosage) derived solely through rules. Our approach is embodied in a novel Semantic Web platform called PREDOSE, which provides search support for complex domain specific information needs in prescription drug abuse epidemiology. When applied to a corpus of over 1 million drug abuse-related web forum posts, our search framework proved effective in retrieving

  12. A Hybrid Approach to Finding Relevant Social Media Content for Complex Domain Specific Information Needs

    PubMed Central

    Cameron, Delroy; Sheth, Amit P.; Jaykumar, Nishita; Thirunarayan, Krishnaprasad; Anand, Gaurish; Smith, Gary A.

    2015-01-01

    While contemporary semantic search systems offer to improve classical keyword-based search, they are not always adequate for complex domain specific information needs. The domain of prescription drug abuse, for example, requires knowledge of both ontological concepts and “intelligible constructs” not typically modeled in ontologies. These intelligible constructs convey essential information that include notions of intensity, frequency, interval, dosage and sentiments, which could be important to the holistic needs of the information seeker. In this paper, we present a hybrid approach to domain specific information retrieval that integrates ontology-driven query interpretation with synonym-based query expansion and domain specific rules, to facilitate search in social media on prescription drug abuse. Our framework is based on a context-free grammar (CFG) that defines the query language of constructs interpretable by the search system. The grammar provides two levels of semantic interpretation: 1) a top-level CFG that facilitates retrieval of diverse textual patterns, which belong to broad templates and 2) a low-level CFG that enables interpretation of specific expressions belonging to such textual patterns. These low-level expressions occur as concepts from four different categories of data: 1) ontological concepts, 2) concepts in lexicons (such as emotions and sentiments), 3) concepts in lexicons with only partial ontology representation, called lexico-ontology concepts (such as side effects and routes of administration (ROA)), and 4) domain specific expressions (such as date, time, interval, frequency and dosage) derived solely through rules. Our approach is embodied in a novel Semantic Web platform called PREDOSE, which provides search support for complex domain specific information needs in prescription drug abuse epidemiology. When applied to a corpus of over 1 million drug abuse-related web forum posts, our search framework proved effective in retrieving

  13. SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity*

    PubMed Central

    Liu, Bernard A.; Jablonowski, Karl; Shah, Eshana E.; Engelmann, Brett W.; Jones, Richard B.; Nash, Piers D.

    2010-01-01

    Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. PMID:20627867

  14. Barriers and Facilitators to Implementing the HEADS-ED: A Rapid Screening Tool for Pediatric Patients in Emergency Departments.

    PubMed

    MacWilliams, Kate; Curran, Janet; Racek, Jakub; Cloutier, Paula; Cappelli, Mario

    2017-12-01

    This study sought to identify barriers and facilitators to the implementation of the HEADS-ED, a screening tool appropriate for use in the emergency department (ED) that facilitates standardized assessments, discharge planning, charting, and linking pediatric mental health patients to appropriate community resources. A qualitative theory-based design was used to identify barriers and facilitators to implementing the HEADS-ED tool. Focus groups were conducted with participants recruited from 6 different ED settings across 2 provinces (Ontario and Nova Scotia). The Theoretical Domains Framework was used as a conceptual framework to guide data collection and to identify themes from focus group discussions. The following themes spanning 12 domains were identified as reflective of participants' beliefs about the barriers and facilitators to implementing the HEADS-ED tool: knowledge, skills, beliefs about capabilities, social professional role and identity, optimism, beliefs about consequences, reinforcement, environmental context and resources, social influences, emotion, behavioral regulation and memory, and attention and decision process. The HEADS-ED has the potential to address the need for better discharge planning, complete charting, and standardized assessments for the increasing population of pediatric mental health patients who present to EDs. This study has identified potential barriers and facilitators, which should be considered when developing an implementation plan for adopting the HEADS-ED tool into practice within EDs.

  15. Evolution of SH2 domains and phosphotyrosine signalling networks

    PubMed Central

    Liu, Bernard A.; Nash, Piers D.

    2012-01-01

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

  16. The Arabidopsis Histone Methyltransferase SUVR4 Binds Ubiquitin via a Domain with a Four-Helix Bundle Structure

    PubMed Central

    Rahman, Mohummad Aminur; Kristiansen, Per E.; Veiseth, Silje V.; Andersen, Jan Terje; Yap, Kyoko L.; Zhou, Ming-Ming; Sandlie, Inger; Thorstensen, Tage; Aalen, Reidunn B.

    2014-01-01

    In eukaryotes, different chromatin states facilitate or repress gene expression and restrict the activity of transposable elements. Post-translational modifications (PTMs) of amino acid residues on the N-terminal tails of histones are suggested to define such states. The histone lysine methyltransferase (HKMTase) SU(VAR)3-9 RELATED4 (SUVR4) of Arabidopsis thaliana functions as a repressor of transposon activity. Binding of ubiquitin by the WIYLD domain facilitates the addition of two methyl groups to monomethylated lysine 9 of histone H3. By using nuclear magnetic resonance (NMR) spectroscopy, we identified SUVR4 WIYLD (S4WIYLD) as a domain with a four-helix bundle structure, in contrast to three-helix bundles of other ubiquitin binding domains. NMR titration analyses showed that residues of helix α1 (Q38, L39, and D40) and helix α4 (N68, T70, A71, V73, D74, I76, S78, and E82) of S4WIYLD and residues between the first and second β-strands (T9 and G10) and on β-strands 3 (R42, G47, K48, and Q49) and 4 (H68, R72, and L73) undergo significant chemical shift changes when the two proteins interact. A model of the complex, generated using HADDOCK, suggests that the N-terminal and C-terminal parts of S4WIYLD constitute a surface that interacts with charged residues close to the hydrophobic patch of ubiquitin. The WIYLD domains of the closely related SUVR1 and SUVR2 Arabidopsis proteins also bind ubiquitin, indicating that this is a general feature of this domain. The question of whether SUVR proteins act as both readers of monoubiquitinated H2B and writers of histone PTMs is discussed. PMID:24625295

  17. Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains*

    PubMed Central

    Borthakur, Susmita; Lee, HyeongJu; Kim, SoonJeung; Wang, Bing-Cheng; Buck, Matthias

    2014-01-01

    The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr921, Tyr930, and Tyr960, has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr921 and Tyr930 enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling. PMID:24825902

  18. Biologic properties of a CH2 domain-deleted recombinant immunoglobulin.

    PubMed

    Slavin-Chiorini, D C; Horan Hand, P H; Kashmiri, S V; Calvo, B; Zaremba, S; Schlom, J

    1993-01-02

    Monoclonal antibody (MAb) B72.3 reacts with TAG-72, a high-molecular-weight mucin expressed on several types of human carcinoma, and is currently being used in clinical trials for the diagnosis and therapy of human carcinoma. An expression construct containing cDNA encoding an immunoglobulin (Ig) heavy chain, with the variable region of murine MAb B72.3 and a human Ig constant region with a deletion of the CH2 domain, was generated. Immunoglobulin from the transfectoma with the highest expression of the TAG-72 immunoreactive antibody was designated MAb chimeric (c) B72.3 delta CH2. The pharmacokinetics of serum clearance of iodine-labeled MAbs cB72.3 delta CH2 and the intact cB72.3 were compared in athymic mice. By 24 hr, less than 1% of the cB72.3 delta CH2 was left in the plasma, while 36% of the cB72.3 still remained. The T1/2 alpha values of the cB72.3 delta CH2 and cB72.3 MAbs were 1.7 and 2.4 hr, respectively. The T1/2 beta values were 7.8 hr for the domain-deleted cMAb and 48.9 hr for cB72.3. Biodistribution studies in athymic mice bearing LS-174T xenografts showed a reduction in the percentage of injected dose per gram in tumor with 131I-cB72.3 delta CH2; however, the 131I-cB72.3 delta CH2 both localized to tumors faster and cleared from the blood faster than the 125I-cB72.3 MAb. Only trace amounts of the 131I-cB72.3 delta CH2 were detected in normal tissues, including kidney. The faster clearance rate, more rapid tumor targeting and lack of metabolic uptake in normal tissues demonstrated with the iodine-labeled CH2 domain-deleted cMAb may be an advantage for certain clinical protocols.

  19. Src binds cortactin through an SH2 domain cystine-mediated linkage.

    PubMed

    Evans, Jason V; Ammer, Amanda G; Jett, John E; Bolcato, Chris A; Breaux, Jason C; Martin, Karen H; Culp, Mark V; Gannett, Peter M; Weed, Scott A

    2012-12-15

    Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

  20. Src binds cortactin through an SH2 domain cystine-mediated linkage

    PubMed Central

    Evans, Jason V.; Ammer, Amanda G.; Jett, John E.; Bolcato, Chris A.; Breaux, Jason C.; Martin, Karen H.; Culp, Mark V.; Gannett, Peter M.; Weed, Scott A.

    2012-01-01

    Summary Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions. PMID:23097045

  1. The PPARγ2 A/B-Domain Plays a Gene-Specific Role in Transactivation and Cofactor Recruitment

    PubMed Central

    Bugge, Anne; Grøntved, Lars; Aagaard, Mads M.; Borup, Rehannah; Mandrup, Susanne

    2009-01-01

    We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5–8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARγ target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARγ-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARγ2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARγ lacking the A/B-domain (PPARγCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARγCDE compared with cells expressing full-length PPARγ2. Using chromatin immunoprecipitation, we demonstrate that PPARγ binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARγ-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARγ2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes. PMID:19282365

  2. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

    PubMed Central

    Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

    2016-01-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

  3. A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

    PubMed

    Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

    2016-12-01

    In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

  4. 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.

  5. Therapeutic targeting of two-pore-domain potassium (K(2P)) channels in the cardiovascular system.

    PubMed

    Wiedmann, Felix; Schmidt, Constanze; Lugenbiel, Patrick; Staudacher, Ingo; Rahm, Ann-Kathrin; Seyler, Claudia; Schweizer, Patrick A; Katus, Hugo A; Thomas, Dierk

    2016-05-01

    The improvement of treatment strategies in cardiovascular medicine is an ongoing process that requires constant optimization. The ability of a therapeutic intervention to prevent cardiovascular pathology largely depends on its capacity to suppress the underlying mechanisms. Attenuation or reversal of disease-specific pathways has emerged as a promising paradigm, providing a mechanistic rationale for patient-tailored therapy. Two-pore-domain K(+) (K(2P)) channels conduct outward K(+) currents that stabilize the resting membrane potential and facilitate action potential repolarization. K(2P) expression in the cardiovascular system and polymodal K2P current regulation suggest functional significance and potential therapeutic roles of the channels. Recent work has focused primarily on K(2P)1.1 [tandem of pore domains in a weak inwardly rectifying K(+) channel (TWIK)-1], K(2P)2.1 [TWIK-related K(+) channel (TREK)-1], and K(2P)3.1 [TWIK-related acid-sensitive K(+) channel (TASK)-1] channels and their role in heart and vessels. K(2P) currents have been implicated in atrial and ventricular arrhythmogenesis and in setting the vascular tone. Furthermore, the association of genetic alterations in K(2P)3.1 channels with atrial fibrillation, cardiac conduction disorders and pulmonary arterial hypertension demonstrates the relevance of the channels in cardiovascular disease. The function, regulation and clinical significance of cardiovascular K(2P) channels are summarized in the present review, and therapeutic options are emphasized. © 2016 Authors; published by Portland Press Limited.

  6. In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

    PubMed

    Machida, Kazuya

    2017-01-01

    Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

  7. Comparative Sequence and X-Inactivation Analyses of a Domain of Escape in Human Xp11.2 and the Conserved Segment in Mouse

    PubMed Central

    Tsuchiya, Karen D.; Greally, John M.; Yi, Yajun; Noel, Kevin P.; Truong, Jean-Pierre; Disteche, Christine M.

    2004-01-01

    We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome. PMID:15197169

  8. Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding

    PubMed Central

    Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael

    2016-01-01

    The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin–MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro. The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin–RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. PMID:26976641

  9. Structural and biophysical investigation of the interaction of a mutant Grb2 SH2 domain (W121G) with its cognate phosphopeptide.

    PubMed

    Papaioannou, Danai; Geibel, Sebastian; Kunze, Micha B A; Kay, Christopher W M; Waksman, Gabriel

    2016-03-01

    The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide. © 2015 The Protein Society.

  10. Facilitators and barriers to quality of care in maternal, newborn and child health: a global situational analysis through metareview.

    PubMed

    Nair, Manisha; Yoshida, Sachiyo; Lambrechts, Thierry; Boschi-Pinto, Cynthia; Bose, Krishna; Mason, Elizabeth Mary; Mathai, Matthews

    2014-05-22

    Conduct a global situational analysis to identify the current facilitators and barriers to improving quality of care (QoC) for pregnant women, newborns and children. Metareview of published and unpublished systematic reviews and meta-analyses conducted between January 2000 and March 2013 in any language. Assessment of Multiple Systematic Reviews (AMSTAR) is used to assess the methodological quality of systematic reviews. Health systems of all countries. Study outcome: QoC measured using surrogate indicators--effective, efficient, accessible, acceptable/patient centred, equitable and safe. Conducted in two phases (1) qualitative synthesis of extracted data to identify and group the facilitators and barriers to improving QoC, for each of the three population groups, into the six domains of WHO's framework and explore new domains and (2) an analysis grid to map the common facilitators and barriers. We included 98 systematic reviews with 110 interventions to improve QoC from countries globally. The facilitators and barriers identified fitted the six domains of WHO's framework--information, patient-population engagement, leadership, regulations and standards, organisational capacity and models of care. Two new domains, 'communication' and 'satisfaction', were generated. Facilitators included active and regular interpersonal communication between users and providers; respect, confidentiality, comfort and support during care provision; engaging users in decision-making; continuity of care and effective audit and feedback mechanisms. Key barriers identified were language barriers in information and communication; power difference between users and providers; health systems not accounting for user satisfaction; variable standards of implementation of standard guidelines; shortage of resources in health facilities and lack of studies assessing the role of leadership in improving QoC. These were common across the three population groups. The barriers to good

  11. The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

    PubMed

    Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

    2009-08-07

    SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

  12. Barriers and Facilitators to Self-Directed Learning in Continuing Professional Development for Physicians in Canada: A Scoping Review.

    PubMed

    Jeong, Dahn; Presseau, Justin; ElChamaa, Rima; Naumann, Danielle N; Mascaro, Colin; Luconi, Francesca; Smith, Karen M; Kitto, Simon

    2018-04-10

    This scoping review explored the barriers and facilitators that influence engagement in and implementation of self-directed learning (SDL) in continuing professional development (CPD) for physicians in Canada. This review followed the six-stage scoping review framework of Arksey and O'Malley and of Daudt et al. In 2015, the authors searched eight online databases for English-language Canadian articles published January 2005-December 2015. To chart and analyze the data from the 17 included studies, they employed two-step analysis process of conventional content analysis followed by directed coding guided by the Theoretical Domains Framework (TDF). Conventional content analysis generated five categories of barriers and facilitators: individual, program, technological, environmental, and workplace/organizational. Directed coding guided by the TDF allowed analysis of barriers and facilitators to behavior change according to two key groups: physicians engaging in SDL and SDL developers designing and implementing SDL programs. Of the 318 total barriers and facilitators coded, 290 (91.2%) were coded for physicians and 28 (8.8%) for SDL developers. The majority (209; 65.7%) were coded in four key TDF domains: environmental context and resources, social influences, beliefs about consequences, and behavioral regulation. This scoping review identified five categories of barriers and facilitators in the literature and four key TDF domains where most factors related to behavior change of physicians and SDL developers regarding SDL programs in CPD were coded. There was a significant gap in the literature about factors that may contribute to SDL developers' capacity to design and implement SDL programs in CPD.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way

  13. Probing SH2-domains using Inhibitor Affinity Purification (IAP).

    PubMed

    Höfener, Michael; Heinzlmeir, Stephanie; Kuster, Bernhard; Sewald, Norbert

    2014-01-01

    Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds. In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain. The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

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

    PubMed

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

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

  15. Lateral diffusion of proteins on supported lipid bilayers: additive friction of synaptotagmin 7 C2A-C2B tandem domains.

    PubMed

    Vasquez, Joseph K; Chantranuvatana, Kan; Giardina, Daniel T; Coffman, Matthew D; Knight, Jefferson D

    2014-12-23

    The synaptotagmin (Syt) family of proteins contains tandem C2 domains, C2A and C2B, which bind membranes in the presence of Ca(2+) to trigger vesicle fusion during exocytosis. Despite recent progress, the role and extent of interdomain interactions between C2A and C2B in membrane binding remain unclear. To test whether the two domains interact on a planar lipid bilayer (i.e., experience thermodynamic interdomain contacts), diffusion of fluorescent-tagged C2A, C2B, and C2AB domains from human Syt7 was measured using total internal reflection fluorescence microscopy with single-particle tracking. The C2AB tandem exhibits a lateral diffusion constant approximately half the value of the isolated single domains and does not change when additional residues are engineered into the C2A-C2B linker. This is the expected result if C2A and C2B are separated when membrane-bound; theory predicts that C2AB diffusion would be faster if the two domains were close enough together to have interdomain contact. Stopped-flow measurements of membrane dissociation kinetics further support an absence of interdomain interactions, as dissociation kinetics of the C2AB tandem remain unchanged when rigid or flexible linker extensions are included. Together, the results suggest that the two C2 domains of Syt7 bind independently to planar membranes, in contrast to reported interdomain cooperativity in Syt1.

  16. Practice-tailored facilitation to improve pediatric preventive care delivery: a randomized trial.

    PubMed

    Meropol, Sharon B; Schiltz, Nicholas K; Sattar, Abdus; Stange, Kurt C; Nevar, Ann H; Davey, Christina; Ferretti, Gerald A; Howell, Diana E; Strosaker, Robyn; Vavrek, Pamela; Bader, Samantha; Ruhe, Mary C; Cuttler, Leona

    2014-06-01

    Evolving primary care models require methods to help practices achieve quality standards. This study assessed the effectiveness of a Practice-Tailored Facilitation Intervention for improving delivery of 3 pediatric preventive services. In this cluster-randomized trial, a practice facilitator implemented practice-tailored rapid-cycle feedback/change strategies for improving obesity screening/counseling, lead screening, and dental fluoride varnish application. Thirty practices were randomized to Early or Late Intervention, and outcomes assessed for 16 419 well-child visits. A multidisciplinary team characterized facilitation processes by using comparative case study methods. Baseline performance was as follows: for Obesity: 3.5% successful performance in Early and 6.3% in Late practices, P = .74; Lead: 62.2% and 77.8% success, respectively, P = .11; and Fluoride: <0.1% success for all practices. Four months after randomization, performance rose in Early practices, to 82.8% for Obesity, 86.3% for Lead, and 89.1% for Fluoride, all P < .001 for improvement compared with Late practices' control time. During the full 6-month intervention, care improved versus baseline in all practices, for Obesity for Early practices to 86.5%, and for Late practices 88.9%; for Lead for Early practices to 87.5% and Late practices 94.5%; and for Fluoride, for Early practices to 78.9% and Late practices 81.9%, all P < .001 compared with baseline. Improvements were sustained 2 months after intervention. Successful facilitation involved multidisciplinary support, rapid-cycle problem solving feedback, and ongoing relationship-building, allowing individualizing facilitation approach and intensity based on 3 levels of practice need. Practice-tailored Facilitation Intervention can lead to substantial, simultaneous, and sustained improvements in 3 domains, and holds promise as a broad-based method to advance pediatric preventive care. Copyright © 2014 by the American Academy of Pediatrics.

  17. Practice-Tailored Facilitation to Improve Pediatric Preventive Care Delivery: A Randomized Trial

    PubMed Central

    Schiltz, Nicholas K.; Sattar, Abdus; Stange, Kurt C.; Nevar, Ann H.; Davey, Christina; Ferretti, Gerald A.; Howell, Diana E.; Strosaker, Robyn; Vavrek, Pamela; Bader, Samantha; Ruhe, Mary C.; Cuttler, Leona

    2014-01-01

    OBJECTIVE: Evolving primary care models require methods to help practices achieve quality standards. This study assessed the effectiveness of a Practice-Tailored Facilitation Intervention for improving delivery of 3 pediatric preventive services. METHODS: In this cluster-randomized trial, a practice facilitator implemented practice-tailored rapid-cycle feedback/change strategies for improving obesity screening/counseling, lead screening, and dental fluoride varnish application. Thirty practices were randomized to Early or Late Intervention, and outcomes assessed for 16 419 well-child visits. A multidisciplinary team characterized facilitation processes by using comparative case study methods. RESULTS: Baseline performance was as follows: for Obesity: 3.5% successful performance in Early and 6.3% in Late practices, P = .74; Lead: 62.2% and 77.8% success, respectively, P = .11; and Fluoride: <0.1% success for all practices. Four months after randomization, performance rose in Early practices, to 82.8% for Obesity, 86.3% for Lead, and 89.1% for Fluoride, all P < .001 for improvement compared with Late practices’ control time. During the full 6-month intervention, care improved versus baseline in all practices, for Obesity for Early practices to 86.5%, and for Late practices 88.9%; for Lead for Early practices to 87.5% and Late practices 94.5%; and for Fluoride, for Early practices to 78.9% and Late practices 81.9%, all P < .001 compared with baseline. Improvements were sustained 2 months after intervention. Successful facilitation involved multidisciplinary support, rapid-cycle problem solving feedback, and ongoing relationship-building, allowing individualizing facilitation approach and intensity based on 3 levels of practice need. CONCLUSIONS: Practice-tailored Facilitation Intervention can lead to substantial, simultaneous, and sustained improvements in 3 domains, and holds promise as a broad-based method to advance pediatric preventive care. PMID:24799539

  18. RBFOX2 protein domains and cellular activities.

    PubMed

    Arya, Anurada D; Wilson, David I; Baralle, Diana; Raponi, Michaela

    2014-08-01

    RBFOX2 (RNA-binding protein, Fox-1 homologue 2)/RBM9 (RNA-binding-motif protein 9)/RTA (repressor of tamoxifen action)/HNRBP2 (hexaribonucleotide-binding protein 2) encodes an RNA-binding protein involved in tissue specific alternative splicing regulation and steroid receptors transcriptional activity. Its ability to regulate specific splicing profiles depending on context has been related to different expression levels of the RBFOX2 protein itself and that of other splicing regulatory proteins involved in the shared modulation of specific genes splicing. However, this cannot be the sole explanation as to why RBFOX2 plays a widespread role in numerous cellular mechanisms from development to cell survival dependent on cell/tissue type. RBFOX2 isoforms with altered protein domains exist. In the present article, we describe the main RBFOX2 protein domains, their importance in the context of splicing and transcriptional regulation and we propose that RBFOX2 isoform distribution may play a fundamental role in RBFOX2-specific cellular effects.

  19. Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew.

    PubMed

    Allard, J D; Herbst, R; Carroll, P M; Simon, M A

    1998-05-22

    The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associated in vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function.

  20. The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

    NASA Astrophysics Data System (ADS)

    Engelmann, Brett Warren

    The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving

  1. Myocardin-Related Transcription Factor A Activation by Competition with WH2 Domain Proteins for Actin Binding.

    PubMed

    Weissbach, Julia; Schikora, Franziska; Weber, Anja; Kessels, Michael; Posern, Guido

    2016-05-15

    The myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF)-mediated gene expression. Activation of MRTF-A occurs in response to alterations in actin dynamics and critically requires the dissociation of repressive G-actin-MRTF-A complexes. However, the mechanism leading to the release of MRTF-A remains unclear. Here we show that WH2 domains compete directly with MRTF-A for actin binding. Actin nucleation-promoting factors, such as N-WASP and WAVE2, as well as isolated WH2 domains, including those of Spire2 and Cobl, activate MRTF-A independently of changes in actin dynamics. Simultaneous inhibition of Arp2-Arp3 or mutation of the CA region only partially reduces MRTF-A activation by N-WASP and WAVE2. Recombinant WH2 domains and the RPEL domain of MRTF-A bind mutually exclusively to cellular and purified G-actin in vitro The competition by different WH2 domains correlates with MRTF-SRF activation. Following serum stimulation, nonpolymerizable actin dissociates from MRTF-A, and de novo formation of the G-actin-RPEL complex is impaired by a transferable factor. Our work demonstrates that WH2 domains activate MRTF-A and contribute to target gene regulation by a competitive mechanism, independently of their role in actin filament formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

    PubMed Central

    Kandachar, Vasundhara; Bai, Ting; Chang, Henry C

    2008-01-01

    Background Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood. Results To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger dAux alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in dAux deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing dAux phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in dAux mutant cells, Delta accumulates at the cell surface. In dAux mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures. Conclusion Our data suggest that dAux mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in dAux mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization. PMID:18466624

  3. A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

    PubMed

    Williams, K P; Shoelson, S E

    1993-03-15

    Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

  4. Facilitators for practice change in Spanish community pharmacy.

    PubMed

    Gastelurrutia, Miguel A; Benrimoj, S I Charlie; Castrillon, Carla C; de Amezua, María J Casado; Fernandez-Llimos, Fernando; Faus, Maria J

    2009-02-01

    To identify and prioritise facilitators for practice change in Spanish community pharmacy. Spanish community pharmacies. Qualitative study. Thirty-three semi-structured interviews were conducted with community pharmacists (n = 15) and pharmacy strategists (n = 18), and the results were examined using the content analysis method. In addition, two nominal groups (seven community pharmacists and seven strategists) were formed to identify and prioritise facilitators. Results of both techniques were then triangulated. Facilitators for practice change. Twelve facilitators were identified and grouped into four domains (D1: Pharmacist; D2: Pharmacy as an organisation; D3: Pharmaceutical profession; D4: Miscellaneous). Facilitators identified in D1 include: the need for more clinical education at both pre- and post-graduate levels; the need for clearer and unequivocal messages from professional leaders about the future of the professional practice; and the need for a change in pharmacists' attitudes. Facilitators in D2 are: the need to change the reimbursement system to accommodate cognitive service delivery as well as dispensing; and the need to change the front office of pharmacies. Facilitators identified in D3 are: the need for the Spanish National Professional Association to take a leadership role in the implementation of cognitive services; the need to reduce administrative workload; and the need for universities to reduce the gap between education and research. Other facilitators identified in this study include: the need to increase patients' demand for cognitive services at pharmacies; the need to improve pharmacist-physician relationships; the need for support from health care authorities; and the need for improved marketing of cognitive services and their benefits to society, including physicians and health care authorities. Twelve facilitators were identified. Strategists considered clinical education and pharmacists' attitude as the most important, and

  5. Facilitation of learning: part 1.

    PubMed

    Warburton, Tyler; Trish, Houghton; Barry, Debbie

    2016-04-06

    This article, the fourth in a series of 11, discusses the context for the facilitation of learning. It outlines the main principles and theories for understanding the process of learning, including examples which link these concepts to practice. The practical aspects of using these theories in a practice setting will be discussed in the fifth article of this series. Together, these two articles will provide mentors and practice teachers with knowledge of the learning process, which will enable them to meet the second domain of the Nursing and Midwifery Council's Standards to Support Learning and Assessment in Practice on facilitation of learning.

  6. A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

    PubMed Central

    Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

    2012-01-01

    SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

  7. PDDL4J: a planning domain description library for java

    NASA Astrophysics Data System (ADS)

    Pellier, D.; Fiorino, H.

    2018-01-01

    PDDL4J (Planning Domain Description Library for Java) is an open source toolkit for Java cross-platform developers meant (1) to provide state-of-the-art planners based on the Pddl language, and (2) to facilitate research works on new planners. In this article, we present an overview of the Automated Planning concepts and languages. We present some planning systems and their most significant applications. Then, we detail the Pddl4j toolkit with an emphasis on the available informative structures, heuristics and search algorithms.

  8. Selecting soluble/foldable protein domains through single-gene or genomic ORF filtering: structure of the head domain of Burkholderia pseudomallei antigen BPSL2063.

    PubMed

    Gourlay, Louise J; Peano, Clelia; Deantonio, Cecilia; Perletti, Lucia; Pietrelli, Alessandro; Villa, Riccardo; Matterazzo, Elena; Lassaux, Patricia; Santoro, Claudio; Puccio, Simone; Sblattero, Daniele; Bolognesi, Martino

    2015-11-01

    The 1.8 Å resolution crystal structure of a conserved domain of the potential Burkholderia pseudomallei antigen and trimeric autotransporter BPSL2063 is presented as a structural vaccinology target for melioidosis vaccine development. Since BPSL2063 (1090 amino acids) hosts only one conserved domain, and the expression/purification of the full-length protein proved to be problematic, a domain-filtering library was generated using β-lactamase as a reporter gene to select further BPSL2063 domains. As a result, two domains (D1 and D2) were identified and produced in soluble form in Escherichia coli. Furthermore, as a general tool, a genomic open reading frame-filtering library from the B. pseudomallei genome was also constructed to facilitate the selection of domain boundaries from the entire ORFeome. Such an approach allowed the selection of three potential protein antigens that were also produced in soluble form. The results imply the further development of ORF-filtering methods as a tool in protein-based research to improve the selection and production of soluble proteins or domains for downstream applications such as X-ray crystallography.

  9. Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain1[OPEN

    PubMed Central

    Villarino, Gonzalo H.; Hu, Qiwen; Flores-Vergara, Miguel; Sehra, Bhupinder; Brumos, Javier; Stepanova, Anna N.; Sundberg, Eva; Heber, Steffen

    2016-01-01

    Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis. PMID:26983993

  10. Molecular Interaction Between Smurfl WW2 Domain and PPXY Motifs of Smadl, Smad5, and Smad6-Modeling and Analysis.

    PubMed

    Sangadala, Sreedhara; Rao Metpally, Raghu Prasad; B Reddy, Boojala Vijay

    2007-08-01

    Abstract The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1- interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smadl, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a

  11. Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

    PubMed

    Tanabe, Atsushi; Konno, Junpei; Tanikawa, Kenya; Sahara, Hiroeki

    2014-02-01

    We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Polar Domain Discovery with Sparkler

    NASA Astrophysics Data System (ADS)

    Duerr, R.; Khalsa, S. J. S.; Mattmann, C. A.; Ottilingam, N. K.; Singh, K.; Lopez, L. A.

    2017-12-01

    The scientific web is vast and ever growing. It encompasses millions of textual, scientific and multimedia documents describing research in a multitude of scientific streams. Most of these documents are hidden behind forms which require user action to retrieve and thus can't be directly accessed by content crawlers. These documents are hosted on web servers across the world, most often on outdated hardware and network infrastructure. Hence it is difficult and time-consuming to aggregate documents from the scientific web, especially those relevant to a specific domain. Thus generating meaningful domain-specific insights is currently difficult. We present an automated discovery system (Figure 1) using Sparkler, an open-source, extensible, horizontally scalable crawler which facilitates high throughput and focused crawling of documents pertinent to a particular domain such as information about polar regions. With this set of highly domain relevant documents, we show that it is possible to answer analytical questions about that domain. Our domain discovery algorithm leverages prior domain knowledge to reach out to commercial/scientific search engines to generate seed URLs. Subject matter experts then annotate these seed URLs manually on a scale from highly relevant to irrelevant. We leverage this annotated dataset to train a machine learning model which predicts the `domain relevance' of a given document. We extend Sparkler with this model to focus crawling on documents relevant to that domain. Sparkler avoids disruption of service by 1) partitioning URLs by hostname such that every node gets a different host to crawl and by 2) inserting delays between subsequent requests. With an NSF-funded supercomputer Wrangler, we scaled our domain discovery pipeline to crawl about 200k polar specific documents from the scientific web, within a day.

  13. Two conformations of the integrin A-domain (I-domain): a pathway for activation?

    PubMed

    Lee, J O; Bankston, L A; Arnaout, M A; Liddington, R C

    1995-12-15

    Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is associated with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. We have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. We now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equivalent of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 A) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-associated acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. Our observations provide the first atomic resolution view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. We propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.

  14. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    PubMed

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  15. The BID Domain of Type IV Secretion Substrates Forms a Conserved Four-Helix Bundle Topped with a Hook.

    PubMed

    Stanger, Frédéric V; de Beer, Tjaart A P; Dranow, David M; Schirmer, Tilman; Phan, Isabelle; Dehio, Christoph

    2017-01-03

    The BID (Bep intracellular delivery) domain functions as secretion signal in a subfamily of protein substrates of bacterial type IV secretion (T4S) systems. It mediates transfer of (1) relaxases and the attached DNA during bacterial conjugation, and (2) numerous Bartonella effector proteins (Beps) during protein transfer into host cells infected by pathogenic Bartonella species. Furthermore, BID domains of Beps have often evolved secondary effector functions within host cells. Here, we provide crystal structures for three representative BID domains and describe a novel conserved fold characterized by a compact, antiparallel four-helix bundle topped with a hook. The conserved hydrophobic core provides a rigid scaffold to a surface that, despite a few conserved exposed residues and similarities in charge distribution, displays significant variability. We propose that the genuine function of BID domains as T4S signal may primarily depend on their rigid structure, while the plasticity of their surface may facilitate adaptation to secondary effector functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

    PubMed Central

    Jin, Lily L.; Wybenga-Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

    2015-01-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y194 impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y194 on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. PMID:25587033

  17. The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding.

    PubMed

    Chen, Shugui; Brier, Sébastien; Smithgall, Thomas E; Engen, John R

    2007-04-01

    The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.

  18. TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism.

    PubMed

    Hori, Roderick T; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

    2004-01-01

    Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules--an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA.

  19. TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism

    PubMed Central

    Hori, Roderick T.; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

    2004-01-01

    Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules—an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA. PMID:15272087

  20. L2 Gender Facilitation and Inhibition in Spoken Word Recognition

    ERIC Educational Resources Information Center

    Behney, Jennifer N.

    2011-01-01

    This dissertation investigates the role of grammatical gender facilitation and inhibition in second language (L2) learners' spoken word recognition. Native speakers of languages that have grammatical gender are sensitive to gender marking when hearing and recognizing a word. Gender facilitation refers to when a given noun that is preceded by an…

  1. A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity.

    PubMed

    Mills, Jamie E; Whitford, Paul C; Shaffer, Jennifer; Onuchic, Jose N; Adams, Joseph A; Jennings, Patricia A

    2007-02-02

    The SH2 domain of the C-terminal Src kinase [Csk] contains a unique disulfide bond that is not present in other known SH2 domains. To investigate whether this unusual disulfide bond serves a novel function, the effects of disulfide bond formation on catalytic activity of the full-length protein and on the structure of the SH2 domain were investigated. The kinase activity of full-length Csk decreases by an order of magnitude upon formation of the disulfide bond in the distal SH2 domain. NMR spectra of the fully oxidized and fully reduced SH2 domains exhibit similar chemical shift patterns and are indicative of similar, well-defined tertiary structures. The solvent-accessible disulfide bond in the isolated SH2 domain is highly stable and far from the small lobe of the kinase domain. However, reduction of this bond results in chemical shift changes of resonances that map to a cluster of residues that extend from the disulfide bond across the molecule to a surface that is in direct contact with the small lobe of the kinase domain in the intact molecule. Normal mode analyses and molecular dynamics calculations suggest that disulfide bond formation has large effects on residues within the kinase domain, most notably within the active-site cleft. Overall, the data indicate that reversible cross-linking of two cysteine residues in the SH2 domain greatly impacts catalytic function and interdomain communication in Csk.

  2. Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

    PubMed Central

    Coussens, Nathan P.; Hayashi, Ryo; Brown, Patrick H.; Balagopalan, Lakshmi; Balbo, Andrea; Akpan, Itoro; Houtman, Jon C. D.; Barr, Valarie A.; Schuck, Peter; Appella, Ettore

    2013-01-01

    The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling. PMID:23979596

  3. Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells.

    PubMed

    Coussens, Nathan P; Hayashi, Ryo; Brown, Patrick H; Balagopalan, Lakshmi; Balbo, Andrea; Akpan, Itoro; Houtman, Jon C D; Barr, Valarie A; Schuck, Peter; Appella, Ettore; Samelson, Lawrence E

    2013-11-01

    The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155-7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.

  4. Creating and Manipulating a Domain-Specific Formal Object Base to Support a Domain-Oriented Application Composition System

    DTIC Science & Technology

    1992-12-01

    and add new attributes as needed (11:129). 2.2.3.2 Feature Oriented Domain Analysis In their Feature-Oriented Domain Analysis ( FODA ) study, the...dissertation, The University of Texas at Austin, Austin Texas, 1990. 12. Kang, Kyo C. and others. Feature-Oriented Domain Analysis ( FODA ) Feasibil- ity Study...2-1 2.2.2 Requirements Languages ..................... 2-2 2.2.3 Domain Analysis ............................ 2-3 2.2.4

  5. Structural insights into SAM domain-mediated tankyrase oligomerization.

    PubMed

    DaRosa, Paul A; Ovchinnikov, Sergey; Xu, Wenqing; Klevit, Rachel E

    2016-09-01

    Tankyrase 1 (TNKS1; a.k.a. ARTD5) and tankyrase 2 (TNKS2; a.k.a ARTD6) are highly homologous poly(ADP-ribose) polymerases (PARPs) that function in a wide variety of cellular processes including Wnt signaling, Src signaling, Akt signaling, Glut4 vesicle translocation, telomere length regulation, and centriole and spindle pole maturation. Tankyrase proteins include a sterile alpha motif (SAM) domain that undergoes oligomerization in vitro and in vivo. However, the SAM domains of TNKS1 and TNKS2 have not been structurally characterized and the mode of oligomerization is not yet defined. Here we model the SAM domain-mediated oligomerization of tankyrase. The structural model, supported by mutagenesis and NMR analysis, demonstrates a helical, homotypic head-to-tail polymer that facilitates TNKS self-association. Furthermore, we show that TNKS1 and TNKS2 can form (TNKS1 SAM-TNKS2 SAM) hetero-oligomeric structures mediated by their SAM domains. Though wild-type tankyrase proteins have very low solubility, model-based mutations of the SAM oligomerization interface residues allowed us to obtain soluble TNKS proteins. These structural insights will be invaluable for the functional and biophysical characterization of TNKS1/2, including the role of TNKS oligomerization in protein poly(ADP-ribosyl)ation (PARylation) and PARylation-dependent ubiquitylation. © 2016 The Protein Society.

  6. A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-delta1.

    PubMed

    Essen, L O; Perisic, O; Lynch, D E; Katan, M; Williams, R L

    1997-03-11

    We have determined the crystal structures of complexes of phosphoinositide-specific phospholipase C-delta1 from rat with calcium, barium, and lanthanum at 2.5-2.6 A resolution. Binding of these metal ions is observed in the active site of the catalytic TIM barrel and in the calcium binding region (CBR) of the C2 domain. The C2 domain of PLC-delta1 is a circularly permuted topological variant (P-variant) of the synaptotagmin I C2A domain (S-variant). On the basis of sequence analysis, we propose that both the S-variant and P-variant topologies are present among other C2 domains. Multiple adjacent binding sites in the C2 domain were observed for calcium and the other metal/enzyme complexes. The maximum number of binding sites observed was for the calcium analogue lanthanum. This complex shows an array-like binding of three lanthanum ions (sites I-III) in a crevice on one end of the C2 beta-sandwich. Residues involved in metal binding are contained in three loops, CBR1, CBR2, and CBR3. Sites I and II are maintained in the calcium and barium complexes, whereas sites II and III coincide with a binary calcium binding site in the C2A domain of synaptotagmin I. Several conformers for CBR1 are observed. The conformation of CBR1 does not appear to be strictly dependent on metal binding; however, metal binding may stabilize certain conformers. No significant structural changes are observed for CBR2 or CBR3. The surface of this ternary binding site provides a cluster of freely accessible liganding positions for putative phospholipid ligands of the C2 domain. It may be that the ternary metal binding site is also a feature of calcium-dependent phospholipid binding in solution. A ternary metal binding site might be a conserved feature among C2 domains that contain the critical calcium ligands in their CBR's. The high cooperativity of calcium-mediated lipid binding by C2 domains described previously is explained by this novel type of calcium binding site.

  7. Tyrosine phosphorylation of the Lyn Src homology 2 (SH2) domain modulates its binding affinity and specificity.

    PubMed

    Jin, Lily L; Wybenga-Groot, Leanne E; Tong, Jiefei; Taylor, Paul; Minden, Mark D; Trudel, Suzanne; McGlade, C Jane; Moran, Michael F

    2015-03-01

    Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y(194) impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y(194) on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

    PubMed

    Schiering, N; Casale, E; Caccia, P; Giordano, P; Battistini, C

    2000-11-07

    Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

  9. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

    PubMed

    Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Domain structure and reorientation in CoF e2O4

    NASA Astrophysics Data System (ADS)

    Abes, M.; Koops, C. T.; Hrkac, S. B.; McCord, J.; Urs, N. O.; Wolff, N.; Kienle, L.; Ren, W. J.; Bouchenoire, L.; Murphy, B. M.; Magnussen, O. M.

    2016-05-01

    The microscopic processes underlying magnetostriction in ferrites were studied for the case of CoF e2O4 single crystals by high-resolution in situ x-ray diffraction and complementary magnetic microscopy techniques. The data support the reports of Yang and Ren [Phys. Rev. B 77, 014407 (2008), 10.1103/PhysRevB.77.014407] that magnetostriction in these materials originates from the switching of crystallographic domains, similar to ferroelastic or ferroelectric domain switching, and reveals the presence of two coexisting tetragonal spinel structures, corresponding to domains of high and of low strain. The latter alternate in the crystal, separated by 90° domain boundaries, and can be explained by the effect of internal stress emerging during the transition into the ferrimagnetic phase. During magnetization of the sample two structural transitions are observed: a conversion of the transversal into axial domains at 1.95 kOe and a growth of the high-strain domains at the cost of the low-strain axial domains at 2.8 kOe. These microscopic changes are in good agreement with the macroscopic magnetization and magnetostriction behavior of CoF e2O4 .

  11. Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

    PubMed

    Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

    2016-11-20

    The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

  12. Atypical binding of the Swa2p UBA domain to ubiquitin.

    PubMed

    Matta-Camacho, Edna; Kozlov, Guennadi; Trempe, Jean-François; Gehring, Kalle

    2009-02-20

    Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix alpha1 and the N-terminal part of helix alpha2 bind to Ub. Mutation of Ala148, a key residue in helix alpha1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices alpha1 and alpha3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix alpha1 of UBA domains involved in membrane protein trafficking.

  13. Barriers and facilitators to disease-modifying antirheumatic drug use in patients with inflammatory rheumatic diseases: a qualitative theory-based study.

    PubMed

    Voshaar, Marieke; Vriezekolk, Johanna; van Dulmen, Sandra; van den Bemt, Bart; van de Laar, Mart

    2016-10-21

    Although disease-modifying anti-rheumatic drugs (DMARDs) are the cornerstone of treatment for inflammatory rheumatic diseases, medication adherence to DMARDs is often suboptimal. Effective interventions to improve adherence to DMARDs are lacking, and new targets are needed to improve adherence. The aim of the present study was to explore patients' barriers and facilitators of optimal DMARD use. These factors might be used as targets for adherence interventions. In a mixed method study design, patients (n = 120) with inflammatory arthritis (IA) completed a questionnaire based on an existing adapted Theoretical Domains Framework (TDF) to identify facilitators and barriers of DMARD use. A subgroup of these patients (n = 21) participated in focus groups to provide insights into their facilitators and barriers. The answers to the questionnaires and responses of the focus groups were thematically coded by three researchers independently and subsequently categorized. The barriers and facilitators that were reported by IA patients presented large inter-individual variations. The identified barriers and facilitators could be captured in the following domains based on an adapted TDF: (i) knowledge, (ii) emotions, (iii) attention, memory, and decision processes, (iv) social influences, (v) beliefs about capability, (vi) beliefs about consequences, (vii) motivation and goals, (viii) goal conflict, (ix) environmental context and resources, and (x) skills. Patients with IA have a variety of barriers and facilitators with regard to their DMARD use. All of these barriers and facilitators could be categorized into adapted domains of the TDF. Interventions that address individual facilitators and barriers, based on capability, opportunity, and motivation, are needed to develop strategies for medication adherence that are tailored to individual patient needs.

  14. Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

    PubMed

    Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

    2002-07-17

    Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

  15. Magnetic domain tuning and the emergence of bubble domains in the bilayer manganite La 2 - 2 x Sr 1 + 2 x Mn 2 O 7 ( x = 0.32 )

    DOE PAGES

    Jeong, Juyoung; Yang, Ilkyu; Yang, Jinho; ...

    2015-08-17

    Here, we report a magnetic force microscopy study of the magnetic domain evolution in the layered manganite La 22xSr 1+2xMn 2O 7 (with x = 0.32). This strongly correlated electron compound is known to exhibit a wide range of magnetic phases, including a recently uncovered biskyrmion phase. We observe a continuous transition from dendritic to stripelike domains, followed by the formation of magnetic bubbles due to a field- and temperature-dependent competition between in-plane and out-of-plane spin alignments. The magnetic bubble phase appears at comparable field and temperature ranges as the biskyrmion phase, suggesting a close relation between both phases. Basedmore » on our real-space images we construct a temperature-field phase diagram for this composition.« less

  16. SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

    PubMed

    Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

    2016-04-07

    The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

    PubMed Central

    Siaud, Nicolas; Lam, Isabel; Christ, Nicole; Schlacher, Katharina; Xia, Bing; Jasin, Maria

    2011-01-01

    The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations. PMID:22194698

  18. A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels.

    PubMed

    Tran, Neil; Proenza, Catherine; Macri, Vincenzo; Petigara, Fiona; Sloan, Erin; Samler, Shannon; Accili, Eric A

    2002-11-15

    Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.

  19. Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

    PubMed Central

    Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

    2010-01-01

    Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

  20. Facilitating Facilitators: Enhancing PBL through a Structured Facilitator Development Program

    ERIC Educational Resources Information Center

    Salinitri, Francine D.; Wilhelm, Sheila M.; Crabtree, Brian L.

    2015-01-01

    With increasing adoption of the problem-based learning (PBL) model, creative approaches to enhancing facilitator training and optimizing resources to maintain effective learning in small groups is essential. We describe a theoretical framework for the development of a PBL facilitator training program that uses the constructivist approach as the…

  1. Oncogenic JAK2V617F requires an intact SH2-like domain for constitutive activation and induction of a myeloproliferative disease in mice.

    PubMed

    Gorantla, Sivahari P; Dechow, Tobias N; Grundler, Rebekka; Illert, Anna Lena; Zum Büschenfelde, Christian Meyer; Kremer, Marcus; Peschel, Christian; Duyster, Justus

    2010-11-25

    The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

  2. Arid5b facilitates chondrogenesis by recruiting the histone demethylase Phf2 to Sox9-regulated genes

    NASA Astrophysics Data System (ADS)

    Hata, Kenji; Takashima, Rikako; Amano, Katsuhiko; Ono, Koichiro; Nakanishi, Masako; Yoshida, Michiko; Wakabayashi, Makoto; Matsuda, Akio; Maeda, Yoshinobu; Suzuki, Yutaka; Sugano, Sumio; Whitson, Robert H.; Nishimura, Riko; Yoneda, Toshiyuki

    2013-11-01

    Histone modification, a critical step for epigenetic regulation, is an important modulator of biological events. Sox9 is a transcription factor critical for endochondral ossification; however, proof of its epigenetic regulation remains elusive. Here we identify AT-rich interactive domain 5b (Arid5b) as a transcriptional co-regulator of Sox9. Arid5b physically associates with Sox9 and synergistically induces chondrogenesis. Growth of Arid5b-/- mice is retarded with delayed endochondral ossification. Sox9-dependent chondrogenesis is attenuated in Arid5b-deficient cells. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrogenic marker genes, H3K9me2 levels are increased in Arid5b-/- chondrocytes. Finally, we show that Phf2 knockdown inhibits Sox9-induced chondrocyte differentiation. Our findings establish an epigenomic mechanism of skeletal development, whereby Arid5b promotes chondrogenesis by facilitating Phf2-mediated histone demethylation of Sox9-regulated chondrogenic gene promoters.

  3. Exercise in patients with Type 2 diabetes: Facilitators and barriers - A qualitative study.

    PubMed

    Advika, T S; Idiculla, Jyothi; Kumari, S Jaya

    2017-01-01

    Diabetes is a major noncommunicable disease affecting more than 65 million Indians. Although treatment algorithms suggest lifestyle measures (diet and exercise) along with medications data regarding adherence to exercise as well as facilitators and barriers to the practice of physical activity in such patients are limited. Hence, this qualitative study was conducted. The objective of this study is to describe the factors which (1) Facilitated and (2) hindered the practice of regular exercise in patients with Type 2 diabetes. The study was conducted on 13 diabetic patients admitted to a tertiary care center in Bengaluru - St. John's Medical College Hospital, to explore factors that acted as facilitators and barriers to physical activity. Data saturation with the coded themes was achieved on interviewing 13 patients, after which, thematic analysis was done, and final themes reported. The age of the study participants (7 males, 6 females) ranged from 40 to 80 years. Among those who did exercise, factors such as awareness regarding the benefits of exercise and complications linked with diabetes, positive family support, and emphasis by nursing staff emerged as facilitators. Lack of time, obligations to others, inability to link exercise with blood sugar control, lack of perception of obesity as a health issue, inadequate emphasis by physicians, social/cultural issues, lack of infrastructure, and physical restriction were the factors that acted as barriers to physical activity. In addition to the above, a clear lack of adherence to standard guidelines, while advising patients by physicians was also noted. A comprehensive approach by both doctors and nurses, based on standard guidelines, could help in implementing adherence to exercise in patients with diabetes.

  4. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

    DOE PAGES

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.; ...

    2016-01-14

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

  5. Unfolding the HIV-1 reverse transcriptase RNase H domain – how to lose a molecular tug-of-war

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

    Zheng, Xunhai; Pedersen, Lars C.; Gabel, Scott A.

    Formation of the mature HIV-1 reverse transcriptase (RT) p66/p51 heterodimer requires subunit-specific processing of the p66/p66' homodimer precursor. Since the ribonuclease H (RH) domain contains an occult cleavage site located near its center, cleavage must occur either prior to folding or subsequent to unfolding. Recent NMR studies have identified a slow, subunit-specific RH domain unfolding process proposed to result from a residue tug-of-war between the polymerase and RH domains on the functionally inactive, p66' subunit. Here, we describe a structural comparison of the isolated RH domain with a domain swapped RH dimer that reveals several intrinsically destabilizing characteristics of themore » isolated domain that facilitate excursions of Tyr427 from its binding pocket and separation of helices B and D. These studies provide independent support for the subunit-selective RH domain unfolding pathway in which instability of the Tyr427 binding pocket facilitates its release followed by domain transfer, acting as a trigger for further RH domain destabilization and subsequent unfolding. As further support for this pathway, NMR studies demonstrate that addition of an RH active site-directed isoquinolone ligand retards the subunit-selective RH' domain unfolding behavior of the p66/p66' homodimer. As a result, this study demonstrates the feasibility of directly targeting RT maturation with therapeutics.« less

  6. Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

    PubMed

    Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

    2009-12-15

    Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

  7. The Experience of Native Peer Facilitators in the Campaign Against Type 2 Diabetes

    PubMed Central

    Struthers, Roxanne; Hodge, Felicia Schanche; De Cora, Lorelei; Geishirt-Cantrell, Betty

    2011-01-01

    Context The use of peer facilitators in health-programs has great potential. One important application is prevention and control of type 2 diabetes among American Indians. Purpose To explore the experience of American Indian facilitators in a culturally appropriate intervention (Talking Circles) on 2 Northern Plains reservations. The Talking Circles offered a forum for educational dialogue on diabetes risk factors and the management of type 2 diabetes. Methods Phenomenology, a qualitative research approach, was used to answer the research question: “What did Native Talking Circle facilitators-experience?” Participants were 4 lay health workers from the intervention reservations who had been trained to present a diabetes curriculum while coordinating and guiding the group discussion. During open-ended, taped interviews, the facilitators shared their experiences conducting the Talking Circles. Analysis categorized the experiences into common themes to explain the phenomena and cultural construction of oral discussions (Talking Circles) of diabetes. Findings Themes included the concept of “a calling” to do the work, which included a self-growth process, a blending of 2 worldviews as a diabetes intervention strategy, the importance of translating educational materials in a liaison role, and commitment to tribal people and communities. Conclusions The experience of the facilitators was positive because they were knowledgeable about American Indian culture arid worldview arid were trained in both Talking Circle facilitation and type 2 diabetes. PMID:12696854

  8. Ring-like oligomers of Synaptotagmins and related C2 domain proteins

    PubMed Central

    Zanetti, Maria N; Bello, Oscar D; Wang, Jing; Coleman, Jeff; Cai, Yiying; Sindelar, Charles V; Rothman, James E; Krishnakumar, Shyam S

    2016-01-01

    We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx. DOI: http://dx.doi.org/10.7554/eLife.17262.001 PMID:27434670

  9. Competition and cooperation among similar representations: toward a unified account of facilitative and inhibitory effects of lexical neighbors.

    PubMed

    Chen, Qi; Mirman, Daniel

    2012-04-01

    One of the core principles of how the mind works is the graded, parallel activation of multiple related or similar representations. Parallel activation of multiple representations has been particularly important in the development of theories and models of language processing, where coactivated representations (neighbors) have been shown to exhibit both facilitative and inhibitory effects on word recognition and production. Researchers generally ascribe these effects to interactive activation and competition, but there is no unified explanation for why the effects are facilitative in some cases and inhibitory in others. We present a series of simulations of a simple domain-general interactive activation and competition model that is broadly consistent with more specialized domain-specific models of lexical processing. The results showed that interactive activation and competition can indeed account for the complex pattern of reversals. Critically, the simulations revealed a core computational principle that determines whether neighbor effects are facilitative or inhibitory: strongly active neighbors exert a net inhibitory effect, and weakly active neighbors exert a net facilitative effect.

  10. Molecular interaction between Smurf1 WW2 domain and PPXY motifs of Smad1, Smad5, and Smad6--modeling and analysis.

    PubMed

    Sangadala, Sreedhara; Metpally, Raghu Prasad Rao; Reddy, Boojala Vijay B

    2007-08-01

    The ubiquitin-proteasome proteolytic pathway is essential for various important biological processes including cell cycle progression, gene transcription, and signal transduction. One of the important regulatory mechanisms by which the bone-inducing activity of the bone morphogenetic protein (BMP) signaling is modulated involves ubiquitin-mediated proteasomal degradation. The BMP induced receptor signal is transmitted intracellularly by phosphorylation of Smad proteins by the activated receptor I. The phosphorylated Smads 1, 5, and 8 (R-Smads) oligomerize with the co-Smad (Smad4). The complex, thus, formed translocates to the nucleus and interacts with other cofactors to regulate the expression of downstream target genes. R-Smads contain PPXY motif in the linker region that interacts with Smad ubiquitin regulatory factor 1 (Smurf1), an E3 ubiquitin ligase that catalyzes ubiquitination of target proteins for proteasomal degradation. Smurf1 contains a HECT domain, a C2 domain, and 2 WW domains (WW1, WW2). The PPXY motif in target proteins and its interaction with Smurf1 may form the basis for regulation of steady-state levels of Smads in controlling BMP-responsiveness of cells. Here, we present a homology-based model of the Smurf1 WW2 domain and the target octa-peptides containing PPXY motif of Smurf1-interacting Smads. We carried out docking of Smurf1 WW2 domain with the PPXY motifs of Smad1, Smad5, and Smad6 and identified the key amino acid residues involved in interaction. Furthermore, we present experimental evidence that WW2 domain of Smurf1 does indeed interact with the Smad proteins and that the deletion of WW2 domain of Smurf1 results in loss of its binding to Smads using the purified recombinant proteins. Finally, we also present data confirming that the deletion of WW2 domain in Smurf1 abolishes its ubiquitination activity on Smad1 in an in vitro ubiquitination assay. It shows that the interaction between the WW domain and Smad PPXY motif is a key step in

  11. Functional innovation from changes in protein domains and their combinations.

    PubMed

    Lees, Jonathan G; Dawson, Natalie L; Sillitoe, Ian; Orengo, Christine A

    2016-06-01

    Domains are the functional building blocks of proteins. In this work we discuss how domains can contribute to the evolution of new functions. Domains themselves can evolve through various mechanisms, altering their intrinsic function. Domains can also facilitate functional innovations by combining with other domains to make novel proteins. We discuss the mechanisms by which domain and domain combinations support functional innovations. We highlight interesting examples where changes in domain combination promote changes at the domain level. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Characterization of substrate binding of the WW domains in human WWP2 protein.

    PubMed

    Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

    2015-07-08

    WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  13. Discovery and Characterization of Cadherin Domains in Saccharophagus degradans 2-40▿ †

    PubMed Central

    Fraiberg, Milana; Borovok, Ilya; Weiner, Ronald M.; Lamed, Raphael

    2010-01-01

    Saccharophagus degradans strain 2-40 is a prominent member of newly discovered group of marine and estuarine bacteria that recycle complex polysaccharides. The S. degradans 2-40 genome codes for 15 extraordinary long polypeptides, ranging from 274 to 1,600 kDa. Five of these contain at least 52 cadherin (CA) and cadherin-like (CADG) domains, the types of which were reported to bind calcium ions and mediate protein/protein interactions in metazoan systems. In order to evaluate adhesive features of these domains, recombinant CA doublet domains (two neighboring domains) from CabC (Sde_3323) and recombinant CADG doublet domains from CabD (Sde_0798) were examined qualitatively and quantitatively for homophilic and heterophilic interactions. In addition, CA and CADG doublet domains were tested for adhesion to the surface of S. degradans 2-40. Results showed obvious homophilic and heterophilic, calcium ion-dependent interactions between CA and CADG doublet domains. Likewise, CA and CADG doublet domains adhered to the S. degradans 2-40 surface of cells that were grown on xylan from birch wood or pectin, respectively, as a sole carbon source. This research shows for the first time that bacterial cadherin homophilic and heterophilic interactions may be similar in their nature to cadherin domains from metazoan lineages. We hypothesize that S. degradans 2-40 cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment. PMID:20023015

  14. Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins

    PubMed Central

    Watada, Hirotaka; Mirmira, Raghavendra G.; Kalamaras, Julie; German, Michael S.

    2000-01-01

    The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein–protein interaction interface. To assess the possibility that the NK2-SD may contribute to DNA-binding specificity, we used a PCR-based approach to identify a consensus DNA-binding sequences for Nkx2.2, an NK-2 family member involved in pancreas and central nervous system development. The consensus sequence (TCTAAGTGAGCTT) is similar to the known binding sequences for other NK-2 homeodomain proteins, but we show that the NK2-SD does not contribute significantly to specific DNA binding to this sequence. To determine whether the NK2-SD contributes to transactivation, we used GAL4-Nkx2.2 fusion constructs to map a powerful transcriptional activation domain in the C-terminal region beyond the conserved NK2-SD. Interestingly, this C-terminal region functions as a transcriptional activator only in the absence of an intact NK2-SD. The NK2-SD also can mask transactivation from the paired homeodomain transcription factor Pax6, but it has no effect on transcription by itself. These results demonstrate that the NK2-SD functions as an intramolecular regulator of the C-terminal activation domain in Nkx2.2 and support a model in which interactions through the NK2-SD regulate the ability of NK-2-class proteins to activate specific genes during development. PMID:10944215

  15. Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling.

    PubMed

    D'Cruz, Akshay A; Kershaw, Nadia J; Chiang, Jessica J; Wang, May K; Nicola, Nicos A; Babon, Jeffrey J; Gack, Michaela U; Nicholson, Sandra E

    2013-12-01

    TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp(488) and Trp(621)) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.

  16. Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling

    PubMed Central

    D'Cruz, Akshay A.; Kershaw, Nadia J.; Chiang, Jessica J.; Wang, May K.; Nicola, Nicos A.; Babon, Jeffrey J.; Gack, Michaela U.; Nicholson, Sandra E.

    2014-01-01

    TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs. PMID:24015671

  17. Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

    PubMed

    Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

    1994-04-01

    We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.

  18. Making cognitive decision support work: Facilitating adoption, knowledge and behavior change through QI.

    PubMed

    Weir, Charlene; Brunker, Cherie; Butler, Jorie; Supiano, Mark A

    2017-07-01

    This paper evaluates the role of facilitation in the successful implementation of Computerized Decision Support (CDS). Facilitation processes include education, specialized computerized decision support, and work process reengineering. These techniques, as well as modeling and feedback enhance self-efficacy, which we propose is one of the factors that mediate the effectiveness of any CDS. In this study, outpatient clinics implemented quality improvement (QI) projects focused on improving geriatric care. Quality Improvement is the systematic process of improving quality through continuous measurement and targeted actions. The program, entitled "Advancing Geriatric Education through Quality Improvement" (AGE QI), consisted of a 6-month, QI based, intervention: (1) 2h didactic session, (2) 1h QI planning session, (3) computerized decision support design and implementation, (4) QI facilitation activities, (5) outcome feedback, and (6) 20h of CME. Specifically, we examined the impact of the QI based program on clinician's perceived self-efficacy in caring for older adults and the relationship of implementation support and facilitation on perceived success. The intervention was implemented at 3 institutions, 27 community healthcare system clinics, and 134 providers. This study reports the results of pre/post surveys for the forty-nine clinicians who completed the full CME program. Self-efficacy ratings for specific clinical behaviors related to care of older adults were assessed using a Likert based instrument. Self-ratings of efficacy improved across the following domains (depression, falls, end-of-life, functional status and medication management) and specifically in QI targeted domains and were associated with overall clinic improvements. Published by Elsevier Inc.

  19. Do You Hear What I Hear? The Impact of a Hearing Voices Simulation on Affective Domain Attributes in Nursing Students.

    PubMed

    Ward, Terry D

    2015-01-01

    Affective domain teaching and learning can facilitate the reduction of stigmatization of clients with mental illness in nursing students. Experiential learning activities such as simulation are regarded as an effective method for facilitating student learning in the affective domain. The project reported here measured the impact of a simulation experience, "Hearing Voices Which Are Distressing," on attitudes, values, and beliefs of accelerated baccalaureate students caring for clients with mental illness who experienced hearing voices.

  20. SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

    PubMed

    Register, A C; Leonard, Stephen E; Maly, Dustin J

    2014-11-11

    Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

  1. Structure of a new crystal form of human Hsp70 ATPase domain.

    PubMed

    Osipiuk, J; Walsh, M A; Freeman, B C; Morimoto, R I; Joachimiak, A

    1999-05-01

    Hsp70 proteins are highly conserved proteins induced by heat shock and other stress conditions. An ATP-binding domain of human Hsp70 protein has been crystallized in two major morphological forms at pH 7.0 in the presence of PEG 8000 and CaCl2. Both crystal forms belong to the orthorhombic space group P212121, but show no resemblance in unit-cell parameters. Analysis of the crystal structures for both forms shows a 1-2 A shift of one of the subdomains of the protein. This conformational change could reflect a 'natural' flexibility of the protein which might be relevant to ATP binding and may facilitate the interaction of other proteins with Hsp70 protein.

  2. In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

    PubMed

    Côté, Jean-François; Vuori, Kristiina

    2006-01-01

    Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

  3. Uniform rotating field network structure to efficiently package a magnetic bubble domain memory

    NASA Technical Reports Server (NTRS)

    Murray, Glen W. (Inventor); Chen, Thomas T. (Inventor); Wolfshagen, Ronald G. (Inventor); Ypma, John E. (Inventor)

    1978-01-01

    A unique and compact open coil rotating magnetic field network structure to efficiently package an array of bubble domain devices is disclosed. The field network has a configuration which effectively enables selected bubble domain devices from the array to be driven in a vertical magnetic field and in an independent and uniform horizontal rotating magnetic field. The field network is suitably adapted to minimize undesirable inductance effects, improve capabilities of heat dissipation, and facilitate repair or replacement of a bubble device.

  4. Conformation switching of AIM2 PYD domain revealed by NMR relaxation and MD simulation.

    PubMed

    Wang, Haobo; Yang, Lijiang; Niu, Xiaogang

    2016-04-29

    Protein absent in melanoma 2 (AIM2) is a double-strand DNA (ds DNA) sensor mainly located in cytoplasm of cell. It includes one N terminal PYD domain and one C terminal HIN domain. When the ds DNA such as DNA viruses and bacteria entered cytoplasm, the HIN domain of AIM2 will recognize and bind to DNA, and the PYD domain will bind to ASC protein which will result in the formation of AIM2 inflammasome. Three AIM2 PYD domain structures have been solved, but every structure yields a unique conformation around the α3 helix region. To understand why different AIM2 PYD structures show different conformations in this region, we use NMR relaxation techniques to study the backbone dynamics of mouse AIM2 PYD domain and perform molecular dynamics (MD) simulations on both mouse and human AIM2 PYD structures. Our results indicate that this region is highly flexible in both mouse and human AIM2 PYD domains, and the PYD domain may exist as a conformation ensemble in solution. Different environment makes the population vary among pre-existing conformational substrates of the ensemble, which may be the reason why different AIM2 PYD structures were observed under different conditions. Further docking analysis reveals that the conformation switching may be important for the autoinhibition of the AIM2 protein. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Life sciences domain analysis model

    PubMed Central

    Freimuth, Robert R; Freund, Elaine T; Schick, Lisa; Sharma, Mukesh K; Stafford, Grace A; Suzek, Baris E; Hernandez, Joyce; Hipp, Jason; Kelley, Jenny M; Rokicki, Konrad; Pan, Sue; Buckler, Andrew; Stokes, Todd H; Fernandez, Anna; Fore, Ian; Buetow, Kenneth H

    2012-01-01

    Objective Meaningful exchange of information is a fundamental challenge in collaborative biomedical research. To help address this, the authors developed the Life Sciences Domain Analysis Model (LS DAM), an information model that provides a framework for communication among domain experts and technical teams developing information systems to support biomedical research. The LS DAM is harmonized with the Biomedical Research Integrated Domain Group (BRIDG) model of protocol-driven clinical research. Together, these models can facilitate data exchange for translational research. Materials and methods The content of the LS DAM was driven by analysis of life sciences and translational research scenarios and the concepts in the model are derived from existing information models, reference models and data exchange formats. The model is represented in the Unified Modeling Language and uses ISO 21090 data types. Results The LS DAM v2.2.1 is comprised of 130 classes and covers several core areas including Experiment, Molecular Biology, Molecular Databases and Specimen. Nearly half of these classes originate from the BRIDG model, emphasizing the semantic harmonization between these models. Validation of the LS DAM against independently derived information models, research scenarios and reference databases supports its general applicability to represent life sciences research. Discussion The LS DAM provides unambiguous definitions for concepts required to describe life sciences research. The processes established to achieve consensus among domain experts will be applied in future iterations and may be broadly applicable to other standardization efforts. Conclusions The LS DAM provides common semantics for life sciences research. Through harmonization with BRIDG, it promotes interoperability in translational science. PMID:22744959

  6. MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

    PubMed

    Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

    2013-01-01

    The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

  7. Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2.

    PubMed

    Zheng, Wang; Cai, Ruiqi; Hofmann, Laura; Nesin, Vasyl; Hu, Qiaolin; Long, Wentong; Fatehi, Mohammad; Liu, Xiong; Hussein, Shaimaa; Kong, Tim; Li, Jingru; Light, Peter E; Tang, Jingfeng; Flockerzi, Veit; Tsiokas, Leonidas; Chen, Xing-Zhen

    2018-02-06

    Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Structure and electrostatic property of cytoplasmic domain of ZntB transporter

    PubMed Central

    Tan, Kemin; Sather, Alicia; Robertson, Janice L; Moy, Shiu; Roux, Benoît; Joachimiak, Andrzej

    2009-01-01

    ZntB is the distant homolog of CorA Mg2+ transporter within the metal ion transporter superfamily. It was early reported that the ZntB from Salmonella typhimurium facilitated efflux of Zn2+ and Cd2+, but not Mg2+. Here, we report the 1.90 Å crystal structure of the intracellular domain of ZntB from Vibrio parahemolyticus. The domain forms a funnel-shaped homopentamer that is similar to the full-length CorA from Thermatoga maritima, but differs from two previously reported dimeric structures of truncated CorA intracellular domains. However, no Zn2+ or Cd2+ binding sites were identified in the high-resolution structure. Instead, 25 well-defined Cl− ions were observed and some of these binding sites are highly conserved within the ZntB family. Continuum electrostatics calculations suggest that the central pore of the funnel is highly attractive for cations, especially divalents. The presence of the bound Cl− ions increases the stability of cations along the pore suggesting they could be important in enhancing cation transport. PMID:19653298

  9. Regulation of Telomere Length Requires a Conserved N-Terminal Domain of Rif2 in Saccharomyces cerevisiae

    PubMed Central

    Kaizer, Hannah; Connelly, Carla J.; Bettridge, Kelsey; Viggiani, Christopher; Greider, Carol W.

    2015-01-01

    The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation. PMID:26294668

  10. Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

    PubMed

    Liu, Bernard A

    2017-01-01

    Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

  11. Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

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

    Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.

    Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturatingmore » conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

  12. A single amino acid difference between the intracellular domains of amyloid precursor protein and amyloid-like precursor protein 2 enables induction of synaptic depression and block of long-term potentiation.

    PubMed

    Trillaud-Doppia, Emilie; Paradis-Isler, Nicolas; Boehm, Jannic

    2016-07-01

    Alzheimer disease (AD) is initially characterized as a disease of the synapse that affects synaptic transmission and synaptic plasticity. While amyloid-beta and tau have been traditionally implicated in causing AD, recent studies suggest that other factors, such as the intracellular domain of the amyloid-precursor protein (APP-ICD), can also play a role in the development of AD. Here, we show that the expression of APP-ICD induces synaptic depression, while the intracellular domain of its homolog amyloid-like precursor protein 2 (APLP2-ICD) does not. We are able to show that this effect by APP-ICD is due to a single alanine vs. proline difference between APP-ICD and APLP2-ICD. The alanine in APP-ICD and the proline in APLP2-ICD lie directly behind a conserved caspase cleavage site. Inhibition of caspase cleavage of APP-ICD prevents the induction of synaptic depression. Finally, we show that the expression of APP-ICD increases and facilitates long-term depression and blocks induction of long-term potentiation. The block in long-term potentiation can be overcome by mutating the aforementioned alanine in APP-ICD to the proline of APLP2. Based on our results, we propose the emergence of a new APP critical domain for the regulation of synaptic plasticity and in consequence for the development of AD. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Synthesis of correlation filters: a generalized space-domain approach for improved filter characteristics

    NASA Astrophysics Data System (ADS)

    Sudharsanan, Subramania I.; Mahalanobis, Abhijit; Sundareshan, Malur K.

    1990-12-01

    Discrete frequency domain design of Minimum Average Correlation Energy filters for optical pattern recognition introduces an implementational limitation of circular correlation. An alternative methodology which uses space domain computations to overcome this problem is presented. The technique is generalized to construct an improved synthetic discriminant function which satisfies the conflicting requirements of reduced noise variance and sharp correlation peaks to facilitate ease of detection. A quantitative evaluation of the performance characteristics of the new filter is conducted and is shown to compare favorably with the well known Minimum Variance Synthetic Discriminant Function and the space domain Minimum Average Correlation Energy filter, which are special cases of the present design.

  14. The Sam Domain of EphA2 Receptor and its Relevance to Cancer: A Novel Challenge for Drug Discovery?

    PubMed

    Mercurio, Flavia A; Leone, Marilisa

    2016-01-01

    Eph receptors play important functions in developmental processes and diseases and among them EphA2 is well known for its controversial role in cancer. Drug discovery strategies are mainly centered on EphA2 extracellular ligand-binding domain however, the receptor also contains a largely unexplored cytosolic Sam (Sterile alpha motif) domain at the C-terminus. EphA2-Sam binds the Sam domain from the lipid phosphatase Ship2 and the first Sam domain of Odin. Sam-Sam interactions may be important to regulate ligand-induced receptor endocytosis and degradation i.e., processes that could be engaged against tumor malignancy. We critically analyzed literature related to a) Eph receptors with particular emphasis on EphA2 and its role in cancer, b) Sam domains, c) heterotypic Sam-Sam interactions involving EphA2-Sam. While literature data indicate that binding of EphA2-Sam to Ship2-Sam should largely generate pro-oncogenic effects in cancer cells, the correlation between EphA2- Sam/Odin-Sam1 complex and the disease is unclear. Recently a few linear peptides encompassing binding interfaces from either Ship2-Sam and Odin-Sam1 have been characterized but failed to efficiently block heterotypic Sam-Sam interactions involving EphA2-Sam due to the lack of a native like fold. Molecule antagonists of heterotypic EphA2-Sam associations could work as potential anticancer agents or be implemented as tools to further clarify receptor functions and eventually validate its role as a novel target in the field of anti-cancer drug discovery. Due to the failure of linear peptides there is a crucial need for novel approaches, based on cyclic or helical molecules, to target Sam-Sam interfaces.

  15. Structural Characterization of Monomeric/Dimeric State of p59fyn SH2 Domain.

    PubMed

    Huculeci, Radu; Kieken, Fabien; Garcia-Pino, Abel; Buts, Lieven; van Nuland, Nico; Lenaerts, Tom

    2017-01-01

    Src homology 2 (SH2) domains are key modulators in various signaling pathways allowing the recognition of phosphotyrosine sites of different proteins. Despite the fact that SH2 domains acquire their biological functions in a monomeric state, a multitude of reports have shown their tendency to dimerize. Here, we provide a technical description on how to isolate and characterize by gel filtration, circular dichroism (CD), and nuclear magnetic resonance (NMR) each conformational state of p59 fyn SH2 domain.

  16. Implementation of the BETTER 2 program: a qualitative study exploring barriers and facilitators of a novel way to improve chronic disease prevention and screening in primary care.

    PubMed

    Sopcak, Nicolette; Aguilar, Carolina; O'Brien, Mary Ann; Nykiforuk, Candace; Aubrey-Bassler, Kris; Cullen, Richard; Grunfeld, Eva; Manca, Donna Patricia

    2016-12-01

    BETTER (Building on Existing Tools to Improve Chronic Disease Prevention and Screening in Primary Care) is a patient-based intervention to improve chronic disease prevention and screening (CDPS) for cardiovascular disease, diabetes, cancer, and associated lifestyle factors in patients aged 40 to 65. The key component of BETTER is a prevention practitioner (PP), a health care professional with specialized skills in CDPS who meets with patients to develop a personalized prevention prescription, using the BETTER toolkit and Brief Action Planning. The purpose of this qualitative study was to understand facilitators and barriers of the implementation of the BETTER 2 program among clinicians, patients, and stakeholders in three (urban, rural, and remote) primary care settings in Newfoundland and Labrador, Canada. We collected and analyzed responses from 20 key informant interviews and 5 focus groups, as well as memos and field notes. Data were organized using Nvivo 10 software and coded using constant comparison methods. We then employed the Consolidated Framework for Implementation Research (CFIR) to focus our analysis on the domains most relevant for program implementation. The following key elements, within the five CFIR domains, were identified as impacting the implementation of BETTER 2: (1) intervention characteristics-complexity and cost of the intervention; (2) outer setting-perception of fit including lack of remuneration, lack of resources, and duplication of services, as well as patients' needs as perceived by physicians and patients; (3) characteristics of prevention practitioners-interest in prevention and ability to support and motivate patients; (4) inner setting-the availability of a local champion and working in a team versus working as a team; and (5) process-planning and engaging, collaboration, and teamwork. The implementation of a novel CDPS program into new primary care settings is a complex, multi-level process. This study identified key elements

  17. Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

    PubMed

    Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

    2017-01-01

    Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

    PubMed Central

    Sherman, A; Keizer, J; Rinzel, J

    1990-01-01

    The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells. PMID:2174274

  19. Occupancy of a C2-C2 type 'zinc-finger' protein domain by copper. Direct observation by electrospray ionization mass spectrometry.

    PubMed

    Hutchens, T W; Allen, M H; Li, C M; Yip, T T

    1992-09-07

    The metal ion specificity of most 'zinc-finger' metal binding domains is unknown. The human estrogen receptor protein contains two different C2-C2 type 'zinc-finger' sequences within its DNA-binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71-residue ERDBD (K180-M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA-binding domain to bind Cu and (ii) document a twofold difference in the Zn- and Cu-binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc-finger domains both in vitro and in vivo.

  20. Formin homology 2 domains occur in multiple contexts in angiosperms

    PubMed Central

    Cvrčková, Fatima; Novotný, Marian; Pícková, Denisa; Žárský, Viktor

    2004-01-01

    Background Involvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions. Results In order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis. Conclusions The plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity. PMID:15256004

  1. The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

    PubMed

    Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

    2012-11-29

    The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

  2. Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2.

    PubMed

    Xu, Emma-Ruoqi; Blythe, Emily E; Fischer, Gerhard; Hyvönen, Marko

    2017-07-28

    Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signaling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. Although the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC/BMP-binding mechanism, other vWC domains may bind to BMP differently. Here, using X-ray crystallography, we present for the first time structures of the vWC domains of two proteins thought to interact with BMP-2: collagen IIA and matricellular protein CCN3. We found that these two vWC domains share a similar N-terminal fold that differs greatly from that in CV-2 vWC, which comprises its BMP-2-binding site. We analyzed the ability of these vWC domains to directly bind to BMP-2 and detected an interaction only between the collagen IIa vWC and BMP-2. Guided by the collagen IIa vWC domain crystal structure and conservation of surface residues among orthologous domains, we mapped the BMP-binding epitope on the subdomain 1 of the vWC domain. This binding site is different from that previously observed in the complex between CV-2 vWC and BMP-2, revealing an alternative mode of interaction between vWC domains and BMPs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Both LOV1 and LOV2 domains of phototropin2 function as the photosensory domain for hypocotyl phototropic responses in Arabidopsis thaliana (Brassicaceae).

    PubMed

    Suetsugu, Noriyuki; Kong, Sam-Geun; Kasahara, Masahiro; Wada, Masamitsu

    2013-01-01

    Phototropins (phot) are blue light receptor proteins that mediate phototropism and control photomovement responses, such as chloroplast photorelocation movement and stomatal opening. Arabidopsis thaliana has two phototropins, phot1 and phot2. Although both phot1 and phot2 redundantly mediate photomovement responses, phot2 uniquely regulates phototropism and the chloroplast avoidance response under high-intensity blue light. However, compared to that of phot1, the mechanistic basis of phot2 function is poorly understood, and in particular, the importance of the LOV2 domain in phot2 function has not been clearly demonstrated. Indeed, photocycle-deficient LOV2 transgenic lines expressing phot2 in a phot1phot2 mutant background retained phototropism, although with less sensitivity than wild-type plants. We isolated 11 alleles of phot2 mutants and determined the molecular lesion in each allele. We analyzed hypocotyl phototropism, chloroplast photorelocation movement, and leaf flattening in the phot2 mutant and the respective phot1phot2 double mutant plants. We demonstrated that unlike the phot2 null mutant, the phot2-10 mutant, which has the defective phot2 LOV2 domain, retained the phototropic response and had unusual chloroplast movement. Mutants phot2-2 and phot2-6, which have a missense mutation in the kinase activation loop of phot2, had the phot2-null mutant phenotype. Furthermore, we convincingly demonstrated that the commonly used phot2-1 mutant allele is a phot2-null mutant. The analyses of the multiple phot2 mutant alleles provided strong evidence for the importance of both LOV domains and the kinase activation loop of phot2 in phototropism and other phot-dependent responses and also demonstrated that phot2-1 allele is a null mutant.

  4. Presence of an SH2 domain in the actin-binding protein tensin.

    PubMed

    Davis, S; Lu, M L; Lo, S H; Lin, S; Butler, J A; Druker, B J; Roberts, T M; An, Q; Chen, L B

    1991-05-03

    The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin bound specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin was also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton.

  5. Ca2+ binding and conformational changes in a calmodulin domain.

    PubMed

    Evenäs, J; Malmendal, A; Thulin, E; Carlström, G; Forsén, S

    1998-09-29

    Calcium activation of the C-terminal domain of calmodulin was studied using 1H and 15N NMR spectroscopy. The important role played by the conserved bidentate glutamate Ca2+ ligand in the binding loops is emphasized by the striking effects resulting from a mutation of this glutamic acid to a glutamine, i.e. E104Q in loop III and E140Q in loop IV. The study involves determination of Ca2+ binding constants, assignments, and structural characterizations of the apo, (Ca2+)1, and (Ca2+)2 states of the E104Q mutant and comparisons to the wild-type protein and the E140Q mutant [Evenäs et al. (1997) Biochemistry 36, 3448-3457]. NMR titration data show sequential Ca2+ binding in the E104Q mutant. The first Ca2+ binds to loop IV and the second to loop III, which is the order reverse to that observed for the E140Q mutant. In both mutants, the major structural changes occur upon Ca2+ binding to loop IV, which implies a different response to Ca2+ binding in the N- and C-terminal EF-hands. Spectral characteristics show that the (Ca2+)1 and (Ca2+)2 states of the E104Q mutant undergo global exchange on a 10-100 micros time scale between conformations seemingly similar to the closed and open structures of this domain in wild-type calmodulin, paralleling earlier observations for the (Ca2+)2 state of the E140Q mutant, indicating that both glutamic acid residues, E104 and E140, are required for stabilization of the open conformation in the (Ca2+)2 state. To verify that the NOE constraints cannot be fulfilled in a single structure, solution structures of the (Ca2+)2 state of the E104Q mutant are calculated. Within the ensemble of structures the precision is good. However, the clearly dynamic nature of the state, a large number of violated distance restraints, ill-defined secondary structural elements, and comparisons to the structures of calmodulin indicate that the ensemble does not provide a good picture of the (Ca2+)2 state of the E104Q mutant but rather represents the distance

  6. The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

    PubMed Central

    Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

    2013-01-01

    Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into

  7. Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

    PubMed

    Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

    2014-01-21

    Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Online interprofessional education facilitation: A scoping review.

    PubMed

    Evans, Sherryn Maree; Ward, Catherine; Reeves, Scott

    2018-04-22

    The use of online media to deliver interprofessional education (IPE) is becoming more prevalent across health professions education settings. Facilitation of IPE activities is known to be critical to the effective delivery of IPE, however, specifics about the nature of online IPE facilitation remains unclear. To explore the health professions education literature to understand the extent, range and nature of research on online IPE facilitation. Scoping review methodology was used to guide a search of four electronic databases for relevant papers. Of the 2095 abstracts initially identified, after screening of both abstracts and full-text papers, 10 studies were selected for inclusion in this review. Following abstraction of key information from each study, a thematic analysis was undertaken. Three key themes emerged to describe the nature of the IPE facilitation literature: (1) types of online IPE facilitation contributions, (2) the experience of online IPE facilitation and (3) personal outcomes of online IPE facilitation. These IPE facilitation themes were particularly focused on facilitation of interprofessional student teams on an asynchronous basis. While the included studies provide some insight into the nature of online IPE facilitation, future research is needed to better understand facilitator contributions, and the facilitation experience and associated outcomes, both relating to synchronous and asynchronous online environments.

  9. MiDas: Automatic Extraction of a Common Domain of Discourse in Sleep Medicine for Multi-center Data Integration

    PubMed Central

    Sahoo, Satya S.; Ogbuji, Chimezie; Luo, Lingyun; Dong, Xiao; Cui, Licong; Redline, Susan S.; Zhang, Guo-Qiang

    2011-01-01

    Clinical studies often use data dictionaries with controlled sets of terms to facilitate data collection, limited interoperability and sharing at a local site. Multi-center retrospective clinical studies require that these data dictionaries, originating from individual participating centers, be harmonized in preparation for the integration of the corresponding clinical research data. Domain ontologies are often used to facilitate multi-center data integration by modeling terms from data dictionaries in a logic-based language, but interoperability among domain ontologies (using automated techniques) is an unresolved issue. Although many upper-level reference ontologies have been proposed to address this challenge, our experience in integrating multi-center sleep medicine data highlights the need for an upper level ontology that models a common set of terms at multiple-levels of abstraction, which is not covered by the existing upper-level ontologies. We introduce a methodology underpinned by a Minimal Domain of Discourse (MiDas) algorithm to automatically extract a minimal common domain of discourse (upper-domain ontology) from an existing domain ontology. Using the Multi-Modality, Multi-Resource Environment for Physiological and Clinical Research (Physio-MIMI) multi-center project in sleep medicine as a use case, we demonstrate the use of MiDas in extracting a minimal domain of discourse for sleep medicine, from Physio-MIMI’s Sleep Domain Ontology (SDO). We then extend the resulting domain of discourse with terms from the data dictionary of the Sleep Heart and Health Study (SHHS) to validate MiDas. To illustrate the wider applicability of MiDas, we automatically extract the respective domains of discourse from 6 sample domain ontologies from the National Center for Biomedical Ontologies (NCBO) and the OBO Foundry. PMID:22195180

  10. MiDas: automatic extraction of a common domain of discourse in sleep medicine for multi-center data integration.

    PubMed

    Sahoo, Satya S; Ogbuji, Chimezie; Luo, Lingyun; Dong, Xiao; Cui, Licong; Redline, Susan S; Zhang, Guo-Qiang

    2011-01-01

    Clinical studies often use data dictionaries with controlled sets of terms to facilitate data collection, limited interoperability and sharing at a local site. Multi-center retrospective clinical studies require that these data dictionaries, originating from individual participating centers, be harmonized in preparation for the integration of the corresponding clinical research data. Domain ontologies are often used to facilitate multi-center data integration by modeling terms from data dictionaries in a logic-based language, but interoperability among domain ontologies (using automated techniques) is an unresolved issue. Although many upper-level reference ontologies have been proposed to address this challenge, our experience in integrating multi-center sleep medicine data highlights the need for an upper level ontology that models a common set of terms at multiple-levels of abstraction, which is not covered by the existing upper-level ontologies. We introduce a methodology underpinned by a Minimal Domain of Discourse (MiDas) algorithm to automatically extract a minimal common domain of discourse (upper-domain ontology) from an existing domain ontology. Using the Multi-Modality, Multi-Resource Environment for Physiological and Clinical Research (Physio-MIMI) multi-center project in sleep medicine as a use case, we demonstrate the use of MiDas in extracting a minimal domain of discourse for sleep medicine, from Physio-MIMI's Sleep Domain Ontology (SDO). We then extend the resulting domain of discourse with terms from the data dictionary of the Sleep Heart and Health Study (SHHS) to validate MiDas. To illustrate the wider applicability of MiDas, we automatically extract the respective domains of discourse from 6 sample domain ontologies from the National Center for Biomedical Ontologies (NCBO) and the OBO Foundry.

  11. Purification of SOCS (Suppressor of Cytokine Signaling) SH2 Domains for Structural and Functional Studies.

    PubMed

    Liau, Nicholas P D; Laktyushin, Artem; Babon, Jeffrey J

    2017-01-01

    Src Homology 2 (SH2) domains are protein domains which have a high binding affinity for specific amino acid sequences containing a phosphorylated tyrosine residue. The Suppressors of Cytokine Signaling (SOCS) proteins use an SH2 domain to bind to components of certain cytokine signaling pathways to downregulate the signaling cascade. The recombinantly produced SH2 domains of various SOCS proteins have been used to undertake structural and functional studies elucidating the method of how such targeting occurs. Here, we describe the protocol for the recombinant production and purification of SOCS SH2 domains, with an emphasis on SOCS3.

  12. Further insight into BRUTUS domain composition and functionality

    PubMed Central

    Matthiadis, Anna; Long, Terri A.

    2016-01-01

    ABSTRACT BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis. PMID:27359166

  13. Further insight into BRUTUS domain composition and functionality.

    PubMed

    Matthiadis, Anna; Long, Terri A

    2016-08-02

    BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.

  14. Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

    PubMed

    Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

    2015-01-01

    EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

  15. Two zinc-binding domains in the transporter AdcA from Streptococcus pyogenes facilitate high-affinity binding and fast transport of zinc.

    PubMed

    Cao, Kun; Li, Nan; Wang, Hongcui; Cao, Xin; He, Jiaojiao; Zhang, Bing; He, Qing-Yu; Zhang, Gong; Sun, Xuesong

    2018-04-20

    Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins due to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes , we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2013-12-01

    The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. © 2013.

  17. End-to-end observatory software modeling using domain specific languages

    NASA Astrophysics Data System (ADS)

    Filgueira, José M.; Bec, Matthieu; Liu, Ning; Peng, Chien; Soto, José

    2014-07-01

    The Giant Magellan Telescope (GMT) is a 25-meter extremely large telescope that is being built by an international consortium of universities and research institutions. Its software and control system is being developed using a set of Domain Specific Languages (DSL) that supports a model driven development methodology integrated with an Agile management process. This approach promotes the use of standardized models that capture the component architecture of the system, that facilitate the construction of technical specifications in a uniform way, that facilitate communication between developers and domain experts and that provide a framework to ensure the successful integration of the software subsystems developed by the GMT partner institutions.

  18. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

    PubMed

    Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

    1991-05-03

    Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

  19. The SLP-76 SH2 domain is required for T cell development and activation

    PubMed Central

    Burns, Jeremy C.; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A.; Kliche, Stefanie; Jordan, Martha S.

    2011-01-01

    The adaptor protein Src homology 2 (SH2) domain containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adaptor proteins that function together to activate second messengers required for TCR signal propagation. The N-terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with the adhesion- and degranulation-promoting adaptor protein (ADAP) and hematopoietic progenitor kinase 1 (HPK1). Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production and phosphorylation of protein kinase D (PKD) and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells. PMID:21949020

  20. Multivariable frequency domain identification via 2-norm minimization

    NASA Technical Reports Server (NTRS)

    Bayard, David S.

    1992-01-01

    The author develops a computational approach to multivariable frequency domain identification, based on 2-norm minimization. In particular, a Gauss-Newton (GN) iteration is developed to minimize the 2-norm of the error between frequency domain data and a matrix fraction transfer function estimate. To improve the global performance of the optimization algorithm, the GN iteration is initialized using the solution to a particular sequentially reweighted least squares problem, denoted as the SK iteration. The least squares problems which arise from both the SK and GN iterations are shown to involve sparse matrices with identical block structure. A sparse matrix QR factorization method is developed to exploit the special block structure, and to efficiently compute the least squares solution. A numerical example involving the identification of a multiple-input multiple-output (MIMO) plant having 286 unknown parameters is given to illustrate the effectiveness of the algorithm.

  1. A qualitative theory guided analysis of stroke survivors' perceived barriers and facilitators to physical activity.

    PubMed

    Nicholson, Sarah L; Donaghy, Marie; Johnston, Marie; Sniehotta, Falko F; van Wijck, Frederike; Johnston, Derek; Greig, Carolyn; McMurdo, Marion E T; Mead, Gillian

    2014-01-01

    After stroke, physical activity and physical fitness levels are low, impacting on health, activity and participation. It is unclear how best to support stroke survivors to increase physical activity. Little is known about the barriers and facilitators to physical activity after stroke. Thus, our aim was to explore stroke survivors' perceived barriers and facilitators to physical activity. Semi-structured interviews with 13 ambulatory stroke survivors exploring perceived barriers and facilitators to physical activity post stroke were conducted in participants' homes, audio-recorded and transcribed verbatim. The Theoretical Domains Framework (TDF) informed content analysis of the interview transcripts. Data saturation was reached after interviews with 13 participants (median age of 76 years (inter-quartile range (IQR) = 69-83 years). The median time since stroke was 345 d (IQR = 316-366 d). The most commonly reported TDF domains were "beliefs about capabilities", "environmental context and resources" and "social influence". The most commonly reported perceived motivators were: social interaction, beliefs of benefits of exercise, high self-efficacy and the necessity of routine behaviours. The most commonly reported perceived barriers were: lack of professional support on discharge from hospital and follow-up, transport issues to structured classes/interventions, lack of control and negative affect. Stroke survivors perceive several different barriers and facilitators to physical activity. Stroke services need to address barriers to physical activity and to build on facilitators to promote physical activity after stroke. Physical activity post stroke can improve physical fitness and function, yet physical activity remains low among stroke survivors. Understanding stroke survivors' perceived barriers and facilitators to physical activity is essential to develop targeted interventions to increase physical activity. Beliefs about capabilities, environmental

  2. BRCT-domain protein BRIT1 influences class switch recombination

    PubMed Central

    Yen, Wei-Feng; Chaudhry, Ashutosh; Vaidyanathan, Bharat; Yewdell, William T.; Pucella, Joseph N.; Sharma, Rahul; Li, Kaiyi; Rudensky, Alexander Y.; Chaudhuri, Jayanta

    2017-01-01

    DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs. PMID:28724724

  3. Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X7 Receptor: A Novel Conformation?

    PubMed Central

    Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; de Freitas, Mônica Santos; Foguel, Débora; Caffarena, Ernesto Raul; Alves, Luiz Anastacio

    2009-01-01

    Scanning experiments have shown that the putative TM2 domain of the P2X7 receptor (P2X7R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X2. These data led us to investigate a new architecture within the P2X receptor family. P2X7R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X7R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X7R (hP2X7R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X7R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X7R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X7R field and/or raise new questions about this issue. PMID:19186133

  4. Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation.

    PubMed Central

    Shoelson, S E; Sivaraja, M; Williams, K P; Hu, P; Schlessinger, J; Weiss, M A

    1993-01-01

    SH2 (src-homology 2) domains define a newly recognized binding motif that mediates the physical association of target phosphotyrosyl proteins with downstream effector enzymes. An example of such phosphoprotein-effector coupling is provided by the association of phosphatidylinositol 3-kinase (PI 3-kinase) with specific phosphorylation sites within the PDGF receptor, the c-Src/polyoma virus middle T antigen complex and the insulin receptor substrate IRS-1. Notably, phosphoprotein association with the SH2 domains of p85 also stimulates an increase in catalytic activity of the PI 3-kinase p110 subunit, which can be mimicked by phosphopeptides corresponding to targeted phosphoprotein phosphorylation sites. To investigate how phosphoprotein binding to the p85 SH2 domain stimulates p110 catalytic activation, we have examined the differential effects of phosphotyrosine and PDGF receptor-, IRS-1- and c-Src-derived phosphopeptides on the conformation of an isolated SH2 domain of PI 3-kinase. Although phosphotyrosine and both activating and non-activating phosphopeptides bind to the SH2 domain, activating phosphopeptides bind with higher affinity and induce a qualitatively distinct conformational change as monitored by CD and NMR spectroscopy. Amide proton exchange and protease protection assays further show that high affinity, specific phosphopeptide binding induces non-local dynamic SH2 domain stabilization. Based on these findings we propose that specific phosphoprotein binding to the p85 subunit induces a change in SH2 domain structure which is transmitted to the p110 subunit and regulates enzymatic activity by an allosteric mechanism. Images PMID:8382612

  5. Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

    PubMed Central

    Graham, Kate L.; Halasz, Peter; Tan, Yan; Hewish, Marilyn J.; Takada, Yoshikazu; Mackow, Erich R.; Robinson, Martyn K.; Coulson, Barbara S.

    2003-01-01

    Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection. PMID:12941907

  6. Trimerization and Triple Helix Stabilization of the Collagen XIX NC2 Domain*

    PubMed Central

    Boudko, Sergei P.; Engel, Jürgen; Bächinger, Hans Peter

    2008-01-01

    The mechanisms of chain selection and assembly of fibril-associated collagens with interrupted triple helices (FACITs) must differ from that of fibrillar collagens, since they lack the characteristic C-propeptide. We analyzed two carboxyl-terminal noncollagenous domains, NC2 and NC1, of collagen XIX as potential trimerization units and found that NC2 forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. In contrast, the NC1 domain requires formation of an adjacent collagen triple helix to form interchain disulfide bridges. The NC2 domain of collagen XIX and probably of other FACITs is responsible for chain selection and trimerization. PMID:18845531

  7. Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

    PubMed

    Thierry, Eric; Guilligay, Delphine; Kosinski, Jan; Bock, Thomas; Gaudon, Stephanie; Round, Adam; Pflug, Alexander; Hengrung, Narin; El Omari, Kamel; Baudin, Florence; Hart, Darren J; Beck, Martin; Cusack, Stephen

    2016-01-07

    Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

    PubMed Central

    Sellers, W R; Rodgers, J W; Kaelin, W G

    1995-01-01

    An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

  9. Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

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

    Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun

    Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminalmore » Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.« less

  10. A comprehensive computational study on pathogenic mis-sense mutations spanning the RING2 and REP domains of Parkin protein.

    PubMed

    Biswas, Ria; Bagchi, Angshuman

    2017-04-30

    Various mutations in PARK2 gene, which encodes the protein parkin, are significantly associated with the onset of autosomal recessive juvenile Parkinson (ARJP) in neuronal cells. Parkin is a multi domain protein, the N-terminal part contains the Ubl and the C-terminal part consists of four zinc coordinating domains, viz., RING0, RING1, in between ring (IBR) and RING2. Disease mutations are spread over all the domains of Parkin, although mutations in some regions may affect the functionality of Parkin more adversely. The mutations in the RING2 domain are seen to abolish the neuroprotective E3 ligase activity of Parkin. In this current work, we carried out detailed in silico analysis to study the extent of pathogenicity of mutations spanning the Parkin RING2 domain and the adjoining REP region by SIFT, Mutation Accessor, PolyPhen2, SNPs and GO, GV/GD and I-mutant. To study the structural and functional implications of these mutations on RING2-REP domain of Parkin, we studied the solvent accessibility (SASA/RSA), hydrophobicity, intra-molecular hydrogen bonding profile and domain analysis by various computational tools. Finally, we analysed the interaction energy profiles of the mutants and compared them to the wild type protein using Discovery studio 2.5. By comparing the various analyses it could be safely concluded that except P437L and A379V mutations, all other mutations were potentially deleterious affecting various structural aspects of RING2 domain architecture. This study is based purely on computational approach which has the potential to identify disease mutations and the information could further be used in treatment of diseases and prognosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. An incoherent feedforward loop facilitates adaptive tuning of gene expression.

    PubMed

    Hong, Jungeui; Brandt, Nathan; Abdul-Rahman, Farah; Yang, Ally; Hughes, Tim; Gresham, David

    2018-04-05

    We studied adaptive evolution of gene expression using long-term experimental evolution of Saccharomyces cerevisiae in ammonium-limited chemostats. We found repeated selection for non-synonymous variation in the DNA binding domain of the transcriptional activator, GAT1, which functions with the repressor, DAL80 in an incoherent type-1 feedforward loop (I1-FFL) to control expression of the high affinity ammonium transporter gene, MEP2. Missense mutations in the DNA binding domain of GAT1 reduce its binding to the GATAA consensus sequence. However, we show experimentally, and using mathematical modeling, that decreases in GAT1 binding result in increased expression of MEP2 as a consequence of properties of I1-FFLs. Our results show that I1-FFLs, one of the most commonly occurring network motifs in transcriptional networks, can facilitate adaptive tuning of gene expression through modulation of transcription factor binding affinities. Our findings highlight the importance of gene regulatory architectures in the evolution of gene expression. © 2018, Hong et al.

  12. Contextualized analysis of a needs assessment using the Theoretical Domains Framework: a case example in endocrinology

    PubMed Central

    2014-01-01

    Background The Theoretical Domains Framework (TDF) is a set of 14 domains of behavior change that provide a framework for the critical issues and factors influencing optimal knowledge translation. Considering that a previous study has identified optimal knowledge translation techniques for each TDF domain, it was hypothesized that the TDF could be used to contextualize and interpret findings from a behavioral and educational needs assessment. To illustrate this hypothesis, findings and recommendations drawn from a 2012 national behavioral and educational needs assessment conducted with healthcare providers who treat and manage Growth and Growth Hormone Disorders, will be discussed using the TDF. Methods This needs assessment utilized a mixed-methods research approach that included a combination of: [a] data sources (Endocrinologists (n:120), Pediatric Endocrinologists (n:53), Pediatricians (n:52)), [b] data collection methods (focus groups, interviews, online survey), [c] analysis methodologies (qualitative - analyzed through thematic analysis, quantitative - analyzed using frequencies, cross-tabulations, and gap analysis). Triangulation was used to generate trustworthy findings on the clinical practice gaps of endocrinologists, pediatric endocrinologists, and general pediatricians in their provision of care to adult patients with adult growth hormone deficiency or acromegaly, or children/teenagers with pediatric growth disorders. The identified gaps were then broken into key underlying determinants, categorized according to the TDF domains, and linked to optimal behavioral change techniques. Results The needs assessment identified 13 gaps, each with one or more underlying determinant(s). Overall, these determinants were mapped to 9 of the 14 TDF domains. The Beliefs about Consequences domain was identified as a contributing determinant to 7 of the 13 challenges. Five of the gaps could be related to the Skills domain, while three were linked to the Knowledge domain

  13. Contextualized analysis of a needs assessment using the Theoretical Domains Framework: a case example in endocrinology.

    PubMed

    Lazure, Patrice; Bartel, Robert C; Biller, Beverly M K; Molitch, Mark E; Rosenthal, Stephen M; Ross, Judith L; Bernsten, Brock D; Hayes, Sean M

    2014-07-24

    The Theoretical Domains Framework (TDF) is a set of 14 domains of behavior change that provide a framework for the critical issues and factors influencing optimal knowledge translation. Considering that a previous study has identified optimal knowledge translation techniques for each TDF domain, it was hypothesized that the TDF could be used to contextualize and interpret findings from a behavioral and educational needs assessment. To illustrate this hypothesis, findings and recommendations drawn from a 2012 national behavioral and educational needs assessment conducted with healthcare providers who treat and manage Growth and Growth Hormone Disorders, will be discussed using the TDF. This needs assessment utilized a mixed-methods research approach that included a combination of: [a] data sources (Endocrinologists (n:120), Pediatric Endocrinologists (n:53), Pediatricians (n:52)), [b] data collection methods (focus groups, interviews, online survey), [c] analysis methodologies (qualitative - analyzed through thematic analysis, quantitative - analyzed using frequencies, cross-tabulations, and gap analysis). Triangulation was used to generate trustworthy findings on the clinical practice gaps of endocrinologists, pediatric endocrinologists, and general pediatricians in their provision of care to adult patients with adult growth hormone deficiency or acromegaly, or children/teenagers with pediatric growth disorders. The identified gaps were then broken into key underlying determinants, categorized according to the TDF domains, and linked to optimal behavioral change techniques. The needs assessment identified 13 gaps, each with one or more underlying determinant(s). Overall, these determinants were mapped to 9 of the 14 TDF domains. The Beliefs about Consequences domain was identified as a contributing determinant to 7 of the 13 challenges. Five of the gaps could be related to the Skills domain, while three were linked to the Knowledge domain. The TDF categorization of

  14. Towards a cross-domain interoperable framework for natural hazards and disaster risk reduction information

    NASA Astrophysics Data System (ADS)

    Tomas, Robert; Harrison, Matthew; Barredo, José I.; Thomas, Florian; Llorente Isidro, Miguel; Cerba, Otakar; Pfeiffer, Manuela

    2014-05-01

    The vast amount of information and data necessary for comprehensive hazard and risk assessment presents many challenges regarding the lack of accessibility, comparability, quality, organisation and dissemination of natural hazards spatial data. In order to mitigate these limitations an interoperable framework has been developed in the framework of the development of legally binding Implementing rules of the EU INSPIRE Directive1* aiming at the establishment of the European Spatial Data Infrastructure. The interoperability framework is described in the Data Specification on Natural risk zones - Technical Guidelines (DS) document2* that was finalized and published on 10.12. 2013. This framework provides means for facilitating access, integration, harmonisation and dissemination of natural hazard data from different domains and sources. The objective of this paper is twofold. Firstly, the paper demonstrates the applicability of the interoperable framework developed in the DS and highlights the key aspects of the interoperability to the various natural hazards communities. Secondly, the paper "translates" into common language the main features and potentiality of the interoperable framework of the DS for a wider audience of scientists and practitioners in the natural hazards domain. Further in this paper the main five aspects of the interoperable framework will be presented. First, the issue of a common terminology for the natural hazards domain will be addressed. A common data model to facilitate cross domain data integration will follow secondly. Thirdly, the common methodology developed to provide qualitative or quantitative assessments of natural hazards will be presented. Fourthly, the extensible classification schema for natural hazards developed from a literature review and key reference documents from the contributing community of practice will be shown. Finally, the applicability of the interoperable framework for the various stakeholder groups will be also

  15. 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.

  16. Barriers and facilitators to health screening in men: A systematic review.

    PubMed

    Teo, Chin Hai; Ng, Chirk Jenn; Booth, Andrew; White, Alan

    2016-09-01

    Men have poorer health status and are less likely to attend health screening compared to women. This systematic review presents current evidence on the barriers and facilitators to engaging men in health screening. We included qualitative, quantitative and mixed-method studies identified through five electronic databases, contact with experts and reference mining. Two researchers selected and appraised the studies independently. Data extraction and synthesis were conducted using the 'best fit' framework synthesis method. 53 qualitative, 44 quantitative and 6 mixed-method studies were included. Factors influencing health screening uptake in men can be categorized into five domains: individual, social, health system, healthcare professional and screening procedure. The most commonly reported barriers are fear of getting the disease and low risk perception; for facilitators, they are perceived risk and benefits of screening. Male-dominant barriers include heterosexual -self-presentation, avoidance of femininity and lack of time. The partner's role is the most common male-dominant facilitator to screening. This systematic review provides a comprehensive overview of barriers and facilitators to health screening in men including the male-dominant factors. The findings are particularly useful for clinicians, researchers and policy makers who are developing interventions and policies to increase screening uptake in men. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

    PubMed

    Scheele, Urte; Alves, Jurgen; Frank, Ronald; Duwel, Michael; Kalthoff, Christoph; Ungewickell, Ernst

    2003-07-11

    Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

  18. Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

    PubMed

    Chaikam, Vijay; Karlson, Dale T

    2010-01-01

    The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

  19. Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

    PubMed

    Weger-Lucarelli, James; Aliota, Matthew T; Wlodarchak, Nathan; Kamlangdee, Attapon; Swanson, Ryan; Osorio, Jorge E

    2015-12-16

    Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about

  20. A theoretical analysis of the barriers and facilitators to the implementation of school-based physical activity policies in Canada: a mixed methods scoping review.

    PubMed

    Weatherson, Katie A; Gainforth, Heather L; Jung, Mary E

    2017-03-27

    Given the potential impact school-based daily physical activity (DPA) policies can have on the health outcomes of Canadian children, it is surprising that such little research has examined the implementation and student-level effectiveness of these policies, and that even less have used theory to understand the barriers and facilitators affecting uptake of this policy by teachers. This review descriptively summarizes the implementation status, approaches used to implement DPA, and the effectiveness of DPA at increasing the physical activity of children at school. In addition, the Theoretical Domains Framework (TDF) was used to explore the barriers and facilitators to DPA implementation. A scoping review of English articles using ERIC, CINAHL, and Google Scholar (2005 to 2016) was conducted. Only studies that evaluated the implementation and/or student-level effectiveness of DPA policies in Canadian elementary schools were included. Only articles that examined DPA implementation barriers and facilitators by teachers, principals, and/or administration were eligible for the TDF analysis. Data on study characteristics and major findings regarding implementation status, implementation approach used, and impact on student's physical activity were extracted and were summarized descriptively, including study quality indicators. Two coders extracted and categorized implementation barriers and facilitators into TDF domains. The search resulted in 66 articles being retrieved and 38 being excluded for not meeting the eligibility criteria, leaving 15 eligible for review (10 of which examined barriers and facilitators to implementation from DPA deliverers' perspective). Eleven of 15 studies examined the Ontario DPA policy, and 2 studies were from both Alberta and British Columbia. Thirteen studies examined implementation, and only two examined effectiveness. DPA implementation status, approaches to delivery, and effectiveness on student's PA levels are inconsistent across the

  1. Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met, c-Kit, ErbB, and Androgen Receptor Interactome*

    PubMed Central

    Leung, Kin K.; Hause, Ronald J.; Barkinge, John L.; Ciaccio, Mark F.; Chuu, Chih-Pin; Jones, Richard B.

    2014-01-01

    Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains. PMID:24728074

  2. Ahcyl2 upregulates NBCe1-B via multiple serine residues of the PEST domain-mediated association.

    PubMed

    Park, Pil Whan; Ahn, Jeong Yeal; Yang, Dongki

    2016-07-01

    Inositol-1,4,5-triphosphate [IP3] receptors binding protein released with IP3 (IRBIT) was previously reported as an activator of NBCe1-B. Recent studies have characterized IRBIT homologue S-Adenosylhomocysteine hydrolase-like 2 (AHCYL2). AHCYL2 is highly homologous to IRBIT (88%) and heteromerizes with IRBIT. The two important domains in the N-terminus of AHCYL2 are a PEST domain and a coiled-coil domain which are highly comparable to those in IRBIT. Therefore, in this study, we tried to identify the role of those domains in mouse AHCYL2 (Ahcyl2), and we succeeded in identifying PEST domain of Ahcyl2 as a regulation region for NBCe1-B activity. Site directed mutagenesis and coimmunoprecipitation assay showed that NBCe1-B binds to the N-terminal Ahcyl2-PEST domain, and its binding is determined by the phosphorylation of 4 critical serine residues (Ser151, Ser154, Ser157, and Ser160) in Ahcyl2 PEST domain. Also we revealed that 4 critical serine residues in Ahcyl2 PEST domain are indispensable for the activation of NBCe1-B using measurement of intracellular pH experiment. Thus, these results suggested that the NBCe1-B is interacted with 4 critical serine residues in Ahcyl2 PEST domain, which play an important role in intracellular pH regulation through NBCe1-B.

  3. Structure of MyTH4-FERM domains in myosin VIIa tail bound to cargo.

    PubMed

    Wu, Lin; Pan, Lifeng; Wei, Zhiyi; Zhang, Mingjie

    2011-02-11

    The unconventional myosin VIIa (MYO7A) is one of the five proteins that form a network of complexes involved in formation of stereocilia. Defects in these proteins cause syndromic deaf-blindness in humans [Usher syndrome I (USH1)]. Many disease-causing mutations occur in myosin tail homology 4-protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) domains in the myosin tail that binds to another USH1 protein, Sans. We report the crystal structure of MYO7A MyTH4-FERM domains in complex with the central domain (CEN) of Sans at 2.8 angstrom resolution. The MyTH4 and FERM domains form an integral structural and functional supramodule binding to two highly conserved segments (CEN1 and 2) of Sans. The MyTH4-FERM/CEN complex structure provides mechanistic explanations for known deafness-causing mutations in MYO7A MyTH4-FERM. The structure will also facilitate mechanistic and functional studies of MyTH4-FERM domains in other myosins.

  4. Identifying the domains of context important to implementation science: a study protocol.

    PubMed

    Squires, Janet E; Graham, Ian D; Hutchinson, Alison M; Michie, Susan; Francis, Jill J; Sales, Anne; Brehaut, Jamie; Curran, Janet; Ivers, Noah; Lavis, John; Linklater, Stefanie; Fenton, Shannon; Noseworthy, Thomas; Vine, Jocelyn; Grimshaw, Jeremy M

    2015-09-28

    There is growing recognition that "context" can and does modify the effects of implementation interventions aimed at increasing healthcare professionals' use of research evidence in clinical practice. However, conceptual clarity about what exactly comprises "context" is lacking. The purpose of this research program is to develop, refine, and validate a framework that identifies the key domains of context (and their features) that can facilitate or hinder (1) healthcare professionals' use of evidence in clinical practice and (2) the effectiveness of implementation interventions. A multi-phased investigation of context using mixed methods will be conducted. The first phase is a concept analysis of context using the Walker and Avant method to distinguish between the defining and irrelevant attributes of context. This phase will result in a preliminary framework for context that identifies its important domains and their features according to the published literature. The second phase is a secondary analysis of qualitative data from 13 studies of interviews with 312 healthcare professionals on the perceived barriers and enablers to their application of research evidence in clinical practice. These data will be analyzed inductively using constant comparative analysis. For the third phase, we will conduct semi-structured interviews with key health system stakeholders and change agents to elicit their knowledge and beliefs about the contextual features that influence the effectiveness of implementation interventions and healthcare professionals' use of evidence in clinical practice. Results from all three phases will be synthesized using a triangulation protocol to refine the context framework drawn from the concept analysis. The framework will then be assessed for content validity using an iterative Delphi approach with international experts (researchers and health system stakeholders/change agents). This research program will result in a framework that identifies the

  5. The tetramerization domain potentiates Kv4 channel function by suppressing closed-state inactivation.

    PubMed

    Tang, Yi-Quan; Zhou, Jing-Heng; Yang, Fan; Zheng, Jie; Wang, KeWei

    2014-09-02

    A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. The SLP-76 Src homology 2 domain is required for T cell development and activation.

    PubMed

    Burns, Jeremy C; Corbo, Evann; Degen, Janine; Gohil, Mercy; Anterasian, Christine; Schraven, Burkart; Koretzky, Gary A; Kliche, Stefanie; Jordan, Martha S

    2011-11-01

    The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.

  7. WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ

    PubMed Central

    Chan, S W; Lim, C J; Huang, C; Chong, Y F; Gunaratne, H J; Hogue, K A; Blackstock, W P; Harvey, K F; Hong, W

    2011-01-01

    The transcriptional co-activators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. YAP and TAZ both possess WW domains, which are important protein–protein interaction modules that mediate interaction with proline-rich motifs, most commonly PPXY. The WW domains of YAP have complex regulatory roles as exemplified by recent reports showing that they can positively or negatively influence YAP activity in a cell and context-specific manner. In this study, we show that the WW domain of TAZ is important for it to transform both MCF10A and NIH3T3 cells and to activate transcription of ITGB2 but not CTGF, as introducing point mutations into the WW domain of TAZ (WWm) abolished its transforming and transcription-promoting ability. Using a proteomic approach, we discovered potential regulatory proteins that interact with TAZ WW domain and identified Wbp2. The interaction of Wbp2 with TAZ is dependent on the WW domain of TAZ and the PPXY-containing C-terminal region of Wbp2. Knockdown of endogenous Wbp2 suppresses, whereas overexpression of Wbp2 enhances, TAZ-driven transformation. Forced interaction of WWm with Wbp2 by direct C-terminal fusion of full-length Wbp2 or its TAZ-interacting C-terminal domain restored the transforming and transcription-promoting ability of TAZ. These results suggest that the WW domain-mediated interaction with Wbp2 promotes the transforming ability of TAZ. PMID:20972459

  8. Vortex Ring Dynamics in Radially Confined Domains

    NASA Astrophysics Data System (ADS)

    Stewart, Kelley; Niebel, Casandra; Jung, Sunghwan; Vlachos, Pavlos

    2010-11-01

    Vortex ring dynamics have been studied extensively in semi-infinite quiescent volumes. However, very little is known about vortex-ring formation in wall-bounded domains where vortex wall interaction will affect both the vortex ring pinch-off and propagation velocity. This study addresses this limitation and studies vortex formation in radially confined domains to analyze the affect of vortex-ring wall interaction on the formation and propagation of the vortex ring. Vortex rings were produced using a pneumatically driven piston cylinder arrangement and were ejected into a long cylindrical tube which defined the confined downstream domain. A range of confinement domains were studied with varying confinement diameters Velocity field measurements were performed using planar Time Resolved Digital Particle Image Velocimetry (TRDPIV) and were processed using an in-house developed cross-correlation PIV algorithm. The experimental analysis was used to facilitate the development of a theoretical model to predict the variations in vortex ring circulation over time within confined domains.

  9. Domain Requirements for DNA Unwinding by Mycobacterial UvrD2, an Essential DNA Helicase†

    PubMed Central

    Sinha, Krishna Murari; Stephanou, Nicolas C.; Unciuleac, Mihaela-Carmen; Glickman, Michael S.; Shuman, Stewart

    2008-01-01

    Mycobacterial UvrD2 is a DNA-dependent ATPase with 3′ to 5′ helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis. PMID:18702526

  10. The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation.

    PubMed

    Basel-Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

    2006-03-01

    The molecular basis of autosomal recessive non-syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I-kappaB kinase/NFkappaB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. A previously unknown signal transduction pathway is important in human cognitive development.

  11. The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

    PubMed

    Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

    2013-07-16

    Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows

  12. Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity

    PubMed Central

    Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

    2018-01-01

    Constitutive activation of the non-receptor tyrosine kinase c-Abl (Abl1) in the Bcr-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukemia. Recent studies have indicated that an interaction between the SH2 domain and the N-lobe of the c-Abl kinase domain has a critical role in leukemogenesis. To dissect the structural basis of this phenomenon we studied c-Abl constructs comprising the SH2 and kinase domains in vitro. We present a crystal structure of an SH2-kinase domain construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the kinase domain. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2-N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the kinase domain. That the effects are small compared to the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the autoinhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity. PMID:25779001

  13. A Unique Phenylalanine in the Transmembrane Domain Strengthens Homodimerization of the Syndecan-2 Transmembrane Domain and Functionally Regulates Syndecan-2*

    PubMed Central

    Kwon, Mi-Jung; Choi, Youngsil; Yun, Ji-Hye; Lee, Weontae; Han, Inn-Oc; Oh, Eok-Soo

    2015-01-01

    The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe167) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe167 was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe167 in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members. PMID:25572401

  14. The SH2 Domain Regulates c-Abl Kinase Activation by a Cyclin-Like Mechanism and Remodulation of the Hinge Motion

    PubMed Central

    Dölker, Nicole; Górna, Maria W.; Sutto, Ludovico; Torralba, Antonio S.; Superti-Furga, Giulio; Gervasio, Francesco L.

    2014-01-01

    Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors. PMID:25299346

  15. The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion.

    PubMed

    Dölker, Nicole; Górna, Maria W; Sutto, Ludovico; Torralba, Antonio S; Superti-Furga, Giulio; Gervasio, Francesco L

    2014-10-01

    Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

  16. Keep your fingers off my DNA: protein-protein interactions mediated by C2H2 zinc finger domains.

    PubMed

    Brayer, Kathryn J; Segal, David J

    2008-01-01

    Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein-protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.

  17. Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

    PubMed Central

    Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

    2016-01-01

    While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding. DOI: http://dx.doi.org/10.7554/eLife.11835.001 PMID:27071344

  18. Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

    PubMed

    Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

    2016-04-12

    While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

  19. Direct EIT reconstructions of complex admittivities on a chest-shaped domain in 2-D.

    PubMed

    Hamilton, Sarah J; Mueller, Jennifer L

    2013-04-01

    Electrical impedance tomography (EIT) is a medical imaging technique in which current is applied on electrodes on the surface of the body, the resulting voltage is measured, and an inverse problem is solved to recover the conductivity and/or permittivity in the interior. Images are then formed from the reconstructed conductivity and permittivity distributions. In the 2-D geometry, EIT is clinically useful for chest imaging. In this work, an implementation of a D-bar method for complex admittivities on a general 2-D domain is presented. In particular, reconstructions are computed on a chest-shaped domain for several realistic phantoms including a simulated pneumothorax, hyperinflation, and pleural effusion. The method demonstrates robustness in the presence of noise. Reconstructions from trigonometric and pairwise current injection patterns are included.

  20. Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase.

    PubMed

    Liu, Dongsheng; Cowburn, David

    2016-01-01

    The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.

  1. Development of an interprofessional lean facilitator assessment scale.

    PubMed

    Bravo-Sanchez, Cindy; Dorazio, Vincent; Denmark, Robert; Heuer, Albert J; Parrott, J Scott

    2018-05-01

    High reliability is important for optimising quality and safety in healthcare organisations. Reliability efforts include interprofessional collaborative practice (IPCP) and Lean quality/process improvement strategies, which require skilful facilitation. Currently, no validated Lean facilitator assessment tool for interprofessional collaboration exists. This article describes the development and pilot evaluation of such a tool; the Interprofessional Lean Facilitator Assessment Scale (ILFAS), which measures both technical and 'soft' skills, which have not been measured in other instruments. The ILFAS was developed using methodologies and principles from Lean/Shingo, IPCP, metacognition research and Bloom's Taxonomy of Learning Domains. A panel of experts confirmed the initial face validity of the instrument. Researchers independently assessed five facilitators, during six Lean sessions. Analysis included quantitative evaluation of rater agreement. Overall inter-rater agreement of the assessment of facilitator performance was high (92%), and discrepancies in the agreement statistics were analysed. Face and content validity were further established, and usability was evaluated, through primary stakeholder post-pilot feedback, uncovering minor concerns, leading to tool revision. The ILFAS appears comprehensive in the assessment of facilitator knowledge, skills, abilities, and may be useful in the discrimination between facilitators of different skill levels. Further study is needed to explore instrument performance and validity.

  2. The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

    PubMed

    Liger, D; Nizard, P; Gaillard, C; vanderSpek, J C; Murphy, J R; Pitard, B; Gillet, D

    1998-11-01

    We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

  3. Purification, crystallization and preliminary crystallographic analysis of the SH2 domain of IL-2-inducible T-cell kinase.

    PubMed

    Joseph, Raji E; Ginder, Nathaniel D; Hoy, Julie A; Nix, Jay C; Honzatko, Richard B; Andreotti, Amy H

    2011-02-01

    Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

  4. Practice Facilitators' and Leaders' Perspectives on a Facilitated Quality Improvement Program.

    PubMed

    McHugh, Megan; Brown, Tiffany; Liss, David T; Walunas, Theresa L; Persell, Stephen D

    2018-04-01

    Practice facilitation is a promising approach to helping practices implement quality improvements. Our purpose was to describe practice facilitators' and practice leaders' perspectives on implementation of a practice facilitator-supported quality improvement program and describe where their perspectives aligned and diverged. We conducted interviews with practice leaders and practice facilitators who participated in a program that included 35 improvement strategies aimed at the ABCS of heart health (aspirin use in high-risk individuals, blood pressure control, cholesterol management, and smoking cessation). Rapid qualitative analysis was used to collect, organize, and analyze the data. We interviewed 17 of the 33 eligible practice leaders, and the 10 practice facilitators assigned to those practices. Practice leaders and practice facilitators both reported value in the program's ability to bring needed, high-quality resources to practices. Practice leaders appreciated being able to set the schedule for facilitation and select among the 35 interventions. According to practice facilitators, however, relying on practice leaders to set the pace of the intervention resulted in a lower level of program intensity than intended. Practice leaders preferred targeted assistance, particularly electronic health record documentation guidance and linkages to state smoking cessation programs. Practice facilitators reported that the easiest interventions were those that did not alter care practices. The dual perspectives of practice leaders and practice facilitators provide a more holistic picture of enablers and barriers to program implementation. There may be greater opportunities to assist small practices through simple, targeted practice facilitator-supported efforts rather than larger, comprehensive quality improvement projects. © 2018 Annals of Family Medicine, Inc.

  5. Structural and functional analysis of the YAP-binding domain of human TEAD2

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

    Tian, Wei; Yu, Jianzhong; Tomchick, Diana R.

    2010-06-15

    The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like {beta}-sandwich fold with two extra helix-turn-helixmore » inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.« less

  6. Fabrication of Large Domain YBa2Cu3O(x) for Magnetic Suspension Applications

    NASA Technical Reports Server (NTRS)

    Sengupta, S.; Corpus, J.; Gaines, J. R., Jr.; Todt, V. R.; Zhang, X.; Miller, D. J.

    1996-01-01

    Large domain YBa2Cu3O(x) levitators have been fabricated using a seeded melt processing technique. Depending upon the seed, either a single or five domained sample can be obtained. The grain boundaries separating each domains in the five domain levitator are found to be 90 degrees. Similar levitation forces can be observed for single and five domained samples. After thermal cycling, however, a small decrease in the levitation force of the five domain levitator was observed as a function of thermal cycles while nearly no change in force was observed in the single domain levitator. Finally, it is shown that both, single and five domain YBCO, behave similarly as a function of sample thickness.

  7. Diversity in peptide recognition by the SH2 domain of SH2B1.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2018-02-01

    SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein-protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate-1 and -2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C-terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high-resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand-binding interface of SH2B1 with high specificity. © 2017 Wiley Periodicals, Inc.

  8. A novel PKD2L1 C-terminal domain critical for trimerization and channel function.

    PubMed

    Zheng, Wang; Hussein, Shaimaa; Yang, JungWoo; Huang, Jun; Zhang, Fan; Hernandez-Anzaldo, Samuel; Fernandez-Patron, Carlos; Cao, Ying; Zeng, Hongbo; Tang, Jingfeng; Chen, Xing-Zhen

    2015-03-30

    As a transient receptor potential (TRP) superfamily member, polycystic kidney disease 2-like-1 (PKD2L1) is also called TRPP3 and has similar membrane topology as voltage-gated cation channels. PKD2L1 is involved in hedgehog signaling, intestinal development, and sour tasting. PKD2L1 and PKD1L3 form heterotetramers with 3:1 stoichiometry. C-terminal coiled-coil-2 (CC2) domain (G699-W743) of PKD2L1 was reported to be important for its trimerization but independent studies showed that CC2 does not affect PKD2L1 channel function. It thus remains unclear how PKD2L1 proteins oligomerize into a functional channel. By SDS-PAGE, blue native PAGE and mutagenesis we here identified a novel C-terminal domain called C1 (K575-T622) involved in stronger homotrimerization than the non-overlapping CC2, and found that the PKD2L1 N-terminus is critical for dimerization. By electrophysiology and Xenopus oocyte expression, we found that C1, but not CC2, is critical for PKD2L1 channel function. Our co-immunoprecipitation and dynamic light scattering experiments further supported involvement of C1 in trimerization. Further, C1 acted as a blocking peptide that inhibits PKD2L1 trimerization as well as PKD2L1 and PKD2L1/PKD1L3 channel function. Thus, our study identified C1 as the first PKD2L1 domain essential for both PKD2L1 trimerization and channel function, and suggest that PKD2L1 and PKD2L1/PKD1L3 channels share the PKD2L1 trimerization process.

  9. Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Kitade, Yukio

    2018-02-01

    RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

    PubMed

    Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

    2002-04-12

    The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

  11. The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

    PubMed

    Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

    2017-08-21

    Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Ultra-thin clay layers facilitate seismic slip in carbonate faults.

    PubMed

    Smeraglia, Luca; Billi, Andrea; Carminati, Eugenio; Cavallo, Andrea; Di Toro, Giulio; Spagnuolo, Elena; Zorzi, Federico

    2017-04-06

    Many earthquakes propagate up to the Earth's surface producing surface ruptures. Seismic slip propagation is facilitated by along-fault low dynamic frictional resistance, which is controlled by a number of physico-chemical lubrication mechanisms. In particular, rotary shear experiments conducted at seismic slip rates (1 ms -1 ) show that phyllosilicates can facilitate co-seismic slip along faults during earthquakes. This evidence is crucial for hazard assessment along oceanic subduction zones, where pelagic clays participate in seismic slip propagation. Conversely, the reason why, in continental domains, co-seismic slip along faults can propagate up to the Earth's surface is still poorly understood. We document the occurrence of micrometer-thick phyllosilicate-bearing layers along a carbonate-hosted seismogenic extensional fault in the central Apennines, Italy. Using friction experiments, we demonstrate that, at seismic slip rates (1 ms -1 ), similar calcite gouges with pre-existing phyllosilicate-bearing (clay content ≤3 wt.%) micro-layers weaken faster than calcite gouges or mixed calcite-phyllosilicate gouges. We thus propose that, within calcite gouge, ultra-low clay content (≤3 wt.%) localized along micrometer-thick layers can facilitate seismic slip propagation during earthquakes in continental domains, possibly enhancing surface displacement.

  13. E. coli derived Von Willebrand Factor-A2 domain FRET proteins that quantify ADAMTS13 activity

    PubMed Central

    Dayananda, Kannayakanahalli M.; Gogia, Shobhit; Neelamegham, Sriram

    2010-01-01

    The cleavage of the A2-domain of Von Willebrand Factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET proteins’, where variants of YFP (Venus) and CFP (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, 77 amino acids) of this domain. These proteins were expressed in E. coli in soluble form, and they exhibited Fluorescence/Förster Resonance Energy Transfer (FRET) properties. Results show that introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13 mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, upon increasing urea concentration. While proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity, and as a tool to study VWF-A2 conformation dynamics. PMID:21146487

  14. Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

    PubMed

    Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

    2008-11-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

  15. Crystal Structure of the Central Coiled-Coil Domain from Human Liprin-[beta]2

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

    Stafford, Ryan L.; Tang, Ming-Yun; Sawaya, Michael R.

    2012-02-07

    Liprins are a conserved family of scaffolding proteins important for the proper regulation and development of neuronal synapses. Humans have four liprin-{alpha}s and two liprin-{beta}s which all contain long coiled-coil domains followed by three tandem SAM domains. Complex interactions between the coiled-coil and SAM domains are thought to create liprin scaffolds, but the structural and biochemical properties of these domains remain largely uncharacterized. In this study we find that the human liprin-{beta}2 coiled-coil forms an extended dimer. Several protease-resistant subdomains within the liprin-{beta}1 and liprin-{beta}2 coiled-coils were also identified. A 2.0 {angstrom} crystal structure of the central, protease-resistant core ofmore » the liprin-{beta}2 coiled-coil reveals a parallel helix orientation. These studies represent an initial step toward determining the overall architecture of liprin scaffolds and understanding the molecular basis for their synaptic functions.« less

  16. Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

    PubMed

    Sha, Fern; Gencer, Emel Basak; Georgeon, Sandrine; Koide, Akiko; Yasui, Norihisa; Koide, Shohei; Hantschel, Oliver

    2013-09-10

    The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

  17. High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

    PubMed

    Engelmann, Brett W

    2017-01-01

    The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

  18. Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers.

    PubMed

    Wen, Wenhsiang; Chen, Wangjuh Sting; Xiao, Nick; Bender, Ryan; Ghazalpour, Anatole; Tan, Zheng; Swensen, Jeffrey; Millis, Sherri Z; Basu, Gargi; Gatalica, Zoran; Press, Michael F

    2015-09-01

    The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  19. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

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

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

  20. Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

    DOE PAGES

    Achar, Yathish Jagadheesh; Balogh, David; Neculai, Dante; ...

    2015-09-08

    Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteinsmore » retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. We suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.« less

  1. Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels*

    PubMed Central

    Magupalli, Venkat G.; Mochida, Sumiko; Yan, Jin; Jiang, Xin; Westenbroek, Ruth E.; Nairn, Angus C.; Scheuer, Todd; Catterall, William A.

    2013-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of CaV2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca2+/CaM stimulus. Autophosphorylated CaMKII can bind the CaV2.1 channel and synapsin-1 simultaneously. CaMKII binding to CaV2.1 channels induces Ca2+-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of CaV2.1 channels by binding of Ca2+/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to CaV2.1 channels. These results define the functional properties of a signaling complex of CaMKII and CaV2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity. PMID:23255606

  2. The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non‐syndromic mental retardation

    PubMed Central

    Basel‐Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

    2006-01-01

    Background The molecular basis of autosomal recessive non‐syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. Objective To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. Results The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I‐κB kinase/NFκB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. Conclusions A previously unknown signal transduction pathway is important in human cognitive development. PMID:16033914

  3. Single domain YBa2Cu3Oy thick films on metallic substrates

    NASA Astrophysics Data System (ADS)

    Reddy, E. S.; Noudem, J. G.; Goodilin, E. A.; Tarka, M.; Schmitz, G. J.

    2003-03-01

    The fabrication of single domain YBa2Cu3Oy (123) thick films (10-100 mum) on metallic substrates is reported. The process involves the formation of the 123 phase by a peritectic reaction between an air-brushed dense Y2BaCuO5 (211) layer on a Ag12Pd substrate and infiltrated liquid phases containing barium cuprates and copper oxides. Single domain growth is achieved by seeding the green films with a c-axis oriented NdBa2Cu3Oy crystal prior to processing. The maximum processing temperatures are lowered to 970 °C by modifying the characteristics of the liquid phases meant for infiltration by addition of Ag powder. The fabrication technique, processing conditions for single domain growth and the resulting microstructures are discussed.

  4. An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

    PubMed

    Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

    2013-01-01

    Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.

  5. Pairwise domain adaptation module for CNN-based 2-D/3-D registration.

    PubMed

    Zheng, Jiannan; Miao, Shun; Jane Wang, Z; Liao, Rui

    2018-04-01

    Accurate two-dimensional to three-dimensional (2-D/3-D) registration of preoperative 3-D data and intraoperative 2-D x-ray images is a key enabler for image-guided therapy. Recent advances in 2-D/3-D registration formulate the problem as a learning-based approach and exploit the modeling power of convolutional neural networks (CNN) to significantly improve the accuracy and efficiency of 2-D/3-D registration. However, for surgery-related applications, collecting a large clinical dataset with accurate annotations for training can be very challenging or impractical. Therefore, deep learning-based 2-D/3-D registration methods are often trained with synthetically generated data, and a performance gap is often observed when testing the trained model on clinical data. We propose a pairwise domain adaptation (PDA) module to adapt the model trained on source domain (i.e., synthetic data) to target domain (i.e., clinical data) by learning domain invariant features with only a few paired real and synthetic data. The PDA module is designed to be flexible for different deep learning-based 2-D/3-D registration frameworks, and it can be plugged into any pretrained CNN model such as a simple Batch-Norm layer. The proposed PDA module has been quantitatively evaluated on two clinical applications using different frameworks of deep networks, demonstrating its significant advantages of generalizability and flexibility for 2-D/3-D medical image registration when a small number of paired real-synthetic data can be obtained.

  6. Library of electrocatalytic sites in nano-structured domains: electrocatalysis of hydrogen peroxide.

    PubMed

    Pandey, Prem C; Singh, Bhupendra

    2008-12-01

    Electrochemical detection of hydrogen peroxide at eight types of ormosil-modified electrodes, referred as hexacyanoferrate-system; Prussian blue systems (PB-1, PB-2, and PB-3), palladium (Pd-) system, graphite (Gr-) system, gold nanoparticle (AuNPs) system and palladium-gold nanoparticle (Pd-AuNPs) system were studied. The results on electrochemical detection suggested that hydrogen peroxide does not undergo homogeneous electrochemical mediation; however, the presence of redox mediator within nano-structured domains facilitates the electro-analysis of the same via redox electrocatalysis. Four approaches causing manipulation in nano-structured domains are described: (a) increase in the molecular size of the components generating nano-structured domains; (b) modulation via chemical reactivity; (c) modulation by non-reactive moieties and known nanoparticles; and (d) modulation by mixed approaches (a-c), all leading to decrease in a nano-structured domains. The results demonstrated that an increase in the size of nano-structured domains or decrease in micro-porous geometry increases the efficiency of electrocatalysis. The basic reaction protocol adopted in generating nano-structured domains, followed by manipulation protocols, supported the introduction of a library for creating electrocatalytic sites with varying electrocatalytic efficiency within the same basic nano-structured platform.

  7. Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3.

    PubMed

    Doan, Ninh; Gettins, Peter G W

    2007-10-01

    Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

  8. Dissection of Swa2p/Auxilin Domain Requirements for Cochaperoning Hsp70 Clathrin-uncoating Activity In Vivo

    PubMed Central

    Xiao, Jing; Kim, Leslie S.

    2006-01-01

    The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly. PMID:16687570

  9. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

    NASA Astrophysics Data System (ADS)

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-04-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

  10. Vertebrate intersectin1 is repurposed to facilitate cortical midline connectivity and higher order cognition.

    PubMed

    Sengar, Ameet S; Ellegood, Jacob; Yiu, Adelaide P; Wang, Hua; Wang, Wei; Juneja, Subhash C; Lerch, Jason P; Josselyn, Sheena A; Henkelman, R Mark; Salter, Michael W; Egan, Sean E

    2013-02-27

    Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.

  11. Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

    PubMed

    Hofmann, Bianca T; Jücker, Manfred

    2012-10-01

    The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

    PubMed

    Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

    2012-07-10

    SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

  13. Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB.

    PubMed

    Zeng, Lei; Kuti, Miklos; Mujtaba, Shiraz; Zhou, Ming-Ming

    2014-07-01

    The fibroblast growth factor receptor (FGFR) substrate 2 (FRS2) family proteins function as scaffolding adapters for receptor tyrosine kinases (RTKs). The FRS2α proteins interact with RTKs through the phosphotyrosine-binding (PTB) domain and transfer signals from the activated receptors to downstream effector proteins. Here, we report the nuclear magnetic resonance structure of the FRS2α PTB domain bound to phosphorylated TrkB. The structure reveals that the FRS2α-PTB domain is comprised of two distinct but adjacent pockets for its mutually exclusive interaction with either nonphosphorylated juxtamembrane region of the FGFR, or tyrosine phosphorylated peptides TrkA and TrkB. The new structural insights suggest rational design of selective small molecules through targeting of the two conjunct pockets in the FRS2α PTB domain. © 2014 Wiley Periodicals, Inc.

  14. Structures of BIR domains from human NAIP and cIAP2.

    PubMed

    Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

    2009-11-01

    The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.

  15. Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

    PubMed Central

    Doan, Ninh; Gettins, Peter G. W.

    2007-01-01

    Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein. PMID:17608619

  16. 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.

  17. Copper-facilitated Suzuki reactions: application to 2-heterocyclic boronates.

    PubMed

    Deng, James Z; Paone, Daniel V; Ginnetti, Anthony T; Kurihara, Hideki; Dreher, Spencer D; Weissman, Steven A; Stauffer, Shaun R; Burgey, Christopher S

    2009-01-15

    The palladium-catalyzed Suzuki-Miyaura reaction has been utilized as one of the most powerful methods for C-C bond formation. However, Suzuki reactions of electron-deficient 2-heterocyclic boronates generally give low conversions and remain challenging. The successful copper(I) facilitated Suzuki coupling of 2-heterocyclic boronates that is broad in scope is reported. Use of this methodology affords greatly enhanced yields of these notoriously difficult couplings. Furthermore, mechanistic investigations suggest a possible role of copper in the catalytic cycle.

  18. APE2 Zf-GRF facilitates 3'-5' resection of DNA damage following oxidative stress

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

    Wallace, Bret D.; Berman, Zachary; Mueller, Geoffrey A.

    The Xenopus laevis APE2 (apurinic/apyrimidinic endonuclease 2) nuclease participates in 3'-5' nucleolytic resection of oxidative DNA damage and activation of the ATR-Chk1 DNA damage response (DDR) pathway via ill-defined mechanisms. Here we report that APE2 resection activity is regulated by DNA interactions in its Zf-GRF domain, a region sharing high homology with DDR proteins Topoisomerase 3α (TOP3α) and NEIL3 (Nei-like DNA glycosylase 3), as well as transcription and RNA regulatory proteins, such as TTF2 (transcription termination factor 2), TFIIS, and RPB9. Biochemical and NMR results establish the nucleic acid-binding activity of the Zf-GRF domain. Moreover, an APE2 Zf-GRF X-ray structuremore » and small-angle X-ray scattering analyses show that the Zf-GRF fold is typified by a crescent-shaped ssDNA binding claw that is flexibly appended to an APE2 endonuclease/exonuclease/phosphatase (EEP) catalytic core. Structure-guided Zf-GRF mutations impact APE2 DNA binding and 3'-5' exonuclease processing, and also prevent efficient APE2-dependent RPA recruitment to damaged chromatin and activation of the ATR-Chk1 DDR pathway in response to oxidative stress in Xenopus egg extracts. Collectively, our data unveil the APE2 Zf-GRF domain as a nucleic acid interaction module in the regulation of a key single-strand break resection function of APE2, and also reveal topologic similarity of the Zf-GRF to the zinc ribbon domains of TFIIS and RPB9.« less

  19. The E2-25K Ubiquitin-associated (UBA) Domain Aids in Polyubiquitin Chain Synthesis and Linkage Specificity

    PubMed Central

    WILSON, Randall C.; EDMONDSON, Stephen P.; FLATT, Justin W.; HELMS, Kimberli; TWIGG, Pamela D.

    2011-01-01

    E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologues represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage. PMID:21281599

  20. Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.

    PubMed

    Monsey, John; Shen, Wei; Schlesinger, Paul; Bose, Ron

    2010-03-05

    Her4 (ErbB-4) and Her2/neu (ErbB-2) are receptor-tyrosine kinases belonging to the epidermal growth factor receptor (EGFR) family. Crystal structures of EGFR and Her4 kinase domains demonstrate kinase dimerization and activation through an allosteric mechanism. The kinase domains form an asymmetric dimer, where the C-lobe surface of one monomer contacts the N-lobe of the other monomer. EGFR kinase dimerization and activation in vitro was previously reported using a nickel-chelating lipid-liposome system, and we now apply this system to all other members of the EGFR family. Polyhistidine-tagged Her4, Her2/neu, and Her3 kinase domains are bound to these nickel-liposomes and are brought to high local concentration, mimicking what happens to full-length receptors in vivo following ligand binding. Addition of nickel-liposomes to Her4 kinase domain results in 40-fold activation in kinase activity and marked enhancement of C-terminal tail autophosphorylation. Activation of Her4 shows a sigmoidal dependence on kinase concentration, consistent with a cooperative process requiring kinase dimerization. Her2/neu kinase activity is also activated by nickel-liposomes, and is increased further by heterodimerization with Her3 or Her4. The ability of Her3 and Her4 to heterodimerize and activate other family members is studied in vitro. Her3 kinase domain readily activates Her2/neu but is a poor activator of Her4, which differs from the prediction made by the asymmetric dimer model. Mutation of Her3 residues (952)ENI(954) to the corresponding sequence in Her4 enhanced the ability of Her3 to activate Her4, demonstrating that sequence differences on the C-lobe surface influence the heterodimerization and activation of ErbB kinase domains.

  1. Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

    PubMed

    Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2014-12-01

    YES-associated protein 2 (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 the 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. © 2014 FEBS.

  2. WAVE2 is regulated by multiple phosphorylation events within its VCA domain.

    PubMed

    Pocha, Shirin M; Cory, Giles O

    2009-01-01

    The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. (c) 2008 Wiley-Liss, Inc.

  3. Magnetic force microscopy study of domain walls in Co{sub 2}Z ferrite

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

    Qin, Lang; Verweij, Henk, E-mail: verweij.1@osu.edu

    2014-03-01

    Graphical abstract: - Highlights: • Hexaferrite Co{sub 2}Z is synthesized through the modified Pechini method. • Magnetic domains are observed in anisotropic Co{sub 2}Z single grain using MFM. • Observed single grain domain thickness is in good agreement with Dotsh model. - Abstract: Hexaferrite Co{sub 2}Z was synthesized through the modified Pechini method. Partially oriented samples were obtained after consolidation with uniaxial pressing and calcination/sintering at 1300 °C/1330 °C. The sample composition and morphology was identified with X-ray diffractometry (XRD) and scanning electron microscopy (SEM) with energy-dispersive X-ray spectrometry (EDS). MFM studies of the single grains revealed a domain structuremore » with 0.7 μm wide. The Co{sub 2}Z static magnetization was measured with a vibrating sample magnetometer (VSM), and was used to calculate a single grain domain with a thickness of 4.8 μm. This result is in good agreement with SEM observations of the single grain thickness.« less

  4. An intact Pms2 ATPase domain is not essential for male fertility

    PubMed Central

    Fischer, Jared M; Dudley, Sandra; Miller, Ashleigh J; Liskay, R Michael

    2016-01-01

    The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3. PMID:26753533

  5. An intact Pms2 ATPase domain is not essential for male fertility.

    PubMed

    Fischer, Jared M; Dudley, Sandra; Miller, Ashleigh J; Liskay, R Michael

    2016-03-01

    The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2(ko/ko)), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2(ko/ko) males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2(cre)), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2(cre/cre) male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2(cre) allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2(ko/ko) mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2(cre/cre) testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. 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

  7. NovelFam3000 – Uncharacterized human protein domains conserved across model organisms

    PubMed Central

    Kemmer, Danielle; Podowski, Raf M; Arenillas, David; Lim, Jonathan; Hodges, Emily; Roth, Peggy; Sonnhammer, Erik LL; Höög, Christer; Wasserman, Wyeth W

    2006-01-01

    Background Despite significant efforts from the research community, an extensive portion of the proteins encoded by human genes lack an assigned cellular function. Most metazoan proteins are composed of structural and/or functional domains, of which many appear in multiple proteins. Once a domain is characterized in one protein, the presence of a similar sequence in an uncharacterized protein serves as a basis for inference of function. Thus knowledge of a domain's function, or the protein within which it arises, can facilitate the analysis of an entire set of proteins. Description From the Pfam domain database, we extracted uncharacterized protein domains represented in proteins from humans, worms, and flies. A data centre was created to facilitate the analysis of the uncharacterized domain-containing proteins. The centre both provides researchers with links to dispersed internet resources containing gene-specific experimental data and enables them to post relevant experimental results or comments. For each human gene in the system, a characterization score is posted, allowing users to track the progress of characterization over time or to identify for study uncharacterized domains in well-characterized genes. As a test of the system, a subset of 39 domains was selected for analysis and the experimental results posted to the NovelFam3000 system. For 25 human protein members of these 39 domain families, detailed sub-cellular localizations were determined. Specific observations are presented based on the analysis of the integrated information provided through the online NovelFam3000 system. Conclusion Consistent experimental results between multiple members of a domain family allow for inferences of the domain's functional role. We unite bioinformatics resources and experimental data in order to accelerate the functional characterization of scarcely annotated domain families. PMID:16533400

  8. Exercise and physical activity in asylum seekers in Northern England; using the theoretical domains framework to identify barriers and facilitators.

    PubMed

    Haith-Cooper, Melanie; Waskett, Catherine; Montague, Jane; Horne, Maria

    2018-06-19

    Many asylum seekers have complex mental health needs which can be exacerbated by the challenging circumstances in which they live and difficulties accessing health services. Regular moderate physical activity can improve mental health and would be a useful strategy to achieve this. Evidence suggests there are barriers to engaging black and minority ethnic groups in physical activity, but there is little research around asylum seekers to address the key barriers and facilitators in this group. A two stage qualitative study used semi-structured interviews underpinned by the Theoretical Domains Framework. The interviews were conducted in voluntary sector groups in four towns/ cities in Northern England. Purposive sampling recruited 36 asylum seekers from 18 different countries. Interviews were audio recorded, transcribed verbatim and subject to framework analysis. Stage two involved a nominal group technique with five key stakeholders including asylum seekers and those that work with them. They followed a four stage process to rank and reach consensus on the key barrier to undertaking physical activity/ exercise that could be addressed locally through a future intervention. A number of barriers and facilitators were identified including a lack of understanding of the term physical activity and recommended levels but knowledge of the health benefits of physical activity/ exercise and the motivation to increase levels having engaged with activities back home. Living as an asylum seeker was considered a barrier due to the stress, poverty and temporary nature of living in an unfamiliar place. The outcome of the nominal group technique was that a lack of knowledge of facilities in the local area was the prevailing barrier that could be addressed. Public health practitioners could develop interventions which capitalise on the motivation and knowledge of asylum seekers to encourage an increase in physical activity which may in turn reduce the breadth and depth of mental

  9. Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

    PubMed Central

    Wu, George

    2011-01-01

    Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGFβ signaling, which attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ. PMID:21212736

  10. Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

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

    Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

    Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

  11. Frequency-domain multiscale quantum mechanics/electromagnetics simulation method

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

    Meng, Lingyi; Yin, Zhenyu; Yam, ChiYung, E-mail: yamcy@yangtze.hku.hk, E-mail: ghc@everest.hku.hk

    A frequency-domain quantum mechanics and electromagnetics (QM/EM) method is developed. Compared with the time-domain QM/EM method [Meng et al., J. Chem. Theory Comput. 8, 1190–1199 (2012)], the newly developed frequency-domain QM/EM method could effectively capture the dynamic properties of electronic devices over a broader range of operating frequencies. The system is divided into QM and EM regions and solved in a self-consistent manner via updating the boundary conditions at the QM and EM interface. The calculated potential distributions and current densities at the interface are taken as the boundary conditions for the QM and EM calculations, respectively, which facilitate themore » information exchange between the QM and EM calculations and ensure that the potential, charge, and current distributions are continuous across the QM/EM interface. Via Fourier transformation, the dynamic admittance calculated from the time-domain and frequency-domain QM/EM methods is compared for a carbon nanotube based molecular device.« less

  12. Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

    PubMed

    Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

    2017-05-05

    The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  13. Domain topology and domain switching kinetics in a hybrid improper ferroelectric

    PubMed Central

    Huang, F. -T.; Xue, F.; Gao, B.; Wang, L. H.; Luo, X.; Cai, W.; Lu, X. -Z.; Rondinelli, J. M.; Chen, L. Q.; Cheong, S. -W.

    2016-01-01

    Charged polar interfaces such as charged ferroelectric walls or heterostructured interfaces of ZnO/(Zn,Mg)O and LaAlO3/SrTiO3, across which the normal component of electric polarization changes suddenly, can host large two-dimensional conduction. Charged ferroelectric walls, which are energetically unfavourable in general, were found to be mysteriously abundant in hybrid improper ferroelectric (Ca,Sr)3Ti2O7 crystals. From the exploration of antiphase boundaries in bilayer-perovskites, here we discover that each of four polarization-direction states is degenerate with two antiphase domains, and these eight structural variants form a Z4 × Z2 domain structure with Z3 vortices and five distinct types of domain walls, whose topology is directly relevant to the presence of abundant charged walls. We also discover a zipper-like nature of antiphase boundaries, which are the reversible creation/annihilation centres of pairs of two types of ferroelectric walls (and also Z3-vortex pairs) in 90° and 180° polarization switching. Our results demonstrate the unexpectedly rich nature of hybrid improper ferroelectricity. PMID:27215944

  14. Blockade of a key region in the extracellular domain inhibits HER2 dimerization and signaling.

    PubMed

    Menendez, Javier A; Schroeder, Barbara; Peirce, Susan K; Vellon, Luciano; Papadimitropoulou, Adriana; Espinoza, Ingrid; Lupu, Ruth

    2015-06-01

    Several treatment strategies target the human epidermal growth factor receptor 2 (HER2) in breast carcinomas, including monoclonal antibodies directed against HER2's extracellular domain (ECD) and small molecule inhibitors of its tyrosine kinase activity. Yet, novel therapies are needed that prevent HER2 dimerization with other HER family members, because current treatments are only partially effective. To test the hypothesis that HER2 activation requires a protein sequence in the HER2-ECD that mediates HER2 homo- and heterodimerization, we introduced a series of deletion mutations in the third subdomain of HER2-ECD. These deletion mutants were retrovirally expressed in breast cancer (BC) cells that naturally overexpress HER2 and in noncancerous, HER2-negative breast epithelial cells. One-factor analysis of variance or Student's t test were used to analyze differences. All statistical tests were two-sided. The smallest deletion in the ECD domain of HER2, which removed only 16 amino acids (HER2-ECDΔ451-466), completely disrupted the oncogenic potential of HER2. In contrast to wild-type HER2, the mutant-inhibited anchorage-independent growth (mean number of colonies: mutant, 70, 95% confidence interval [CI] = 55 to 85; wild-type, 400, 95% CI = 320 to 480, P < .001) increased sensitivity to paclitaxel treatment in both transformed and nontransformed cells. Overexpression of HER2Δ451-466 efficiently inhibited activation of HER1, HER2, and HER3 in all cell lines tested. These findings reveal that an essential "activating" sequence exists in the extracellular domain of HER2. Disruption of this sequence disables the HER2 dimerization loop, blocks subsequent activation of HER2-driven oncogenic signaling, and generates a dominant-negative form of HER2. Reagents specifically against this molecular activation switch may represent a novel targeted approach for the management of HER2-overexpressing carcinomas. © The Author 2015. Published by Oxford University Press. All

  15. Facilitating Decision Making, Re-Use and Collaboration: A Knowledge Management Approach for System Self-Awareness

    DTIC Science & Technology

    2009-10-01

    FACILITATING DECISION MAKING, RE-USE AND COLLABORATION: A KNOWLEDGE MANAGEMENT APPROACH FOR SYSTEM SELF- AWARENESS Shelley P. Gallup, Douglas J... Information Systems Experimentation (DISE) Group Naval Postgraduate School, Monterey, CA 93943 Keywords: Program self- awareness , decision making...decision makers express in obtaining constant awareness of what is going on in their domains of decision making because information that is needed

  16. ECOD: An Evolutionary Classification of Protein Domains

    PubMed Central

    Kinch, Lisa N.; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V.

    2014-01-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. PMID:25474468

  17. ECOD: an evolutionary classification of protein domains.

    PubMed

    Cheng, Hua; Schaeffer, R Dustin; Liao, Yuxing; Kinch, Lisa N; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V

    2014-12-01

    Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.

  18. Artificial ligand binding within the HIF2[alpha] PAS-B domain of the HIF2 transcription factor

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

    Scheuermann, Thomas H.; Tomchick, Diana R.; Machius, Mischa

    2009-05-12

    The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2{alpha} and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2{alpha} PAS-B domain contains a large internal cavity that accommodates ligands identified frommore » a small-molecule screen. Binding one of these ligands to HIF2{alpha} PAS-B modulates the affinity of the HIF2{alpha}:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.« less

  19. Chemical shift assignments of the partially deuterated Fyn SH2-SH3 domain.

    PubMed

    Kieken, Fabien; Loth, Karine; van Nuland, Nico; Tompa, Peter; Lenaerts, Tom

    2018-04-01

    Src Homology 2 and 3 (SH2 and SH3) are two key protein interaction modules involved in regulating the activity of many proteins such as tyrosine kinases and phosphatases by respective recognition of phosphotyrosine and proline-rich regions. In the Src family kinases, the inactive state of the protein is the direct result of the interaction of the SH2 and the SH3 domain with intra-molecular regions, leading to a closed structure incompetent with substrate modification. Here, we report the 1 H, 15 N and 13 C backbone- and side-chain chemical shift assignments of the partially deuterated Fyn SH3-SH2 domain and structural differences between tandem and single domains. The BMRB accession number is 27165.

  20. Shear at Twin Domain Boundaries in YBa2Cu3O7-x

    NASA Astrophysics Data System (ADS)

    Caldwell, W. A.; Tamura, N.; Celestre, R. S.; MacDowell, A. A.; Padmore, H. A.; Geballe, T. H.; Koster, G.; Batterman, B. W.; Patel, J. R.

    2004-05-01

    The microstructure and strain state of twin domains in YBa2Cu3O7-x are discussed based upon synchrotron white-beam x-ray microdiffraction measurements. Intensity variations of the fourfold twin splitting of Laue diffraction peaks are used to determine the twin domain structure. Strain analysis shows that interfaces between neighboring twin domains are strained in shear, whereas the interior of these domains are regions of low strain. These measurements are consistent with the orientation relationships of twin boundaries within and across domains and show that basal plane shear stresses can exceed 100MPa where twin domains meet. Our results support stress field pinning of magnetic flux vortices by twin domain boundaries.

  1. Facilitating an L2 Book Club: A Conversation-Analytic Study of Task Management

    ERIC Educational Resources Information Center

    Ro, Eunseok

    2018-01-01

    This study employs conversation analysis to examine a facilitator's interactional practices in the post-expansion phase of students' presentations in the context of a book club for second language learning. The analysis shows how the facilitator establishes intersubjectivity with regard to the ongoing task and manages students' task performance.…

  2. Barriers and Facilitators to Exercise Participation in People with Hip and/or Knee Osteoarthritis: Synthesis of the Literature Using Behavior Change Theory.

    PubMed

    Dobson, Fiona; Bennell, Kim L; French, Simon D; Nicolson, Philippa J A; Klaasman, Remco N; Holden, Melanie A; Atkins, Lou; Hinman, Rana S

    2016-05-01

    Exercise is recommended for hip and knee osteoarthritis (OA). Patient initiation of, and adherence to, exercise is key to the success of managing symptoms. This study aimed to (1) identify modifiable barriers and facilitators to participation in intentional exercise in hip and/or knee OA, and (2) synthesize findings using behavior change theory. A scoping review with systematic searches was conducted through March 2015. Two reviewers screened studies for eligibility. Barriers and facilitators were extracted and synthesized according to the Theoretical Domains Framework (TDF) by two independent reviewers. Twenty-three studies (total of 4633 participants) were included. The greatest number of unique barriers and facilitators mapped to the Environmental Context and Resources domain. Many barriers were related to Beliefs about Consequences and Beliefs about Capabilities, whereas many facilitators were related to Reinforcement. Clinicians should take a proactive role in facilitating exercise uptake and adherence, rather than trusting patients to independently overcome barriers to exercise. Strategies that may be useful include a personalized approach to exercise prescription, considering environmental context and available resources, personalized education about beneficial consequences of exercise and reassurance about exercise capability, and use of reinforcement strategies. Future research should investigate the effectiveness of behavior change interventions that specifically target these factors.

  3. Critical Role of the Src Homology 2 (SH2) Domain of Neuronal SH2B1 in the Regulation of Body Weight and Glucose Homeostasis in Mice

    PubMed Central

    Morris, David L.; Cho, Kae Won; Rui, Liangyou

    2010-01-01

    SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (ΔN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/ΔN503 compound mutant mice. R555E had a replacement of Arg555 with Glu within the SH2 domain. ΔN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or ΔN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner. PMID:20484460

  4. Critical role of the Src homology 2 (SH2) domain of neuronal SH2B1 in the regulation of body weight and glucose homeostasis in mice.

    PubMed

    Morris, David L; Cho, Kae Won; Rui, Liangyou

    2010-08-01

    SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (DeltaN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/DeltaN503 compound mutant mice. R555E had a replacement of Arg(555) with Glu within the SH2 domain. DeltaN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or DeltaN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner.

  5. Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

    PubMed

    Boëda, Batiste; Knowles, Phillip P; Briggs, David C; Murray-Rust, Judith; Soriano, Erika; Garvalov, Boyan K; McDonald, Neil Q; Way, Michael

    2011-04-01

    Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

  6. Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

    PubMed

    Ambaye, Nigus D; Gunzburg, Menachem J; Traore, Daouda A K; Del Borgo, Mark P; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

    2014-02-01

    Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

  7. Human Protein-disulfide Isomerase Is a Redox-regulated Chaperone Activated by Oxidation of Domain a′*

    PubMed Central

    Wang, Chao; Yu, Jiang; Huo, Lin; Wang, Lei; Feng, Wei; Wang, Chih-chen

    2012-01-01

    Protein-disulfide isomerase (PDI), with domains arranged as abb′xa′c, is a key enzyme and chaperone localized in the endoplasmic reticulum (ER) catalyzing oxidative folding and preventing misfolding/aggregation of proteins. It has been controversial whether the chaperone activity of PDI is redox-regulated, and the molecular basis is unclear. Here, we show that both the chaperone activity and the overall conformation of human PDI are redox-regulated. We further demonstrate that the conformational changes are triggered by the active site of domain a′, and the minimum redox-regulated cassette is located in b′xa′. The structure of the reduced bb′xa′ reveals for the first time that domain a′ packs tightly with both domain b′ and linker x to form one compact structural module. Oxidation of domain a′ releases the compact conformation and exposes the shielded hydrophobic areas to facilitate its high chaperone activity. Thus, the study unequivocally provides mechanistic insights into the redox-regulated chaperone activity of human PDI. PMID:22090031

  8. WAVE2 is regulated by multiple phosphorylation events within its VCA domain

    PubMed Central

    Pocha, Shirin M; Cory, Giles O

    2009-01-01

    The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. PMID:19012317

  9. Structural Basis and Function of XRN2-Binding by XTB Domains

    PubMed Central

    Richter, Hannes; Katic, Iskra; Gut, Heinz; Großhans, Helge

    2016-01-01

    The ribonuclease XRN2 is an essential player in RNA metabolism. In Caenorhabditis elegans, XRN2 functions with PAXT-1, which shares a putative XRN2-binding domain (XTBD) with otherwise unrelated mammalian proteins. Here, we characterize structure and function of an XTBD – XRN2 complex. Although XTBD stably interconnects two XRN2 domains through numerous interacting residues, mutation of a single critical residue suffices to disrupt XTBD – XRN2 complexes in vitro, and recapitulates paxt-1 null mutant phenotypes in vivo. Demonstrating conservation of function, vertebrate XTBD-containing proteins bind XRN2 in vitro, and human CDKN2AIPNL (C2AIL) can substitute for PAXT-1 in vivo. In vertebrates, where three distinct XTBD-containing proteins exist, XRN2 may partition to distinct stable heterodimeric complexes, likely differing in subcellular localization or function. In C. elegans, complex formation with the unique PAXT-1 serves to preserve the stability of XRN2 in the absence of substrate. PMID:26779609

  10. The study of co-citation analysis and knowledge structure on healthcare domain

    NASA Astrophysics Data System (ADS)

    Chu, Kuo-Chung; Liu, Wen-I.; Tsai, Ming-Yu

    2012-11-01

    With the prevalence of Internet and digital archives, the online e-journal database facilitates scholars to search literature in a research domain, or to cross-search an inter-disciplined field; the key literature can be efficiently traced out. This study intends to build a Web-based citation analysis system, which consists of four modules, they are: 1) literature search module; (2) statistics module; (3) articles analysis module; and (4) co-citation analysis module. The system focuses on PubMed Central dataset that has 170,000 records. In a research domain, a specific keyword searches in terms of authors, journals, and core issues. In addition, we use data mining techniques for co-citation analysis. The results assist researchers with in-depth understanding of the domain knowledge. Having an automated system for co-citation analysis, it helps to understand changes, trends, and knowledge structure of research domain. For the best of our knowledge, the proposed system differentiates from existing online electronic retrieval database analysis function. Perhaps, the proposed system is going to be a value-added database of healthcare domain, and hope to contribute the researchers.

  11. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

    PubMed Central

    Cooper, J A; Kashishian, A

    1993-01-01

    We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

  12. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation.

    PubMed

    Friberg, Anders; Thumann, Sybille; Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D; Sattler, Michael; Kempkes, Bettina

    2015-05-01

    Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics.

  13. Direct visualization of phase separation between superconducting and nematic domains in Co-doped CaFe2As2 close to a first-order phase transition

    NASA Astrophysics Data System (ADS)

    Fente, Antón; Correa-Orellana, Alexandre; Böhmer, Anna E.; Kreyssig, Andreas; Ran, S.; Bud'ko, Sergey L.; Canfield, Paul C.; Mompean, Federico J.; García-Hernández, Mar; Munuera, Carmen; Guillamón, Isabel; Suderow, Hermann

    2018-01-01

    We show that biaxial strain induces alternating tetragonal superconducting and orthorhombic nematic domains in Co-substituted CaFe2As2 . We use atomic force, magnetic force, and scanning tunneling microscopy to identify the domains and characterize their properties, finding in particular that tetragonal superconducting domains are very elongated, more than several tens of micrometers long and about 30 nm wide; have the same Tc as unstrained samples; and hold vortices in a magnetic field. Thus, biaxial strain produces a phase-separated state, where each phase is equivalent to what is found on either side of the first-order phase transition between antiferromagnetic orthorhombic and superconducting tetragonal phases found in unstrained samples when changing Co concentration. Having such alternating superconducting domains separated by normal conducting domains with sizes of the order of the coherence length opens opportunities to build Josephson junction networks or vortex pinning arrays and suggests that first-order quantum phase transitions lead to nanometric-size phase separation under the influence of strain.

  14. Examining, Documenting, and Modeling the Problem Space of a Variable Domain

    DTIC Science & Technology

    2002-06-14

    Feature-Oriented Domain Analysis ( FODA ) .............................................................................................. 9...development of this proposed process include: Feature-Oriented Domain Analysis ( FODA ) [3,4], Organization Domain Modeling (ODM) [2,5,6], Family-Oriented...configuration knowledge using generators [2]. 8 Existing Methods of Domain Engineering Feature-Oriented Domain Analysis ( FODA ) FODA is a domain

  15. Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

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

    Faravelli,A.; Dimasi, N.

    The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

  16. Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

    PubMed Central

    Park, Jeong Eun; Son, Alexander I.; Hua, Rui; Wang, Lianqing; Zhang, Xue; Zhou, Renping

    2012-01-01

    The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity. PMID:22570727

  17. Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer Through the Receiver Domain

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

    S Menon; S Wang

    The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving {alpha}4-{beta}5-{alpha}5, a common interface for activated receiver domain dimers. However, themore » switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.« less

  18. Roles of the SH2 and SH3 domains in the regulation of neuronal Src kinase functions.

    PubMed

    Groveman, Bradley R; Xue, Sheng; Marin, Vedrana; Xu, Jindong; Ali, Mohammad K; Bienkiewicz, Ewa A; Yu, Xian-Min

    2011-02-01

    Previous studies demonstrated that intra-domain interactions between Src family kinases (SFKs), stabilized by binding of the phosphorylated C-terminus to the SH2 domain and/or binding of the SH2 kinase linker to the SH3 domain, lock the molecules in a closed conformation, disrupt the kinase active site, and inactivate SFKs. Here we report that the up-regulation of N-methyl-D-aspartate receptors (NMDARs) induced by expression of constitutively active neuronal Src (n-Src), in which the C-terminus tyrosine is mutated to phenylalanine (n-Src/Y535F), is significantly reduced by dysfunctions of the SH2 and/or SH3 domains of the protein. Furthermore, we found that dysfunctions of SH2 and/or SH3 domains reduce auto-phosphorylation of the kinase activation loop, depress kinase activity, and decrease NMDAR phosphorylation. The SH2 domain plays a greater regulatory role than the SH3 domain. Our data also show that n-Src binds directly to the C-terminus of the NMDAR NR2A subunit in vitro, with a K(D) of 108.2 ± 13.3 nM. This binding is not Src kinase activity-dependent, and dysfunctions of the SH2 and/or SH3 domains do not significantly affect the binding. These data indicate that the SH2 and SH3 domains may function to promote the catalytic activity of active n-Src, which is important in the regulation of NMDAR functions. © 2010 The Authors Journal compilation © 2010 FEBS.

  19. Interaction of the Transactivation Domain of B-Myb with the TAZ2 Domain of the Coactivator p300: Molecular Features and Properties of the Complex

    PubMed Central

    Oka, Ojore; Waters, Lorna C.; Strong, Sarah L.; Dosanjh, Nuvjeevan S.; Veverka, Vaclav; Muskett, Frederick W.; Renshaw, Philip S.; Klempnauer, Karl-Heinz; Carr, Mark D.

    2012-01-01

    The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD) and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (Kd ∼1.0–10 µM) complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (∼1200 Å2). The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1), which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2. PMID:23300815

  20. 15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

    PubMed

    Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

    2005-04-01

    This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

  1. 2.5-D frequency-domain viscoelastic wave modelling using finite-element method

    NASA Astrophysics Data System (ADS)

    Zhao, Jian-guo; Huang, Xing-xing; Liu, Wei-fang; Zhao, Wei-jun; Song, Jian-yong; Xiong, Bin; Wang, Shang-xu

    2017-10-01

    2-D seismic modelling has notable dynamic information discrepancies with field data because of the implicit line-source assumption, whereas 3-D modelling suffers from a huge computational burden. The 2.5-D approach is able to overcome both of the aforementioned limitations. In general, the earth model is treated as an elastic material, but the real media is viscous. In this study, we develop an accurate and efficient frequency-domain finite-element method (FEM) for modelling 2.5-D viscoelastic wave propagation. To perform the 2.5-D approach, we assume that the 2-D viscoelastic media are based on the Kelvin-Voigt rheological model and a 3-D point source. The viscoelastic wave equation is temporally and spatially Fourier transformed into the frequency-wavenumber domain. Then, we systematically derive the weak form and its spatial discretization of 2.5-D viscoelastic wave equations in the frequency-wavenumber domain through the Galerkin weighted residual method for FEM. Fixing a frequency, the 2-D problem for each wavenumber is solved by FEM. Subsequently, a composite Simpson formula is adopted to estimate the inverse Fourier integration to obtain the 3-D wavefield. We implement the stiffness reduction method (SRM) to suppress artificial boundary reflections. The results show that this absorbing boundary condition is valid and efficient in the frequency-wavenumber domain. Finally, three numerical models, an unbounded homogeneous medium, a half-space layered medium and an undulating topography medium, are established. Numerical results validate the accuracy and stability of 2.5-D solutions and present the adaptability of finite-element method to complicated geographic conditions. The proposed 2.5-D modelling strategy has the potential to address modelling studies on wave propagation in real earth media in an accurate and efficient way.

  2. Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling

    NASA Astrophysics Data System (ADS)

    Egea-Jimenez, Antonio Luis; Gallardo, Rodrigo; Garcia-Pino, Abel; Ivarsson, Ylva; Wawrzyniak, Anna Maria; Kashyap, Rudra; Loris, Remy; Schymkowitz, Joost; Rousseau, Frederic; Zimmermann, Pascale

    2016-07-01

    PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP2). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP2-specific recognition. Experiments with cells support the importance of the syntenin-PIP2 interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.

  3. The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation*

    PubMed Central

    Ho, Tina; Watt, Brenda; Spruce, Lynn A.; Seeholzer, Steven H.; Marks, Michael S.

    2016-01-01

    The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils. PMID:26694611

  4. A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain.

    PubMed

    Alexandre, Maxime T A; Arents, Jos C; van Grondelle, Rienk; Hellingwerf, Klaas J; Kennis, John T M

    2007-03-20

    Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a

  5. Characterization of Runella slithyformis HD-Pnk, a bifunctional DNA/RNA end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase domain.

    PubMed

    Munir, Annum; Shuman, Stewart

    2016-11-28

    5' and 3' end healing are key steps in nucleic acid break repair in which 5' -OH ends are phosphorylated by a polynucleotide kinase and 3' -PO 4 or 2',3' -cyclic-PO 4 ends are hydrolyzed by a phosphoesterase to generate the 5' -PO 4 and 3' -OH termini required for sealing by classic polynucleotide ligases. End healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2',3' -phosphoesterase HD domain and a C-terminal 5' -OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5' -OH polynucleotides (9-mers or longer) in the presence of magnesium and any NTP donor. HD-Pnk dephosphorylates RNA 2',3' -cyclic phosphate, RNA 3' -phosphate, RNA 2' -phosphate, and DNA 3' -phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper or cobalt. HD-Pnkp homologs are present in genera from eleven bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. The present study provides insights to the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnkp as the exemplar of a novel clade of dual 5' and 3' end-healing enzymes that phosphorylate 5' -OH termini and dephosphorylate 2',3' -cyclic-PO 4 , 3' -PO 4 , and 2' -PO 4 ends. The distinctive feature of HD-Pnk is its domain composition: a fusion of an N-terminal HD phosphohydrolase module to a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, domain order, and similar polypeptide size are distributed widely among genera from eleven bacterial phyla. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. The elusive role of the SPRY2 domain in RyR1

    PubMed Central

    Willemse, Hermia; Mirza, Shamaruh; Gallant, Esther M; Board, Philip G

    2011-01-01

    The second of three SPRY domains (SPRY2, S1085-V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ∼10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells. PMID:21239886

  7. Identification of a linear neutralization domain in the protein VP2 of African horse sickness virus.

    PubMed

    Martínez-Torrecuadrada, J L; Casal, J I

    1995-07-10

    Overlapping fragments of the outermost capsid protein VP2 of African horse sickness virus serotype 4 (AHSV-4) have been expressed in Escherichia coli. Horse sera from infected and vaccinated animals, rabbit sera, and mice monoclonal antibodies specific for AHSV were used to screen these fragments for antigenic regions. The screening revealed that the major antigenic domain of the AHSV-4 VP2 is localized in a central region (amino acids 200 to 413) and that both the N-terminal region (aa 1-159) and the half C-terminal region (aa 414-1060) are not immunogenic. All the fragments containing a region between amino acids 253 and 413 (fragment H) were able to elicit consistently high titers of neutralizing antibodies. The ability of several subfragments of this region to evoke neutralizing antibodies indicates the presence of several sites inside this domain. However, neutralizing antibodies in sera of horse infected or vaccinated with attenuated viruses were not absorbed by fragment H, indicating that this domain is not immunodominant in AHSV. This information might be useful in designing a subunit vaccine against AHSV infection.

  8. Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

    PubMed

    Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

    2008-09-05

    The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

  9. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

    PubMed

    Sancho, Rosa M; Law, Bernard M H; Harvey, Kirsten

    2009-10-15

    Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

  10. Understanding Facilitation: Theory and Principles.

    ERIC Educational Resources Information Center

    Hogan, Christine

    This book introduces newcomers to the concept of facilitation, and it presents a critical analysis of established and current theory on facilitation for existing practitioners. The following are among the topics discussed: (1) emergence of the field of facilitation; (2) development of facilitation in management; (3) development of facilitation in…

  11. A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

    PubMed

    Morris, Gavin; Fanucchi, Sylvia

    2016-04-05

    Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

  12. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    PubMed Central

    Liu, Zhongle; Moran, Gary P.; Myers, Lawrence C.

    2016-01-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of ‘free,’ non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large ‘free’ pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the

  13. Amplification of TLO Mediator Subunit Genes Facilitate Filamentous Growth in Candida Spp.

    PubMed

    Liu, Zhongle; Moran, Gary P; Sullivan, Derek J; MacCallum, Donna M; Myers, Lawrence C

    2016-10-01

    Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation

  14. RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

    PubMed Central

    Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

    2013-01-01

    In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

  15. Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain

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

    Kim, Daehwan; Yeom, Ji-Hyun; Lee, Boeun

    The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation andmore » invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer. - Highlights: • TM-JM1/2 peptides are efficiently delivered into cells by AuNP-Apt-conjugates. • TM-JM1/2 peptides loaded onto AuNP-Apt conjugates inhibit DDR2 activation. • Inhibition of DDR2 activation by TM-JM1/2 peptides decreases tumor progression.« less

  16. Nanoscopic studies of domain structure dynamics in ferroelectric La:HfO2 capacitors

    NASA Astrophysics Data System (ADS)

    Buragohain, P.; Richter, C.; Schenk, T.; Lu, H.; Mikolajick, T.; Schroeder, U.; Gruverman, A.

    2018-05-01

    Visualization of domain structure evolution under an electrical bias has been carried out in ferroelectric La:HfO2 capacitors by a combination of Piezoresponse Force Microscopy (PFM) and pulse switching techniques to study the nanoscopic mechanism of polarization reversal and the wake-up process. It has been directly shown that the main mechanism behind the transformation of the polarization hysteretic behavior and an increase in the remanent polarization value upon the alternating current cycling is electrically induced domain de-pinning. PFM imaging and local spectroscopy revealed asymmetric switching in the La:HfO2 capacitors due to a significant imprint likely caused by the different boundary conditions at the top and bottom interfaces. Domain switching kinetics can be well-described by the nucleation limited switching model characterized by a broad distribution of the local switching times. It has been found that the domain velocity varies significantly throughout the switching process indicating strong interaction with structural defects.

  17. Toward a Data Scalable Solution for Facilitating Discovery of Science Resources

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

    Weaver, Jesse R.; Castellana, Vito G.; Morari, Alessandro

    Science is increasingly motivated by the need to process larger quantities of data. It is facing severe challenges in data collection, management, and processing, so much so that the computational demands of “data scaling” are competing with, and in many fields surpassing, the traditional objective of decreasing processing time. Example domains with large datasets include astronomy, biology, genomics, climate/weather, and material sciences. This paper presents a real-world use case in which we wish to answer queries pro- vided by domain scientists in order to facilitate discovery of relevant science resources. The problem is that the metadata for these science resourcesmore » is very large and is growing quickly, rapidly increasing the need for a data scaling solution. We propose a system – SGEM – designed for answering graph-based queries over large datasets on cluster architectures, and we re- port performance results for queries on the current RDESC dataset of nearly 1.4 billion triples, and on the well-known BSBM SPARQL query benchmark.« less

  18. Solution structure of leptospiral LigA4 Big domain

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

    Mei, Song; Zhang, Jiahai; Zhang, Xuecheng

    Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Bigmore » domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca{sup 2+} binding property of LigA4 Big domain. - Highlights: • Determining the solution structure of a bacterial immunoglobulin-like domain from a surface protein of Leptospira. • The solution structure shows some structural characteristics significantly different from the classic Ig-like domains. • A potential Ca{sup 2+}-binding site was identified by strains-all and NMR chemical shift perturbation.« less

  19. Superdiffusive motion of membrane-targeting C2 domains

    NASA Astrophysics Data System (ADS)

    Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

    2015-12-01

    Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

  20. Binding Assays Using Recombinant SH2 Domains: Far-Western, Pull-Down, and Fluorescence Polarization.

    PubMed

    Machida, Kazuya; Liu, Bernard

    2017-01-01

    Recognition of phosphotyrosine-containing sequences by SH2 domains confers specificity in tyrosine kinase pathways. By assessing interactions between isolated SH2 domains and their binding proteins, it is possible to gain insight into otherwise inaccessible complex cellular systems. Far-Western, pull-down, and fluorescence polarization (FP) have been frequently used for characterization of phosphotyrosine signaling. Here, we outline standard protocols for these established assays using recombinant SH2 domain, emphasizing the importance of appropriate sample preparation and assay controls.

  1. WW domain-binding protein 2: an adaptor protein closely linked to the development of breast cancer.

    PubMed

    Chen, Shuai; Wang, Han; Huang, Yu-Fan; Li, Ming-Li; Cheng, Jiang-Hong; Hu, Peng; Lu, Chuan-Hui; Zhang, Ya; Liu, Na; Tzeng, Chi-Meng; Zhang, Zhi-Ming

    2017-07-19

    The WW domain is composed of 38 to 40 semi-conserved amino acids shared with structural, regulatory, and signaling proteins. WW domain-binding protein 2 (WBP2), as a binding partner of WW domain protein, interacts with several WW-domain-containing proteins, such as Yes kinase-associated protein (Yap), paired box gene 8 (Pax8), WW-domain-containing transcription regulator protein 1 (TAZ), and WW-domain-containing oxidoreductase (WWOX) through its PPxY motifs within C-terminal region, and further triggers the downstream signaling pathway in vitro and in vivo. Studies have confirmed that phosphorylated form of WBP2 can move into nuclei and activate the transcription of estrogen receptor (ER) and progesterone receptor (PR), whose expression were the indicators of breast cancer development, indicating that WBP2 may participate in the progression of breast cancer. Both overexpression of WBP2 and activation of tyrosine phosphorylation upregulate the signal cascades in the cross-regulation of the Wnt and ER signaling pathways in breast cancer. Following the binding of WBP2 to the WW domain region of TAZ which can accelerate migration, invasion and is required for the transformed phenotypes of breast cancer cells, the transformation of epithelial to mesenchymal of MCF10A is activated, suggesting that WBP2 is a key player in regulating cell migration. When WBP2 binds with WWOX, a tumor suppressor, ER transactivation and tumor growth can be suppressed. Thus, WBP2 may serve as a molecular on/off switch that controls the crosstalk between E2, WWOX, Wnt, TAZ, and other oncogenic signaling pathways. This review interprets the relationship between WBP2 and breast cancer, and provides comprehensive views about the function of WBP2 in the regulation of the pathogenesis of breast cancer and endocrine therapy in breast cancer treatment.

  2. Sequence analysis of DBL2β domain of vargene of Indonesian Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Sulistyaningsih, E.; Romadhon, B. D.; Palupi, I.; Hidayah, F.; Dewi, R.; Prasetyo, A.

    2018-03-01

    Malaria is a major health problem in tropical countries including Indonesia. The most deadly agent is Plasmodium falciparum. In P. falciparum infection, PfEMP1 is supposed to play an important role in the pathogenesis of malaria. PfEMP1 is encoded by var gene family, it is a polymorphic protein where the extra-cellular portion contains of three distinct binding domains: Duffy binding-like (DBL), Cysteine-rich interdomain regions (CIDR) and C2. PfEMP1 varies in domain composition and binding specificity. The study explored the characteristic of Indonesian DBL2β-var genes and investigated its role to the malaria outcome. Twenty blood samples from clinically mild to severe malaria patients in Jember, East Java were collected for DNA extraction. Diagnosis was confirmed by Giemsa-stained thick blood smear. PCR was conducted using specific primer targeting on the full-length of DBL2ß and resulted approximately single band of 1,7 kb in a sample. This band was observed only from severe malaria sample. Sequence analysis directly from PCR product showed 74-99% similarities with previous sequences in Gene Bank. In conclusion, the DBL2β domain of vargene of Indonesian isolates was 1603 nucleotides in length and there was a possible association of the existence of DBL2β domain with the severity of malaria outcome.

  3. Goal-Based Domain Modeling as a Basis for Cross-Disciplinary Systems Engineering

    NASA Astrophysics Data System (ADS)

    Jarke, Matthias; Nissen, Hans W.; Rose, Thomas; Schmitz, Dominik

    Small and medium-sized enterprises (SMEs) are important drivers for innovation. In particular, project-driven SMEs that closely cooperate with their customers have specific needs in regard to information engineering of their development process. They need a fast requirements capture since this is most often included in the (unpaid) offer development phase. At the same time, they need to maintain and reuse the knowledge and experiences they have gathered in previous projects extensively as it is their core asset. The situation is complicated further if the application field crosses disciplinary boundaries. To bridge the gaps and perspectives, we focus on shared goals and dependencies captured in models at a conceptual level. Such a model-based approach also offers a smarter connection to subsequent development stages, including a high share of automated code generation. In the approach presented here, the agent- and goal-oriented formalism i * is therefore extended by domain models to facilitate information organization. This extension permits a domain model-based similarity search, and a model-based transformation towards subsequent development stages. Our approach also addresses the evolution of domain models reflecting the experiences from completed projects. The approach is illustrated with a case study on software-intensive control systems in an SME of the automotive domain.

  4. The PYRIN domain: A member of the death domain-fold superfamily

    PubMed Central

    Fairbrother, Wayne J.; Gordon, Nathaniel C.; Humke, Eric W.; O'Rourke, Karen M.; Starovasnik, Melissa A.; Yin, Jian-Ping; Dixit, Vishva M.

    2001-01-01

    PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction. PMID:11514682

  5. Direct visualization of phase separation between superconducting and nematic domains in Co-doped CaFe 2 As 2 close to a first-order phase transition

    DOE PAGES

    Fente, Antón; Correa-Orellana, Alexandre; Böhmer, Anna E.; ...

    2018-01-09

    We show that biaxial strain induces alternating tetragonal superconducting and orthorhombic nematic domains in Co substituted CaFe 2As 2. We use Atomic Force, Magnetic Force and Scanning Tunneling Microscopy (AFM, MFM and STM) to identify the domains and characterize their properties, nding in particular that tetragonal superconducting domains are very elongated, more than several tens of μm long and about 30 nm wide, have the same Tc than unstrained samples and hold vortices in a magnetic eld. Thus, biaxial strain produces a phase separated state, where each phase is equivalent to what is found at either side of the rstmore » order phase transition between antiferromagnetic orthorhombic and superconducting tetragonal phases found in unstrained samples when changing Co concentration. Having such alternating superconducting domains separated by normal conducting domains with sizes of order of the coherence length opens opportunities to build Josephson junction networks or vortex pinning arrays and suggests that first order quantum phase transitions lead to nanometric size phase separation under the influence of strain.« less

  6. Magnetic Force Microscopy Investigation of Magnetic Domains in Nd2Fe14B

    NASA Astrophysics Data System (ADS)

    Talari, Mahesh Kumar; Markandeyulu, G.; Rao, K. Prasad

    2010-07-01

    Remenance and coercivity in Nd2Fe14B materials are strongly dependent on the microstructural aspects like phases morphology and grain size. The coercivity (Hc) of a magnetic material varies inversely with the grain size (D) and there is a critical size below which Hc∝D6. Domain wall pinning by grain boundaries and foreign phases is the important mechanism in explaining the improvement in coercivity and remenance. Nd2Fe14B intermetallic compound with stochiometric composition was prepared from pure elements (Nd -99.5%, Fe—99.95%, B -99.99%) by arc melting in argon atmosphere. Magnetic Force Microscope (MFM) gives high-resolution magnetic domain structural information of ferromagnetic samples. DI-3100 Scanning Probe Microscope with MESP probes was used For MFM characterization of the samples. Magnetic domains observed in cast ingots were very long (up to 40 μm were observed) and approximately 1-5 μm wide due to high anisotropy of the compounds. Magnetic domains have displayed different image contrast and morphologies at different locations of the samples. The domain morphologies and image contrast obtained in this analysis were explained in this paper.

  7. Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells.

    PubMed

    Mänz, Benjamin; de Graaf, Miranda; Mögling, Ramona; Richard, Mathilde; Bestebroer, Theo M; Rimmelzwaan, Guus F; Fouchier, Ron A M

    2016-07-01

    A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts. Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions-most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2-have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

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

    Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta

    2015-04-01

    Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of proteinmore » belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.« less

  9. A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects

    PubMed Central

    Simon-Vecsei, Zsófia; Király, Róbert; Bagossi, Péter; Tóth, Boglárka; Dahlbom, Ingrid; Caja, Sergio; Csősz, Éva; Lindfors, Katri; Sblattero, Daniele; Nemes, Éva; Mäki, Markku; Fésüs, László; Korponay-Szabó, Ilma R.

    2012-01-01

    The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1–2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits. PMID:22198767

  10. Synthesis, purification and crystallographic studies of the C-terminal sterol carrier protein type 2 (SCP-2) domain of human hydroxysteroid dehydrogenase-like protein 2.

    PubMed

    Cheng, Zhong; Li, Yao; Sui, Chun; Sun, Xiaobo; Xie, Yong

    2015-07-01

    Human hydroxysteroid dehydrogenase-like protein 2 (HSDL2) is a member of the short-chain dehydrogenase/reductase (SDR) subfamily of oxidoreductases and contains an N-terminal catalytic domain and a C-termianl sterol carrier protein type 2 (SCP-2) domain. In this study, the C-terminal SCP-2 domain of human HSDL2, including residues Lys318-Arg416, was produced in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to 2.10 Å resolution. The crystal belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a = b = 70.4, c = 60.6 Å, α = β = 90, γ = 120°. Two protein molecules are present in the asymmetric unit, resulting in a Matthews coefficient of 2.16 Å(3) Da(-1) and an approximate solvent content of 43%.

  11. The Measurement and Role of Ecological Resilience Systems Theory Across Domain-Specific Outcomes: The Domain-Specific Resilient Systems Scales.

    PubMed

    Maltby, John; Day, Liz; Hall, Sophie S; Chivers, Sally

    2017-10-01

    Research suggests that trait resilience may be best understood within an ecological resilient systems theory, comprising engineering, ecological, and adaptive capacity resilience. However, there is no evidence as to how this theory translates to specific life domains. Data from two samples (the United States, n = 1,278; the United Kingdom, n = 211) facilitated five studies that introduce the Domain-Specific Resilient Systems Scales for assessing ecological resilient systems theory within work, health, marriage, friendships, and education. The Domain-Specific Resilient Systems Scales are found to predict unique variance in job satisfaction, lower job burnout, quality-of-life following illness, marriage commitment, and educational engagement, while controlling for factors including sex, age, personality, cognitive ability, and trait resilience. The findings also suggest a distinction between the three resilience dimensions in terms of the types of systems to which they contribute. Engineering resilience may contribute most to life domains where an established system needs to be maintained, for example, one's health. Ecological resilience may contribute most to life domains where the system needs sustainability in terms of present and future goal orientation, for example, one's work. Adaptive Capacity may contribute most to life domains where the system needs to be retained, preventing it from reaching a crisis state, for example, work burnout.

  12. Analytical time-domain Green’s functions for power-law media

    PubMed Central

    Kelly, James F.; McGough, Robert J.; Meerschaert, Mark M.

    2008-01-01

    Frequency-dependent loss and dispersion are typically modeled with a power-law attenuation coefficient, where the power-law exponent ranges from 0 to 2. To facilitate analytical solution, a fractional partial differential equation is derived that exactly describes power-law attenuation and the Szabo wave equation [“Time domain wave-equations for lossy media obeying a frequency power-law,” J. Acoust. Soc. Am. 96, 491–500 (1994)] is an approximation to this equation. This paper derives analytical time-domain Green’s functions in power-law media for exponents in this range. To construct solutions, stable law probability distributions are utilized. For exponents equal to 0, 1∕3, 1∕2, 2∕3, 3∕2, and 2, the Green’s function is expressed in terms of Dirac delta, exponential, Airy, hypergeometric, and Gaussian functions. For exponents strictly less than 1, the Green’s functions are expressed as Fox functions and are causal. For exponents greater than or equal than 1, the Green’s functions are expressed as Fox and Wright functions and are noncausal. However, numerical computations demonstrate that for observation points only one wavelength from the radiating source, the Green’s function is effectively causal for power-law exponents greater than or equal to 1. The analytical time-domain Green’s function is numerically verified against the material impulse response function, and the results demonstrate excellent agreement. PMID:19045774

  13. A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

    PubMed

    Anosova, Irina; Melnik, Svitlana; Tripsianes, Konstantinos; Kateb, Fatiha; Grummt, Ingrid; Sattler, Michael

    2015-05-26

    The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Cell Signaling by a Novel SH2 Domain Protein that is Overexpressed with Her2 in Breast Cancer

    DTIC Science & Technology

    1998-01-01

    1990) Methods Enzymol. 185, 51. Wary, K. K, Mainiero, F ., Isakoff, S. J., Marcantonio , E. E., and Giancotti 527-537 F . G. (1996) Cell 87, 733-743...family of SH2 domain proteins and acts cell nonautonomously in excretory canal development. Dev. Biol. 184:150-164. 3. Pelicci, G., L. Lanfrancone, F ...Grignani, J. McGlade, F . Cavallo, G. Forni, I. Nicoletti, T. Pawson, and P. G. Pelicci. 1992. A novel transforming protein (SHC) with an SH2

  15. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways

    PubMed Central

    Sancho, Rosa M.; Law, Bernard M.H.; Harvey, Kirsten

    2009-01-01

    Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2–DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2–DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2–DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2–DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease. PMID:19625296

  16. Barriers and facilitators to healthcare professional behaviour change in clinical trials using the Theoretical Domains Framework: a case study of a trial of individualized temperature-reduced haemodialysis.

    PubMed

    Presseau, Justin; Mutsaers, Brittany; Al-Jaishi, Ahmed A; Squires, Janet; McIntyre, Christopher W; Garg, Amit X; Sood, Manish M; Grimshaw, Jeremy M

    2017-05-22

    Implementing the treatment arm of a clinical trial often requires changes to healthcare practices. Barriers to such changes may undermine the delivery of the treatment making it more likely that the trial will demonstrate no treatment effect. The 'Major outcomes with personalized dialysate temperature' (MyTEMP) is a cluster-randomised trial to be conducted in 84 haemodialysis centres across Ontario, Canada to investigate whether there is a difference in major outcomes with an individualized dialysis temperature (IDT) of 0.5 °C below a patient's body temperature measured at the beginning of each haemodialysis session, compared to a standard dialysis temperature of 36.5 °C. To inform how to deploy the IDT across many haemodialysis centres, we assessed haemodialysis physicians' and nurses' perceived barriers and enablers to IDT use. We developed two topic guides using the Theoretical Domains Framework (TDF) to assess perceived barriers and enablers to IDT ordering and IDT setting (physician and nurse behaviours, respectively). We recruited a purposive sample of haemodialysis physicians and nurses from across Ontario and conducted in-person or telephone interviews. We used directed content analysis to double-code transcribed utterances into TDF domains, and inductive thematic analysis to develop themes. We interviewed nine physicians and nine nurses from 11 Ontario haemodialysis centres. We identified seven themes of potential barriers and facilitators to implementing IDTs: (1) awareness of clinical guidelines and how IDT fits with local policies (knowledge; goals), (2) benefits and motivation to use IDT (beliefs about consequences; optimism; reinforcement; intention; goals), (3) alignment of IDTs with usual practice and roles (social/professional role and identity; nature of the behaviour; beliefs about capabilities), (4) thermometer availability/accuracy and dialysis machine characteristics (environmental context and resources), (5) impact on workload (beliefs

  17. 1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

    PubMed

    Hörnig, Christina; Albert, Dana; Fischer, Lutz; Hörnig, Michael; Rådmark, Olof; Steinhilber, Dieter; Werz, Oliver

    2005-07-22

    5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

  18. TREatment of ATopic eczema (TREAT) Registry Taskforce: An international Delphi exercise to identify a core set of domains and domain items for national atopic eczema photo- and systemic therapy registries.

    PubMed

    Gerbens, L A A; Apfelbacher, C J; Irvine, A D; Barbarot, S; de Booij, R J; Boyce, A E; Deleuran, M; Eichenfield, L F; Hof, M H; Middelkamp-Hup, M A; Roberts, A; Schmitt, J; Vestergaard, C; Wall, D; Weidinger, S; Williamson, P R; Flohr, C; Spuls, P I

    2018-05-15

    Evidence of immunomodulatory therapies to guide clinical management for atopic eczema (AE) is scarce, despite frequent and often off-label use. Patient registries provide valuable evidence for the effects of treatments under real world conditions which can inform treatment guidelines, give the opportunity for health economic evaluation and the evaluation of quality of care, as well as pharmacogenetic and -dynamic research which cannot be adequately addressed in clinical trials. The TREatment of ATopic eczema (TREAT) Registry Taskforce aims to seek international consensus on a core set of domains and items ('what to measure') for AE research registries, using a Delphi approach. Participants from six stakeholder groups were included: doctors, nurses, non-clinical researchers, patients, industry and regulatory body representatives. The eDelphi comprised 3 sequential online rounds, requesting participants to rate the importance of each proposed domain item. Participants could add domain items to the proposed list in round 1. A final consensus meeting was held to ratify the core set. 479 participants from 36 countries accessed the eDelphi platform, of whom 86%, 79% and 74% completed rounds 1, 2, and 3 respectively. At the face-to-face consensus meeting attended by 42 participants the final core set was established containing 19 domains with 69 domain items (49 baseline and 20 follow-up items). This core set of domains and items to be captured by national AE systemic therapy registries will standardise data collection and thereby allow direct comparability across registries and facilitate data pooling between countries. Ultimately, it will provide greater insight into the effectiveness, safety and cost-effectiveness of photo- and systemic immunomodulatory therapies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  19. The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

    PubMed

    Maurizy, Chloé; Quinternet, Marc; Abel, Yoann; Verheggen, Céline; Santo, Paulo E; Bourguet, Maxime; C F Paiva, Ana; Bragantini, Benoît; Chagot, Marie-Eve; Robert, Marie-Cécile; Abeza, Claire; Fabre, Philippe; Fort, Philippe; Vandermoere, Franck; M F Sousa, Pedro; Rain, Jean-Christophe; Charpentier, Bruno; Cianférani, Sarah; Bandeiras, Tiago M; Pradet-Balade, Bérengère; Manival, Xavier; Bertrand, Edouard

    2018-05-29

    R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

  20. Ribosomal small subunit domains radiate from a central core

    NASA Astrophysics Data System (ADS)

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O'Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-02-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2‧OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit.

  1. Developing facilitation skills--a narrative.

    PubMed

    Newton, Jennifer M

    2003-07-01

    Effective facilitation has been identified in the literature as one of three elements, along with context and evidence, that have a dynamic and coexisting relationship to enable the successful uptake of evidence into practice. This paper presents an overview of the concept of facilitation within the context of practice development, ahead of a personal and professional reflective account of a 'developing facilitator'. In the summer of 2001, the author was instrumental in organising the first Practice Development School in Melbourne. Thrown in at the deep end, she found herself co-facilitating with an experienced practice developer from the United Kingdom. Having never facilitated in the arena of an action learning group, nor worked in the field of practice development, there was initially a sense of impending overload and drowning in the new knowledge and skills that needed to be acquired. Drawing upon the work of narrative inquiry the author shares her experiences in the anticipation that in telling her story it will assist others in their journey of becoming a facilitator.

  2. Participants' perspectives on making and maintaining behavioural changes in a lifestyle intervention for type 2 diabetes prevention: a qualitative study using the theory domain framework.

    PubMed

    Penn, Linda; Dombrowski, Stephan U; Sniehotta, Falko F; White, Martin

    2013-06-28

    In a qualitative substudy, we sought to elicit participants' perspectives of their behavioural change and maintenance of new behaviours towards intervention optimisation. The intervention was delivered in leisure and community settings in a local authority, which according to the UK government statistics ranks as 1 of the 10 most socioeconomically deprived areas in England. We recruited 218 adults aged 40-65 years at elevated risk of type 2 diabetes (Finnish Diabetes Risk Score≥11) to the intervention. Follow-up at 12 months was completed by 134 (62%). We recruited 15 participants, purposively sampled for physical activity increase, to the qualitative substudy. Lifestyle intervention can prevent type 2 diabetes, but translation to service provision remains challenging. The 'New life, New you' intervention aimed to promote physical activity, healthy eating and weight loss, and included supervised group physical activity sessions. Behavioural change and weight loss at 12-month follow-up were encouraging. We conducted 15 individual semistructured interviews. The Framework approach, with a comparison of emerging themes, was used in analysis of the transcribed data and complemented by the Theory Domains Framework. Themes emerging from the data were grouped as perceptions that promoted initiating, enacting and maintaining behavioural change. The data were then categorised in accordance with the Theory Domains Framework: intentions and goals; reinforcement; knowledge; social role and identity; social influences; skills and beliefs about capabilities; behavioural regulation, memory, emotion, attention and decision processes and environmental context and resources. Participant perceptions of intervention features that facilitated behavioural change processes were then similarly analysed. Social influences, reference to social role and identity (eg, peer support), and intentions and goals (eg, to lose weight) were dominant themes across the three phases of behavioural

  3. Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

    PubMed Central

    Gunawardana, Dilantha; Cheng, Heung-Chin; Gayler, Kenwyn R.

    2008-01-01

    The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B. PMID:18025047

  4. EphA2 transmembrane domain is uniquely required for keratinocyte migration by regulating ephrin-A1 levels.

    PubMed

    Ventrella, Rosa; Kaplan, Nihal; Hoover, Paul; Perez White, Bethany E; Lavker, Robert M; Getsios, Spiro

    2018-04-26

    EphA2 receptor tyrosine kinase (RTK) is activated by ephrin-A1 ligand, which harbors a GPI-anchor that enhances lipid raft localization. While EphA2 and ephrin-A1 modulate keratinocyte migration and differentiation, the ability of this cell-cell communication complex to localize to different membrane regions in keratinocytes remains unknown. Using a combination of biochemical and imaging approaches, we provide evidence that ephrin-A1 and a ligand-activated form of EphA2 partition outside of lipid raft domains in response to calcium-mediated cell-cell contact stabilization in normal human epidermal keratinocytes (NHEKs). EphA2 transmembrane domain (TMD) swapping with a shorter and molecularly distinct TMD of EphA1 resulted in decreased localization of this RTK at cell-cell junctions and increased expression of ephrin-A1, which is a negative regulator of keratinocyte migration. Accordingly, altered EphA2 membrane distribution at cell-cell contacts limited the ability of keratinocytes to seal linear scratch wounds in vitro in an ephrin-A1-dependent manner. Collectively, these studies highlight a key role for the EphA2 TMD in receptor-ligand membrane distribution at cell-cell contacts that modulates ephrin-A1 levels to allow for efficient keratinocyte migration with relevance for cutaneous wound healing. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  5. MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.

    PubMed

    Rebustini, Ivan T; Myers, Christopher; Lassiter, Keyonica S; Surmak, Andrew; Szabova, Ludmila; Holmbeck, Kenn; Pedchenko, Vadim; Hudson, Billy G; Hoffman, Matthew P

    2009-10-01

    Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via beta1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis.

  6. XML Based Markup Languages for Specific Domains

    NASA Astrophysics Data System (ADS)

    Varde, Aparna; Rundensteiner, Elke; Fahrenholz, Sally

    A challenging area in web based support systems is the study of human activities in connection with the web, especially with reference to certain domains. This includes capturing human reasoning in information retrieval, facilitating the exchange of domain-specific knowledge through a common platform and developing tools for the analysis of data on the web from a domain expert's angle. Among the techniques and standards related to such work, we have XML, the eXtensible Markup Language. This serves as a medium of communication for storing and publishing textual, numeric and other forms of data seamlessly. XML tag sets are such that they preserve semantics and simplify the understanding of stored information by users. Often domain-specific markup languages are designed using XML, with a user-centric perspective. Standardization bodies and research communities may extend these to include additional semantics of areas within and related to the domain. This chapter outlines the issues to be considered in developing domain-specific markup languages: the motivation for development, the semantic considerations, the syntactic constraints and other relevant aspects, especially taking into account human factors. Illustrating examples are provided from domains such as Medicine, Finance and Materials Science. Particular emphasis in these examples is on the Materials Markup Language MatML and the semantics of one of its areas, namely, the Heat Treating of Materials. The focus of this chapter, however, is not the design of one particular language but rather the generic issues concerning the development of domain-specific markup languages.

  7. Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca2+ -calmodulin at mossy fiber-CA3 synapses.

    PubMed

    Andrade-Talavera, Yuniesky; Duque-Feria, Paloma; Negrete-Díaz, José Vicente; Sihra, Talvinder S; Flores, Gonzalo; Rodríguez-Moreno, Antonio

    2012-09-01

    Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  8. Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2

    PubMed Central

    Koehnke, Jesko; Jin, Xiangshu; Budreck, Elaine C.; Posy, Shoshana; Scheiffele, Peter; Honig, Barry; Shapiro, Lawrence

    2008-01-01

    Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3-Å crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site region differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions. PMID:18250328

  9. CoMoDo: identifying dynamic protein domains based on covariances of motion.

    PubMed

    Wieninger, Silke A; Ullmann, G Matthias

    2015-06-09

    Most large proteins are built of several domains, compact units which enable functional protein motions. Different domain assignment approaches exist, which mostly rely on concepts of stability, folding, and evolution. We describe the automatic assignment method CoMoDo, which identifies domains based on protein dynamics. Covariances of atomic fluctuations, here calculated by an Elastic Network Model, are used to group residues into domains of different hierarchical levels. The so-called dynamic domains facilitate the study of functional protein motions involved in biological processes like ligand binding and signal transduction. By applying CoMoDo to a large number of proteins, we demonstrate that dynamic domains exhibit features absent in the commonly assigned structural domains, which can deliver insight into the interactions between domains and between subunits of multimeric proteins. CoMoDo is distributed as free open source software at www.bisb.uni-bayreuth.de/CoMoDo.html .

  10. Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

    PubMed

    Ramirez, Ursula D; Nikonova, Anna S; Liu, Hanqing; Pecherskaya, Anna; Lawrence, Sarah H; Serebriiskii, Ilya G; Zhou, Yan; Robinson, Matthew K; Einarson, Margret B; Golemis, Erica A; Jaffe, Eileen K

    2015-05-28

    Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

  11. A two-dimensional time domain near zone to far zone transformation

    NASA Technical Reports Server (NTRS)

    Luebbers, Raymond J.; Ryan, Deirdre; Beggs, John H.; Kunz, Karl S.

    1991-01-01

    In a previous paper, a time domain transformation useful for extrapolating 3-D near zone finite difference time domain (FDTD) results to the far zone was presented. In this paper, the corresponding 2-D transform is outlined. While the 3-D transformation produced a physically observable far zone time domain field, this is not convenient to do directly in 2-D, since a convolution would be required. However, a representative 2-D far zone time domain result can be obtained directly. This result can then be transformed to the frequency domain using a Fast Fourier Transform, corrected with a simple multiplicative factor, and used, for example, to calculate the complex wideband scattering width of a target. If an actual time domain far zone result is required it can be obtained by inverse Fourier transform of the final frequency domain result.

  12. Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2*

    PubMed Central

    Ver Heul, Aaron M.; Fowler, C. Andrew; Ramaswamy, S.; Piper, Robert C.

    2013-01-01

    NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2. PMID:23300079

  13. Questioning skills of clinical facilitators supporting undergraduate nursing students.

    PubMed

    Phillips, Nicole M; Duke, Maxine M; Weerasuriya, Rona

    2017-12-01

    To report on a study investigating questioning skills of clinical facilitators who support the learning of undergraduate nursing students. The ability to think critically is integral to decision-making and the provision of safe and quality patient care. Developing students' critical thinking skills is expected of those who supervise and facilitate student learning in the clinical setting. Models used to facilitate student learning in the clinical setting have changed over the years with clinicians having dual responsibility for patient care and facilitating student learning. Many of these nurses have no preparation for the educative role. This study adapted a comparative study conducted over fifteen years ago. Descriptive online survey including three acute care patient scenarios involving an undergraduate nursing student. Participants were required to identify the questions they would ask the student in relation to the scenario. A total of 133 clinical facilitators including clinical teachers, clinical educators and preceptors from five large partner healthcare organisations of one Australian university participated. The majority of questions asked were knowledge questions, the lowest category in the cognitive domain requiring only simple recall of information. Facilitators who had undertaken an education-related course/workshop or formal qualification asked significantly more questions from the higher cognitive level. The study provides some evidence that nursing facilitators in the clinical setting ask students predominantly low-level questions. Further research is needed to identify strategies that develop the capacity of facilitators to ask higher level cognitive questions. Clinical facilitators should undertake targeted education that focuses on how to frame questions for students that demand application, analysis, synthesis and evaluation. © 2017 John Wiley & Sons Ltd.

  14. Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

    PubMed Central

    Vélez-Bermúdez, Isabel C.; Carretero-Paulet, Lorenzo; Lumbreras, Victoria; Pagès, Montserrat

    2011-01-01

    Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes. PMID:21789193

  15. DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

    PubMed

    Keyamura, Kenji; Katayama, Tsutomu

    2011-08-19

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

  16. DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

    PubMed Central

    Keyamura, Kenji; Katayama, Tsutomu

    2011-01-01

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

  17. Si(111) strained layers on Ge(111): Evidence for c (2 ×4 ) domains

    NASA Astrophysics Data System (ADS)

    Zhachuk, R.; Coutinho, J.; Dolbak, A.; Cherepanov, V.; Voigtländer, B.

    2017-08-01

    The tensile-strained Si (111 ) layers grown on top of Ge (111 ) substrates are studied by combining scanning tunneling microscopy, low-energy electron diffraction, and first-principles calculations. It is shown that the layers exhibit c (2 ×4 ) domains, which are separated by domain walls along <1 ¯10 > directions. A model structure for the c (2 ×4 ) domains is proposed, which shows low formation energy and good agreement with the experimental data. The results of our calculations suggest that Ge atoms are likely to replace Si atoms with dangling bonds on the surface (rest-atoms and adatoms), thus significantly lowering the surface energy and inducing the formation of domain walls. The experiments and calculations demonstrate that when surface strain changes from compressive to tensile, the (111) reconstruction converts from dimer-adatom-stacking fault-based to adatom-based structures.

  18. Dominant von Willebrand disease type 2A groups I and II due to missense mutations in the A2 domain of the von Willebrand factor gene: diagnosis and management.

    PubMed

    Michiels, Jan Jacques; van Vliet, Huub H D M

    2009-01-01

    Pertinent findings in patients with von Willebrand disease (VWD) type 2A include prolonged bleeding time (BT), consistently low von Willebrand factor (VWF):ristocetin cofactor activity (RCo)/antigen concentration (Ag) and VWF:collagen binding (CB)/Ag ratios, absence of high, and (depending on severity) intermediate and large VWF multimers, the presence of pronounced triplet structure of individual bands and increased VWF degradation products due to increased proteolysis caused by mutations in the A2 domain of VWF. Two categories of VWD type 2A can be distinguished: group I with severe and group II with mild VWD. A minority of VWD type 2A have mild VWD characterized by near normal to prolonged BT, normal factor VIII coagulant activity and VWF:Ag, low VWF:RCo and VWF:CB, a normal ristocetin-induced platelet aggregation and complete but transient correction of BT and functional VWF parameters to normal levels for only a few hours due to short half-lives for VWF:RCo and CWF:CB. Such transient complete responses to desmopressin (DDAVP) lasting only a few hours may facilitate treatment and prophylaxis of minor bleedings, but may not be able to prevent bleeding during minor and major surgery. Most VWD type 2A patients have pronounced VWD with very low VWF:RCo, prolonged BT, PFA-100 closure times >250 s, and response to DDAVP is only transient, minor, poor or absent, with no correction of the BT despite some increase in VWF:RCo, thus being candidates for factor VIII-VWF concentrate substitution for the acute and prophylactic treatment of bleeding symptoms. Copyright (c) 2009 S. Karger AG, Basel.

  19. An exploration of tutors' experiences of facilitating problem-based learning. Part 2--implications for the facilitation of problem based learning.

    PubMed

    Haith-Cooper, Melanie

    2003-01-01

    This paper is the second of two parts exploring a study that was undertaken to investigate the role of the tutor in facilitating problem-based learning (PBL). The first part focussed on the methodological underpinnings of the study. This paper aims to focus on the findings of the study and their implications for the facilitation of PBL. Six essential themes emerged from the findings that described the facilitation role. The tutors believed that their facilitation role was essentially structured around the decision of when to intervene and how to intervene in the PBL process. Modelling and non-verbal communication were seen as essential strategies for the facilitator. Underpinning these decisions was the need to trust in the philosophy of PBL. However, within many of the themes, there was a divergence of opinion as to how the role should actually be undertaken. Despite this, these findings have implications for the future role of PBL facilitators in Health Professional Education.

  20. Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE

    PubMed Central

    Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G.; Zheng, Hongjin; Gonen, Tamir

    2014-01-01

    The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins. PMID:25088546

  1. IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85.

    PubMed Central

    Myers, M G; Backer, J M; Sun, X J; Shoelson, S; Hu, P; Schlessinger, J; Yoakim, M; Schaffhausen, B; White, M F

    1992-01-01

    IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains. Images PMID:1332046

  2. Crystal structure of the Rasputin NTF2-like domain from Drosophila melanogaster

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

    Vognsen, Tina, E-mail: tv@farma.ku.dk; Kristensen, Ole, E-mail: ok@farma.ku.dk

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer The crystal structure of the NTF2-like domain of Rasputin protein is presented. Black-Right-Pointing-Pointer Differences to known ligand binding sites of nuclear transport factor 2 are discussed. Black-Right-Pointing-Pointer A new ligand binding site for the Rasputin and G3BP proteins is proposed. -- Abstract: The crystal structure of the NTF2-like domain of the Drosophila homolog of Ras GTPase SH3 Binding Protein (G3BP), Rasputin, was determined at 2.7 A resolution. The overall structure is highly similar to nuclear transport factor 2: It is a homodimer comprised of a {beta}-sheet and three {alpha}-helices forming a cone-like shape. However, known binding sites formore » RanGDP and FxFG containing peptides show electrostatic and steric differences compared to nuclear transport factor 2. A HEPES molecule bound in the structure suggests a new, and possibly physiologically relevant, ligand binding site.« less

  3. L1 Use in L2 Vocabulary Learning: Facilitator or Barrier

    ERIC Educational Resources Information Center

    Liu, Jing

    2008-01-01

    Based on empirical research and qualitative analysis, this paper aims to explore the effects of L1 use on L2 vocabulary teaching. The results show that, during L2 vocabulary teaching process, the proper application of L1 can effectively facilitate the memorization of new words, and the bilingual method (both English explanation and Chinese…

  4. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

    PubMed

    Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

    1993-05-06

    Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

  5. Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation.

    PubMed

    Aguirre, Jacob D; Dunkerley, Karen M; Mercier, Pascal; Shaw, Gary S

    2017-01-10

    Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential.

  6. EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

    PubMed Central

    Ling, P D; Ryon, J J; Hayward, S D

    1993-01-01

    EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the

  7. EBNA-2 of herpesvirus papio diverges significantly from the type A and type B EBNA-2 proteins of Epstein-Barr virus but retains an efficient transactivation domain with a conserved hydrophobic motif.

    PubMed

    Ling, P D; Ryon, J J; Hayward, S D

    1993-06-01

    EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (HVP). All human EBV isolates have either a type A or type B EBNA-2 gene. However, the HVP EBNA-2 gene falls into neither the type A category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA-2. To define the domains of EBNA-2 required for transcriptional activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong transcriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the

  8. Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

    PubMed

    Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

    2017-05-01

    Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

  9. Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

    PubMed Central

    2008-01-01

    Background Shuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures. Results The Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) formins that define a specific branch (Class III) of the formin family. Conclusion We propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms. PMID:18430232

  10. Structural basis for the interaction between Pyk2-FAT domain and leupaxin LD repeats

    DOE PAGES

    Vanarotti, Murugendra S.; Finkelstein, David B.; Guibao, Cristina D.; ...

    2016-02-11

    Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has fourmore » leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.« less

  11. Structural basis for the interaction between Pyk2-FAT domain and leupaxin LD repeats

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

    Vanarotti, Murugendra S.; Finkelstein, David B.; Guibao, Cristina D.

    Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor tyrosine kinase and belongs to the focal adhesion kinase (FAK) family. Like FAK, the C-terminal focal adhesion-targeting (FAT) domain of Pyk2 binds to paxillin, a scaffold protein in focal adhesions; however, the interaction between the FAT domain of Pyk2 and paxillin is dynamic and unstable. Leupaxin is another member in the paxillin family and was suggested to be the native binding partner of Pyk2; Pyk2 gene expression is strongly correlated with that of leupaxin in many tissues including primary breast cancer. Here, we report that leupaxin interacts with Pyk2-FAT. Leupaxin has fourmore » leucine–aspartate (LD) motifs. The first and third LD motifs of leupaxin preferably target the two LD-binding sites on the Pyk2-FAT domain, respectively. Moreover, the full-length leupaxin binds to Pyk2-FAT as a stable one-to-one complex. Together, we propose that there is an underlying selectivity between leupaxin and paxillin for Pyk2, which may influence the differing behavior of the two proteins at focal adhesion sites.« less

  12. Domain Evolution and Functional Diversification of Sulfite Reductases

    NASA Astrophysics Data System (ADS)

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

  13. Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

    PubMed Central

    Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

    2013-01-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

  14. Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

    PubMed

    Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

    2013-12-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

  15. MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

    PubMed Central

    Rebustini, Ivan T.; Myers, Christopher; Lassiter, Keyonica S.; Surmak, Andrew; Szabova, Ludmila; Holmbeck, Kenn; Pedchenko, Vadim; Hudson, Billy G.; Hoffman, Matthew P.

    2009-01-01

    Summary Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Importantly, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA-treatment, increasing MT-MMP and pro-proliferative gene expression via β1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA-treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis. PMID:19853562

  16. Cross Domain Deterrence: Livermore Technical Report, 2014-2016

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

    Barnes, Peter D.; Bahney, Ben; Matarazzo, Celeste

    2016-08-03

    Lawrence Livermore National Laboratory (LLNL) is an original collaborator on the project titled “Deterring Complex Threats: The Effects of Asymmetry, Interdependence, and Multi-polarity on International Strategy,” (CDD Project) led by the UC Institute on Global Conflict and Cooperation at UCSD under PIs Jon Lindsay and Erik Gartzke , and funded through the DoD Minerva Research Initiative. In addition to participating in workshops and facilitating interaction among UC social scientists, LLNL is leading the computational modeling effort and assisting with empirical case studies to probe the viability of analytic, modeling and data analysis concepts. This report summarizes LLNL work on themore » CDD Project to date, primarily in Project Years 1-2, corresponding to Federal fiscal year 2015. LLNL brings two unique domains of expertise to bear on this Project: (1) access to scientific expertise on the technical dimensions of emerging threat technology, and (2) high performance computing (HPC) expertise, required for analyzing the complexity of bargaining interactions in the envisioned threat models. In addition, we have a small group of researchers trained as social scientists who are intimately familiar with the International Relations research. We find that pairing simulation scientists, who are typically trained in computer science, with domain experts, social scientists in this case, is the most effective route to developing powerful new simulation tools capable of representing domain concepts accurately and answering challenging questions in the field.« less

  17. The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

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

    S Lee; Y Xue; J Hulbert

    2011-12-31

    Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, onemore » from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.« less

  18. VAR2CSA domains expressed in Escherichia coli induce cross-reactive antibodies to native protein.

    PubMed

    Oleinikov, Andrew V; Francis, Susan E; Dorfman, Jeffrey R; Rossnagle, Eddie; Balcaitis, Stephanie; Getz, Tony; Avril, Marion; Gose, Severin; Smith, Joseph D; Fried, Michal; Duffy, Patrick E

    2008-04-15

    The variant surface antigen VAR2CSA is a pregnancy malaria vaccine candidate, but its size and polymorphism are obstacles to development. We expressed 3D7-type VAR2CSA domains in Escherichia coli as insoluble His-tagged proteins (Duffy binding-like [DBL] domains DBL1, DBL3, DBL4, and DBL5) that were denatured and refolded or as soluble glutathione S-transferase-tagged protein (DBL6). Anti-DBL5 antiserum cross-reacted with surface proteins of chondroitin sulfate A (CSA)-binding laboratory strains (3D7-CSA and FCR3-CSA) and a clinical pregnancy malaria isolate, whereas anti-DBL6 antiserum reacted only to 3D7 surface protein. This is the first report that E. coli-expressed VAR2CSA domains induce antibody to native VAR2CSA.

  19. Creating and Manipulating Formalized Software Architectures to Support a Domain-Oriented Application Composition System

    DTIC Science & Technology

    1992-12-01

    OOD) Paradigm ...... .... 2-7 2.4.3 Feature-Oriented Domain Analysis ( FODA ) ..... 2-7 2.4.4 Hierarchical Software Systems .................. 2-7...domain analysis ( FODA ) is one approach to domain analysis whose primary goal is to make domain products reusable (20:47). A domain model describes 2-5...7), among others. 2.4.3 Feature-Oriented Domain Analysis ( FODA ) Kang and others used the com- plete FODA methodology to successfully develop a window

  20. Structures of BIR domains from human NAIP and cIAP2

    PubMed Central

    Herman, Maria Dolores; Moche, Martin; Flodin, Susanne; Welin, Martin; Trésaugues, Lionel; Johansson, Ida; Nilsson, Martina; Nordlund, Pär; Nyman, Tomas

    2009-01-01

    The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1′–P4′ side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3′ position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2′ and P4′ pockets make similar interactions to those seen in other BIR domain–peptide complexes. The structures also reveal how a serine in the P1′ position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins. PMID:19923725

  1. Crystal Structure of the Extracellular Cholinesterase-Like Domain from Neuroligin-2

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

    Koehnke,J.; Jin, X.; Budreck, E.

    Neuroligins (NLs) are catalytically inactive members of a family of cholinesterase-like transmembrane proteins that mediate cell adhesion at neuronal synapses. Postsynaptic neuroligins engage in Ca2+-dependent transsynaptic interactions via their extracellular cholinesterase domain with presynaptic neurexins (NRXs). These interactions may be regulated by two short splice insertions (termed A and B) in the NL cholinesterase domain. Here, we present the 3.3- Angstroms crystal structure of the ectodomain from NL2 containing splice insertion A (NL2A). The overall structure of NL2A resembles that of cholinesterases, but several structural features are unique to the NL proteins. First, structural elements surrounding the esterase active-site regionmore » differ significantly between active esterases and NL2A. On the opposite surface of the NL2A molecule, the positions of the A and B splice insertions identify a candidate NRX interaction site of the NL protein. Finally, sequence comparisons of NL isoforms allow for mapping the location of residues of previously identified mutations in NL3 and NL4 found in patients with autism spectrum disorders. Overall, the NL2 structure promises to provide a valuable model for dissecting NL isoform- and synapse-specific functions.« less

  2. Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

    PubMed Central

    Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel; Prendergast, James G.; Mati, Ioulia K.; Eijk, Paul P.; Cockroft, Scott L.; Bradley, Mark; Ylstra, Bauke; Gilbert, Nick

    2013-01-01

    DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. PMID:23416946

  3. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    NASA Technical Reports Server (NTRS)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  4. Automated hierarchical classification of protein domain subfamilies based on functionally-divergent residue signatures

    PubMed Central

    2012-01-01

    Background The NCBI Conserved Domain Database (CDD) consists of a collection of multiple sequence alignments of protein domains that are at various stages of being manually curated into evolutionary hierarchies based on conserved and divergent sequence and structural features. These domain models are annotated to provide insights into the relationships between sequence, structure and function via web-based BLAST searches. Results Here we automate the generation of conserved domain (CD) hierarchies using a combination of heuristic and Markov chain Monte Carlo (MCMC) sampling procedures and starting from a (typically very large) multiple sequence alignment. This procedure relies on statistical criteria to define each hierarchy based on the conserved and divergent sequence patterns associated with protein functional-specialization. At the same time this facilitates the sequence and structural annotation of residues that are functionally important. These statistical criteria also provide a means to objectively assess the quality of CD hierarchies, a non-trivial task considering that the protein subgroups are often very distantly related—a situation in which standard phylogenetic methods can be unreliable. Our aim here is to automatically generate (typically sub-optimal) hierarchies that, based on statistical criteria and visual comparisons, are comparable to manually curated hierarchies; this serves as the first step toward the ultimate goal of obtaining optimal hierarchical classifications. A plot of runtimes for the most time-intensive (non-parallelizable) part of the algorithm indicates a nearly linear time complexity so that, even for the extremely large Rossmann fold protein class, results were obtained in about a day. Conclusions This approach automates the rapid creation of protein domain hierarchies and thus will eliminate one of the most time consuming aspects of conserved domain database curation. At the same time, it also facilitates protein domain

  5. Ferroelectric-Domain-Patterning-Controlled Schottky Junction State in Monolayer MoS 2

    DOE PAGES

    Xiao, Zhiyong; Song, Jingfeng; Ferry, David K.; ...

    2017-06-08

    Here, we exploit scanning probe controlled domain patterning in a ferroelectric top-layer to induce nonvolatile modulation of the conduction characteristic of monolayer MoS 2 between a transistor and a junction state. In the presence of a domain wall, MoS 2 exhibits rectified I-V that is well described by the thermionic emission model. The induced Schottky barrier height Φ eff Β varies from 0.38 eV to 0.57 eV and is tunabe by a SiO 2 global back-gate, while the tuning range of Φ eff Β the barrier height depends sensitively on the conduction band tail trapping states. Our work points tomore » a new route to achieve programmable functionalities in van der Waals materials and sheds light on the critical performance limiting factors in these hybrid systems.« less

  6. Ferroelectric-Domain-Patterning-Controlled Schottky Junction State in Monolayer MoS 2

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

    Xiao, Zhiyong; Song, Jingfeng; Ferry, David K.

    Here, we exploit scanning probe controlled domain patterning in a ferroelectric top-layer to induce nonvolatile modulation of the conduction characteristic of monolayer MoS 2 between a transistor and a junction state. In the presence of a domain wall, MoS 2 exhibits rectified I-V that is well described by the thermionic emission model. The induced Schottky barrier height Φ eff Β varies from 0.38 eV to 0.57 eV and is tunabe by a SiO 2 global back-gate, while the tuning range of Φ eff Β the barrier height depends sensitively on the conduction band tail trapping states. Our work points tomore » a new route to achieve programmable functionalities in van der Waals materials and sheds light on the critical performance limiting factors in these hybrid systems.« less

  7. Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.

    PubMed

    Engen, J R; Smithgall, T E; Gmeiner, W H; Smith, D L

    1999-04-02

    Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains. Copyright 1999 Academic Press.

  8. Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2′,3′-Phosphoesterase HD Domain and a C-Terminal 5′-OH Polynucleotide Kinase Domain

    PubMed Central

    Munir, Annum

    2016-01-01

    ABSTRACT 5′- and 3′-end-healing reactions are key steps in nucleic acid break repair in which 5′-OH ends are phosphorylated by a polynucleotide kinase (Pnk) and 3′-PO4 or 2′,3′-cyclic-PO4 ends are hydrolyzed by a phosphoesterase to generate the 5′-PO4 and 3′-OH termini required for sealing by classic polynucleotide ligases. End-healing and sealing enzymes are present in diverse bacterial taxa, often organized as modular units within a single multifunctional polypeptide or as subunits of a repair complex. Here we identify and characterize Runella slithyformis HD-Pnk as a novel bifunctional end-healing enzyme composed of an N-terminal 2′,3′-phosphoesterase HD domain and a C-terminal 5′-OH polynucleotide kinase P-loop domain. HD-Pnk phosphorylates 5′-OH polynucleotides (9-mers or longer) in the presence of magnesium and any nucleoside triphosphate donor. HD-Pnk dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt. HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a putative ATP-dependent polynucleotide ligase. IMPORTANCE The present study provides insights regarding the diversity of nucleic acid repair strategies via the characterization of Runella slithyformis HD-Pnk as the exemplar of a novel clade of dual 5′- and 3′-end-healing enzymes that phosphorylate 5′-OH termini and dephosphorylate 2′,3′-cyclic-PO4, 3′-PO4, and 2′-PO4 ends. The distinctive feature of HD-Pnk is its domain composition, i.e., a fusion of an N-terminal HD phosphohydrolase module and a C-terminal P-loop polynucleotide kinase module. Homologs of Runella HD-Pnk with the same domain composition, same domain order, and similar polypeptide sizes are distributed widely among genera from 11 bacterial phyla. PMID:27895092

  9. Computation Of Facilitated Transport of O2 In Hemoglobin

    NASA Technical Reports Server (NTRS)

    Davis, Sanford

    1991-01-01

    Report describes computations of unsteady facilitated transport of oxygen through liquid membrane of hemoglobin. Used here, "facilitated transport" means diffusion of permeant through membrane in which that diffusion enhanced by reversible chemical reaction between permeant and membrane. In this case, reversible reactions between hemoglobin and oxygen.

  10. 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.

  11. 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

  12. Direct observation of charged domain walls in hybrid improper ferroelectric (Ca,Sr)3Ti2O7

    NASA Astrophysics Data System (ADS)

    Kurushima, Kousuke; Yoshimoto, Wataru; Ishii, Yui; Cheong, Sang-Wook; Mori, Shigeo

    2017-10-01

    We investigated ferroelectric (FE) domain wall structures including “charged domain walls” of hybrid improper FE (Ca,Sr)3Ti2O7 at the subatomic resolution by dark-field transmission electron microscopy (TEM) and high-resolution state-of-the-art aberration-corrected high-angle annular-dark-field (HAADF) scanning transmission electron microscopy (STEM). Dark-field TEM and high-resolution HAADF-STEM images obtained in the FE phase of single crystals of Ca2.46Sr0.54Ti2O7 revealed the formation of abundant charged domain walls with the head-to-head and tail-to-tail configurations in the FE domain structure, in addition to the FE 180° domain structure. The charged domain walls with the head-to-head and tail-to-tail FE polarizations exist stably and can be characterized as the unique double arc-type displacement of Ca/Sr ions in a unit cell without charge accumulation.

  13. Predicting detection performance with model observers: Fourier domain or spatial domain?

    PubMed

    Chen, Baiyu; Yu, Lifeng; Leng, Shuai; Kofler, James; Favazza, Christopher; Vrieze, Thomas; McCollough, Cynthia

    2016-02-27

    The use of Fourier domain model observer is challenged by iterative reconstruction (IR), because IR algorithms are nonlinear and IR images have noise texture different from that of FBP. A modified Fourier domain model observer, which incorporates nonlinear noise and resolution properties, has been proposed for IR and needs to be validated with human detection performance. On the other hand, the spatial domain model observer is theoretically applicable to IR, but more computationally intensive than the Fourier domain method. The purpose of this study is to compare the modified Fourier domain model observer to the spatial domain model observer with both FBP and IR images, using human detection performance as the gold standard. A phantom with inserts of various low contrast levels and sizes was repeatedly scanned 100 times on a third-generation, dual-source CT scanner at 5 dose levels and reconstructed using FBP and IR algorithms. The human detection performance of the inserts was measured via a 2-alternative-forced-choice (2AFC) test. In addition, two model observer performances were calculated, including a Fourier domain non-prewhitening model observer and a spatial domain channelized Hotelling observer. The performance of these two mode observers was compared in terms of how well they correlated with human observer performance. Our results demonstrated that the spatial domain model observer correlated well with human observers across various dose levels, object contrast levels, and object sizes. The Fourier domain observer correlated well with human observers using FBP images, but overestimated the detection performance using IR images.

  14. Predicting detection performance with model observers: Fourier domain or spatial domain?

    PubMed Central

    Chen, Baiyu; Yu, Lifeng; Leng, Shuai; Kofler, James; Favazza, Christopher; Vrieze, Thomas; McCollough, Cynthia

    2016-01-01

    The use of Fourier domain model observer is challenged by iterative reconstruction (IR), because IR algorithms are nonlinear and IR images have noise texture different from that of FBP. A modified Fourier domain model observer, which incorporates nonlinear noise and resolution properties, has been proposed for IR and needs to be validated with human detection performance. On the other hand, the spatial domain model observer is theoretically applicable to IR, but more computationally intensive than the Fourier domain method. The purpose of this study is to compare the modified Fourier domain model observer to the spatial domain model observer with both FBP and IR images, using human detection performance as the gold standard. A phantom with inserts of various low contrast levels and sizes was repeatedly scanned 100 times on a third-generation, dual-source CT scanner at 5 dose levels and reconstructed using FBP and IR algorithms. The human detection performance of the inserts was measured via a 2-alternative-forced-choice (2AFC) test. In addition, two model observer performances were calculated, including a Fourier domain non-prewhitening model observer and a spatial domain channelized Hotelling observer. The performance of these two mode observers was compared in terms of how well they correlated with human observer performance. Our results demonstrated that the spatial domain model observer correlated well with human observers across various dose levels, object contrast levels, and object sizes. The Fourier domain observer correlated well with human observers using FBP images, but overestimated the detection performance using IR images. PMID:27239086

  15. Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice

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

    Kawai, Ikuma; Hisaki, Tomoka; Sugiura, Koji

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase. Black-Right-Pointing-Pointer DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. Black-Right-Pointing-Pointer We produced in vitro and in vivo model to better understand the role of DDR2. Black-Right-Pointing-Pointer DDR2 might play an inhibitory role in the proliferation of chondrocyte. -- Abstract: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens. DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. The decrement of endogenous DDR2 represses osteoblastic marker gene expression and osteogenic differentiation in murine preosteoblastic cells, but themore » functions of DDR2 in chondrogenic cellular proliferation remain unclear. To better understand the role of DDR2 signaling in cellular proliferation in endochondral ossification, we inhibited Ddr2 expression via the inhibitory effect of miRNA on Ddr2 mRNA (miDdr2) and analyzed the cellular proliferation and differentiation in the prechondrocyte ATDC5 cell lines. To investigate DDR2's molecular role in endochondral cellular proliferation in vivo, we also produced transgenic mice in which the expression of truncated, kinase dead (KD) DDR2 protein is induced, and evaluated the DDR2 function in cellular proliferation in chondrocytes. Although the miDdr2-transfected ATDC5 cell lines retained normal differentiation ability, DDR2 reduction finally promoted cellular proliferation in proportion to the decreasing ratio of Ddr2 expression, and it also promoted earlier differentiation to cartilage cells by insulin induction. The layer of hypertrophic chondrocytes in KD Ddr2 transgenic mice was not significantly thicker than that of normal littermates, but the layer of proliferative chondrocytes in KD-Ddr2 transgenic mice was significantly thicker than that of normal

  16. Mutation-Linked Defective Inter-Domain Interactions within Ryanodine Receptor Cause Aberrant Ca2+ Release Leading to Catecholaminergic Polymorphic Ventricular Tachycardia

    PubMed Central

    Suetomi, Takeshi; Yano, Masafumi; Uchinoumi, Hitoshi; Fukuda, Masakazu; Hino, Akihiro; Ono, Makoto; Xu, Xiaojuan; Tateishi, Hiroki; Okuda, Shinichi; Doi, Masahiro; Kobayashi, Shigeki; Ikeda, Yasuhiho; Yamamoto, Takeshi; Ikemoto, Noriaki; Matsuzaki, Masunori

    2011-01-01

    Background The molecular mechanism by which catecholaminergic polymorphic ventricular tachycardia (CPVT) is induced by single amino acid mutations within the cardiac ryanodine receptor (RyR2) remains elusive. Here, we investigated mutation-induced conformational defects of RyR2 using a knock-in (KI) mouse model expressing the human CPVT-associated RyR2 mutant (S2246L; Serine to Leucine mutation at the residue 2246). Methods and Results All KI mice we examined produced VT after exercise on a treadmill. cAMP-dependent increase in the frequency of Ca2+ sparks was more pronounced in saponin-permeabilized KI cardiomyocytes than in WT cardiomyocytes. Site-directed fluorescent labeling and quartz microbalance assays of the specific binding of DP2246 (a peptide corresponding to the 2232–2266 region: the 2246 domain) showed that DP2246 binds with the K201-binding sequence of RyR2 (1741– 2270). Introduction of S2246L mutation into the DP2246 increased the affinity of peptide binding. Fluorescence quench assays of inter-domain interactions within RyR2 showed that tight interaction of the 2246 domain/K201-binding domain is coupled with domain unzipping of the N-terminal (1-600)/central (2000–2500) domain pair in an allosteric manner. Dantrolene corrected the mutation-caused domain unzipping of the domain switch, and stopped the exercise-induced ventricular tachycardia. Conclusions The CPVT-linked mutation of RyR2, S2246L, causes an abnormally tight local sub-domain/sub-domain interaction within the central domain involving the mutation site, which induces defective interaction between the N-terminal and central domains. This results in an erroneous activation of Ca2+ channel in a diastolic state reflecting on the increased Ca2+ spark frequency, which then leads to lethal arrhythmia. PMID:21768539

  17. Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding Domain I in mismatch recognition.

    PubMed Central

    Lee, Susan D.; Surtees, Jennifer A.; Alani, Eric

    2007-01-01

    In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. In this study we showed that the msh2Δ1 mutation, containing a complete deletion of the conserved mismatch recognition Domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Δ1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of Domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that Domain I in MSH2 contributed a non-specific DNA binding activity while Domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA-binding. These observations reveal distinct requirements for the MSH2 DNA binding Domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding. PMID:17157869

  18. Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition.

    PubMed

    Lee, Susan D; Surtees, Jennifer A; Alani, Eric

    2007-02-09

    In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.

  19. The HIV-1 late domain-2 S40A polymorphism in antiretroviral (or ART)-exposed individuals influences protease inhibitor susceptibility.

    PubMed

    Watanabe, Susan M; Simon, Viviana; Durham, Natasha D; Kemp, Brittney R; Machihara, Satoshi; Kemal, Kimdar Sherefa; Shi, Binshan; Foley, Brian; Li, Hongru; Chen, Benjamin K; Weiser, Barbara; Burger, Harold; Anastos, Kathryn; Chen, Chaoping; Carter, Carol A

    2016-09-06

    The p6 region of the HIV-1 structural precursor polyprotein, Gag, contains two motifs, P7TAP11 and L35YPLXSL41, designated as late (L) domain-1 and -2, respectively. These motifs bind the ESCRT-I factor Tsg101 and the ESCRT adaptor Alix, respectively, and are critical for efficient budding of virus particles from the plasma membrane. L domain-2 is thought to be functionally redundant to PTAP. To identify possible other functions of L domain-2, we examined this motif in dominant viruses that emerged in a group of 14 women who had detectable levels of HIV-1 in both plasma and genital tract despite a history of current or previous antiretroviral therapy. Remarkably, variants possessing mutations or rare polymorphisms in the highly conserved L domain-2 were identified in seven of these women. A mutation in a conserved residue (S40A) that does not reduce Gag interaction with Alix and therefore did not reduce budding efficiency was further investigated. This mutation causes a simultaneous change in the Pol reading frame but exhibits little deficiency in Gag processing and virion maturation. Whether introduced into the HIV-1 NL4-3 strain genome or a model protease (PR) precursor, S40A reduced production of mature PR. This same mutation also led to high level detection of two extended forms of PR that were fairly stable compared to the WT in the presence of IDV at various concentrations; one of the extended forms was effective in trans processing even at micromolar IDV. Our results indicate that L domain-2, considered redundant in vitro, can undergo mutations in vivo that significantly alter PR function. These may contribute fitness benefits in both the absence and presence of PR inhibitor.

  20. Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

    PubMed Central

    Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

    2008-01-01

    DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

  1. Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

    PubMed

    Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

    2008-02-01

    Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

  2. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

    PubMed Central

    Kandasamy, Suresh K.

    2016-01-01

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing. PMID:27872309

  3. Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

    PubMed Central

    Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

    2015-01-01

    Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

  4. Target recognition of beta2-glycoprotein I (beta2GPI)-dependent anticardiolipin antibodies: evidence for involvement of the fourth domain of beta2GPI in antibody binding.

    PubMed

    George, J; Gilburd, B; Hojnik, M; Levy, Y; Langevitz, P; Matsuura, E; Koike, T; Shoenfeld, Y

    1998-04-15

    Beta2-glycoprotein I (beta2GPI) is an absolute requirement for the binding of autoimmune anticardiolipin Abs (aCL) to cardiolipin (CL). We evaluated the target recognition of human beta2GPI by IgG derived from two patients with primary and two with secondary antiphospholipid syndrome. The total IgG serum fractions and beta2GPI affinity-purified IgGs were assessed by using various domain-deleted mutants (DM) of human beta2GPI (DMs: I-III, I-IV, II-V, III-V, IV-V, and V) and mouse mAbs against individual beta2GPI domains. The four IgGs bound slightly to CL in the absence of beta2GPI and showed increased binding in the beta2GPI presence. Following affinity purification of the IgGs on a beta2GPI column, reactivity toward CL was absent. DMs containing domain V inhibited the binding of biotinylated beta2GPI to CL. The addition to CL-coated plates of DM V, but not the other DMs, reduced the binding of all four IgGs. The anti-beta2GPI IgGs bound only to complete beta2GPI and DM I-IV coated on the plates. The binding to plate-adsorbed beta2GPI could be inhibited by complete beta2GPI and DM I-IV, the latter being a more efficient inhibitor. Further, the human anti-beta2GPI IgGs could compete with the binding to beta2GPI of Cof-21 mouse mAb (directed at domain IV), but not with the two other mouse mAbs. The results suggest that some "autoimmune:" beta2GPI-dependent anticardiolipin Abs recognize a beta2GPI target that is distinct from the CL-binding site in domain V. The target site for some antiphospholipid syndrome IgGs appear to reside in domain IV of beta2GPI.

  5. A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

    PubMed

    Håvik, B; Ragnhildstveit, E; Lorens, J B; Saelemyr, K; Fauske, O; Knudsen, L K; Fjose, A

    1999-12-20

    The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences. Copyright 1999 Academic Press.

  6. Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

    PubMed Central

    Yang, Xiaodan; Hu, Zhulong; Fan, Shanshan; Zhang, Qiang; Zhong, Yi; Guo, Dong; Qin, Yali

    2018-01-01

    Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation. PMID:29415027

  7. Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

    PubMed

    Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

    2016-10-01

    Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy. © 2016 American Heart Association, Inc.

  8. The TIR domain of TIR-NB-LRR resistance proteins is a signaling domain involved in cell death induction.

    PubMed

    Swiderski, Michal R; Birker, Doris; Jones, Jonathan D G

    2009-02-01

    In plants, the TIR (toll interleukin 1 receptor) domain is found almost exclusively in nucleotide-binding (NB) leucine-rich repeat resistance proteins and their truncated homologs, and has been proposed to play a signaling role during resistance responses mediated by TIR containing R proteins. Transient expression in Nicotiana benthamiana leaves of "TIR + 80", the RPS4 truncation without the NB-ARC domain, leads to EDS1-, SGT1-, and HSP90-dependent cell death. Transgenic Arabidopsis plants expressing the RPS4 TIR+80 from either dexamethasone or estradiol-inducible promoters display inducer-dependent cell death. Cell death is also elicited by transient expression of similarly truncated constructs from two other R proteins, RPP1A and At4g19530, but is not elicited by similar constructs representing RPP2A and RPP2B proteins. Site-directed mutagenesis of the RPS4 TIR domain identified many loss-of-function mutations but also revealed several gain-of function substitutions. Lack of cell death induction by the E160A substitution suggests that amino acids outside of the TIR domain contribute to cell death signaling in addition to the TIR domain itself. This is consistent with previous observations that the TIR domain itself is insufficient to induce cell death upon transient expression.

  9. Parental Provision of Structure: Implementation and Correlates in Three Domains

    ERIC Educational Resources Information Center

    Grolnick, Wendy S.; Raftery-Helmer, Jacquelyn N.; Marbell, Kristine N; Flamm, Elizabeth S.; Cardemil, Esteban V.

    2014-01-01

    This study examined parents' provision of "structure," defined as the organization of the environment to facilitate competence, and the degree to which it supports versus controls children's autonomy, in the domains of homework and studying, unsupervised time, and responsibilities in a diverse sample of sixth-grade children and their…

  10. Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

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

    Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

    2010-11-05

    Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

  11. Study of the individual cytochrome b5 and cytochrome b5 reductase domains of Ncb5or reveals a unique heme pocket and a possible role of the CS domain.

    PubMed

    Deng, Bin; Parthasarathy, Sudharsan; Wang, WenFang; Gibney, Brian R; Battaile, Kevin P; Lovell, Scott; Benson, David R; Zhu, Hao

    2010-09-24

    NADH cytochrome b(5) oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b(5) (b(5)), CHORD-SGT1 (CS), and cytochrome b(5) reductase (b(5)R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b(5) and b(5)R domains (Ncb5or-b(5) and Ncb5or-b(5)R, respectively) and compared them with human microsomal b(5) (Cyb5A) and b(5)R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b(5) reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His(89) and His(112), consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b(5) family shown to have such a heme environment. Like other b(5) family members, Ncb5or-b(5) has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b(5) differs from Cyb5A with respect to location of the second heme ligand (His(112)) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b(5)R to Ncb5or-b(5) is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b(5) and b(5)R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b(5)R domains suggest that the CS domain facilitates docking of the b(5) and b(5)R domains. Trp(114) is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b(5)R domain to the b(5) domain.

  12. Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

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

    Sampathkumar, Parthasarathy; Kim, Seung Joong; Manglicmot, Danalyn

    2012-10-23

    The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of {approx}456 polypeptide chains contributed by {approx}30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal 'FG' repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain ofmore » Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 {angstrom} resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed.« less

  13. Two-dimensional Kinetics Regulation of αLβ2-ICAM-1 Interaction by Conformational Changes of the αL-Inserted Domain*

    PubMed Central

    Zhang, Fang; Marcus, Warren D.; Goyal, Nimita H.; Selvaraj, Periasamy; Springer, Timothy A.; Zhu, Cheng

    2006-01-01

    The leukocyte integrin αLβ2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of αLβ2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with αLβ2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by ~8000- and ~30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type αLβ2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the αLβ2, is required for affinity and on-rate up-regulation. PMID:16234238

  14. Comparative Effectiveness of a Technology-Facilitated Depression Care Management Model in Safety-Net Primary Care Patients With Type 2 Diabetes: 6-Month Outcomes of a Large Clinical Trial

    PubMed Central

    Ell, Kathleen; Jin, Haomiao; Vidyanti, Irene; Chou, Chih-Ping; Lee, Pey-Jiuan; Gross-Schulman, Sandra; Sklaroff, Laura Myerchin; Belson, David; Nezu, Arthur M; Hay, Joel; Wang, Chien-Ju; Scheib, Geoffrey; Di Capua, Paul; Hawkins, Caitlin; Liu, Pai; Ramirez, Magaly; Wu, Brian W; Richman, Mark; Myers, Caitlin; Agustines, Davin; Dasher, Robert; Kopelowicz, Alex; Allevato, Joseph; Roybal, Mike; Ipp, Eli; Haider, Uzma; Graham, Sharon; Mahabadi, Vahid; Guterman, Jeffrey

    2018-01-01

    Background Comorbid depression is a significant challenge for safety-net primary care systems. Team-based collaborative depression care is effective, but complex system factors in safety-net organizations impede adoption and result in persistent disparities in outcomes. Diabetes-Depression Care-management Adoption Trial (DCAT) evaluated whether depression care could be significantly improved by harnessing information and communication technologies to automate routine screening and monitoring of patient symptoms and treatment adherence and allow timely communication with providers. Objective The aim of this study was to compare 6-month outcomes of a technology-facilitated care model with a usual care model and a supported care model that involved team-based collaborative depression care for safety-net primary care adult patients with type 2 diabetes. Methods DCAT is a translational study in collaboration with Los Angeles County Department of Health Services, the second largest safety-net care system in the United States. A comparative effectiveness study with quasi-experimental design was conducted in three groups of adult patients with type 2 diabetes to compare three delivery models: usual care, supported care, and technology-facilitated care. Six-month outcomes included depression and diabetes care measures and patient-reported outcomes. Comparative treatment effects were estimated by linear or logistic regression models that used generalized propensity scores to adjust for sampling bias inherent in the nonrandomized design. Results DCAT enrolled 1406 patients (484 in usual care, 480 in supported care, and 442 in technology-facilitated care), most of whom were Hispanic or Latino and female. Compared with usual care, both the supported care and technology-facilitated care groups were associated with significant reduction in depressive symptoms measured by scores on the 9-item Patient Health Questionnaire (least squares estimate, LSE: usual care=6.35, supported

  15. 2D Covalent Metals: A New Materials Domain of Electrochemical CO2 Conversion with Broken Scaling Relationship.

    PubMed

    Shin, Hyeyoung; Ha, Yoonhoo; Kim, Hyungjun

    2016-10-04

    Toward a sustainable carbon cycle, electrochemical conversion of CO 2 into valuable fuels has drawn much attention. However, sluggish kinetics and a substantial overpotential, originating from the strong correlation between the adsorption energies of intermediates and products, are key obstacles of electrochemical CO 2 conversion. Here we show that 2D covalent metals with a zero band gap can overcome the intrinsic limitation of conventional metals and metal alloys and thereby substantially decrease the overpotential for CO 2 reduction because of their covalent characteristics. From first-principles-based high-throughput screening results on 61 2D covalent metals, we find that the strong correlation between the adsorption energies of COOH and CO can be entirely broken. This leads to the computational design of CO 2 -to-CO and CO 2 -to-CH 4 conversion catalysts in addition to hydrogen-evolution-reaction catalysts. Toward efficient electrochemical catalysts for CO 2 reduction, this work suggests a new materials domain having two contradictory properties in a single material: covalent nature and electrical conductance.

  16. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites.

    PubMed

    Ammerman, Michelle L; Presnyak, Vladimir; Fisk, John C; Foda, Bardees M; Read, Laurie K

    2010-11-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.

  17. Inhibitor-induced structural change in the HCV IRES domain IIa RNA

    PubMed Central

    Paulsen, Ryan B.; Seth, Punit P.; Swayze, Eric E.; Griffey, Richard H.; Skalicky, Jack J.; Cheatham, Thomas E.; Davis, Darrell R.

    2010-01-01

    Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5′ untranslated region (5′ UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90° helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class. PMID:20360559

  18. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

    PubMed Central

    Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

    2014-01-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

  19. Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

    PubMed

    Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

    2017-03-01

    Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. TU-CD-304-11: Veritas 2.0: A Cloud-Based Tool to Facilitate Research and Innovation

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

    Mishra, P; Patankar, A; Etmektzoglou, A

    Purpose: We introduce Veritas 2.0, a cloud-based, non-clinical research portal, to facilitate translation of radiotherapy research ideas to new delivery techniques. The ecosystem of research tools includes web apps for a research beam builder for TrueBeam Developer Mode, an image reader for compressed and uncompressed XIM files, and a trajectory log file based QA/beam delivery analyzer. Methods: The research beam builder can generate TrueBeam readable XML file either from scratch or from pre-existing DICOM-RT plans. DICOM-RT plan is first converted to XML format and then researcher can interactively modify or add control points to them. Delivered beam can be verifiedmore » via reading generated images and analyzing trajectory log files. Image reader can read both uncompressed and HND-compressed XIM images. The trajectory log analyzer lets researchers plot expected vs. actual values and deviations among 30 mechanical axes. The analyzer gives an animated view of MLC patterns for the beam delivery. Veritas 2.0 is freely available and its advantages versus standalone software are i) No software installation or maintenance needed, ii) easy accessibility across all devices iii) seamless upgrades and iv) OS independence. Veritas is written using open-source tools like twitter bootstrap, jQuery, flask, and Python-based modules. Results: In the first experiment, an anonymized 7-beam DICOM-RT IMRT plan was converted to XML beam containing 1400 control points. kV and MV imaging points were inserted into this XML beam. In another experiment, a binary log file was analyzed to compare actual vs expected values and deviations among axes. Conclusions: Veritas 2.0 is a public cloud-based web app that hosts a pool of research tools for facilitating research from conceptualization to verification. It is aimed at providing a platform for facilitating research and collaboration. I am full time employee at Varian Medical systems, Palo Alto.« less

  1. The glycosylated IgII extracellular domain of EMMPRIN is implicated in the induction of MMP-2.

    PubMed

    Papadimitropoulou, Adriana; Mamalaki, Avgi

    2013-07-01

    EMMPRIN is a widely expressed transmembrane glycoprotein that plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. It stimulates the production of matrix metalloproteinase (MMPs) by tumor-associated fibroblasts. In the present study, our aim was to (a) to investigate if the IgII loop domain of the extracellular domain (ECD) of EMMPRIN contributes to the MMP production by fibroblasts and (b) to evaluate the significance of glycosylation in this process. For this purpose, we expressed the ECD, IgI, or IgII domains of EMMPRIN, in their glycosylated and non-glycosylated forms, in the heterologous expression systems of P. pastoris and E. coli, respectively. Dermal fibroblasts were treated with purified recombinant domains and proteins from cell extracts and supernatants were analyzed by Western blot and zymography assays. Fibroblasts treated with ECD-, IgI-, and IgII-glycosylated domains of EMMPRIN significantly stimulated the gelatinolytic activity of MMP-2, compared to untreated fibroblasts, whereas no significant effect was observed after treatment with the non-glycosylated ECD, IgI, and IgII domains. Western blot analysis from cell extracts and supernatants revealed that only the glycosylated forms were able to stimulate MMP-2 production and secretion, respectively. Quantitative PCR revealed that this effect was not attributed to transcriptional alterations. This study showed that N-glycosylation was a prerequisite for efficient MMP-2 production, with the IgII loop domain contributing significantly to this process. Perturbation of the function of IgII-EMMPRIN loop could have potential therapeutic value in the inhibition of MMP-2-dependent cancer cell invasion and metastasis.

  2. Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression

    PubMed Central

    Libbrecht, Maxwell W.; Ay, Ferhat; Hoffman, Michael M.; Gilbert, David M.; Bilmes, Jeffrey A.; Noble, William Stafford

    2015-01-01

    The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term “specific expression domains.” We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. PMID:25677182

  3. Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression.

    PubMed

    Libbrecht, Maxwell W; Ay, Ferhat; Hoffman, Michael M; Gilbert, David M; Bilmes, Jeffrey A; Noble, William Stafford

    2015-04-01

    The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term "specific expression domains." We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. © 2015 Libbrecht et al.; Published by Cold Spring Harbor Laboratory Press.

  4. A new principle technic for the transformation from frequency domain to time domain

    NASA Astrophysics Data System (ADS)

    Gao, Ben-Qing

    2017-03-01

    A principle technic for the transformation from frequency domain to time domain is presented. Firstly, a special type of frequency domain transcendental equation is obtained for an expected frequency domain parameter which is a rational or irrational fraction expression. Secondly, the inverse Laplace transformation is performed. When the two time-domain factors corresponding to the two frequency domain factors at two sides of frequency domain transcendental equation are known quantities, a time domain transcendental equation is reached. At last, the expected time domain parameter corresponding to the expected frequency domain parameter can be solved by the inverse convolution process. Proceeding from rational or irrational fraction expression, all solving process is provided. In the meantime, the property of time domain sequence is analyzed and the strategy for choosing the parameter values is described. Numerical examples are presented to verify the proposed theory and technic. Except for rational or irrational fraction expressions, examples of complex relative permittivity of water and plasma are used as verification method. The principle method proposed in the paper can easily solve problems which are difficult to be solved by Laplace transformation.

  5. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  6. Imaging and engineering the nanoscale-domain structure of a Sr0.61Ba0.39Nb2O6 crystal using a scanning force microscope

    NASA Astrophysics Data System (ADS)

    Terabe, K.; Takekawa, S.; Nakamura, M.; Kitamura, K.; Higuchi, S.; Gotoh, Y.; Gruverman, A.

    2002-09-01

    We have investigated the ferroelectric domain structure formed in a Sr0.61Ba0.39Nb2O6 single crystal by cooling the crystal through the Curie point. Imaging the etched surface structure using a scanning force microscope (SFM) in both the topographic mode and the piezoresponse mode revealed that a multidomain structure of nanoscale islandlike domains was formed. The islandlike domains could be inverted by applying an appropriate voltage using a conductive SFM tip. Furthermore, a nanoscale periodically inverted-domain structure was artificially fabricated using the crystal which underwent poling treatment.

  7. Interactions of the Auxilin-1 PTEN-like Domain with Model Membranes Result in Nanoclustering of Phosphatidyl Inositol Phosphates

    PubMed Central

    Kalli, Antreas C.; Morgan, Gareth; Sansom, Mark S.P.

    2013-01-01

    Auxilin-1 is a neuron-specific membrane-binding protein involved in a late stage of clathrin-mediated endocytosis. It recruits Hsc70, thus initiating uncoating of the clathrin-coated vesicles. Interactions of auxilin-1 with the vesicle membrane are crucial for this function and are mediated via an N-terminal PTEN-like domain. We have used multiscale molecular dynamics simulations to probe the interactions of the auxilin-1 PTEN-like domain with lipid bilayers containing differing phospholipid composition, including bilayers containing phosphatidyl inositol phosphates. Our results suggest a novel, to our knowledge, model for the auxilin/membrane encounter and subsequent interactions. Negatively charged lipids (especially PIP2) enhance binding of auxilin to lipid bilayers and facilitate its correct orientation relative to the membrane. Mutations in three basic residues (R301E/R307E/K311E) of the C2 subdomain of the PTEN-like domain perturbed its interaction with the bilayer, changing its orientation. The interaction of membrane-bound auxilin-1 PTEN-like domain with negatively charged lipid headgroups results in nanoclustering of PIP2 molecules in the adjacent bilayer leaflet. PMID:23823232

  8. CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling

    PubMed Central

    Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C.; Reth, Michael; Nitschke, Lars

    2013-01-01

    A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity. PMID:23836650

  9. CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling.

    PubMed

    Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C; Reth, Michael; Nitschke, Lars

    2013-07-23

    A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

  10. Origin of band bending at domain boundaries of MoS2: First-principles study

    NASA Astrophysics Data System (ADS)

    Kaneko, Tomoaki; Saito, Riichiro

    2018-04-01

    Using first-principles calculations based on density functional theory, the energetics and electronic structure of domain boundaries of MoS2, in which the same polar edges face each other, are investigated. We find that the interface model with homoelemental bonds is not energetically preferred in this system. The domain boundaries have defect levels that have wide distributions inside the band gap of MoS2. The upshift (or downshift) of the MoS2 energy band occurs around the domain boundaries when the occupation number of electrons in the defect levels increases (or decreases). The charge transfer of electrons from the graphite substrate plays an important role in band bending, which is observed in the recent experiments by scanning tunneling microscopy/spectroscopy.

  11. Effect of the SH3-SH2 domain linker sequence on the structure of Hck kinase.

    PubMed

    Meiselbach, Heike; Sticht, Heinrich

    2011-08-01

    The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family tyrosine kinases, which are found in many signaling pathways, differ in their physiological function despite their high overall structural similarity. In this context, the differences in the SH3-SH2 domain linkers might play a role for differential regulation, but the structural consequences of linker sequence remain poorly understood. We have therefore performed comparative molecular dynamics simulations of wildtype Hck and of a mutant Hck in which the SH3-SH2 domain linker is replaced by the corresponding sequence from the homologous kinase Lck. These simulations reveal that linker replacement not only affects the orientation of the SH3 domain itself, but also leads to an alternative conformation of the activation segment in the Hck kinase domain. The sequence of the SH3-SH2 domain linker thus exerts a remote effect on the active site geometry and might therefore play a role in modulating the structure of the inactive kinase or in fine-tuning the activation process itself.

  12. The Bro1-Domain Protein, EGO-2, Promotes Notch Signaling in Caenorhabditis elegans

    PubMed Central

    Liu, Ying; Maine, Eleanor M.

    2007-01-01

    In Caenorhabditis elegans, as in other animals, Notch-type signaling mediates numerous inductive events during development. The mechanism of Notch-type signaling involves proteolytic cleavage of the receptor and subsequent transport of the receptor intracellular domain to the nucleus, where it acts as a transcriptional regulator. Notch-type signaling activity is modulated by post-translational modifications and endocytosis of ligand and receptor. We previously identified the ego-2 (enhancer of glp-1) gene as a positive regulator of germline proliferation that interacts genetically with the GLP-1/Notch signaling pathway in the germline. Here, we show that ego-2 positively regulates signaling in various tissues via both GLP-1 and the second C. elegans Notch-type receptor, LIN-12. ego-2 activity also promotes aspects of development not known to require GLP-1 or LIN-12. The EGO-2 protein contains a Bro1 domain, which is known in other systems to localize to certain endosomal compartments. EGO-2 activity in the soma promotes GLP-1 signaling in the germline, consistent with a role for EGO-2 in production of active ligand. Another C. elegans Bro1-domain protein, ALX-1, is known to interact physically with LIN-12/Notch. We document a complex phenotypic interaction between ego-2 and alx-1, consistent with their relationship being antagonistic with respect to some developmental processes and agonistic with respect to others. PMID:17603118

  13. A 2D Daubechies finite wavelet domain method for transient wave response analysis in shear deformable laminated composite plates

    NASA Astrophysics Data System (ADS)

    Nastos, C. V.; Theodosiou, T. C.; Rekatsinas, C. S.; Saravanos, D. A.

    2018-03-01

    An efficient numerical method is developed for the simulation of dynamic response and the prediction of the wave propagation in composite plate structures. The method is termed finite wavelet domain method and takes advantage of the outstanding properties of compactly supported 2D Daubechies wavelet scaling functions for the spatial interpolation of displacements in a finite domain of a plate structure. The development of the 2D wavelet element, based on the first order shear deformation laminated plate theory is described and equivalent stiffness, mass matrices and force vectors are calculated and synthesized in the wavelet domain. The transient response is predicted using the explicit central difference time integration scheme. Numerical results for the simulation of wave propagation in isotropic, quasi-isotropic and cross-ply laminated plates are presented and demonstrate the high spatial convergence and problem size reduction obtained by the present method.

  14. The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets and Nuclei

    PubMed Central

    Xiang, Siheng; Kato, Masato; Wu, Leeju; Lin, Yi; Ding, Ming; Zhang, Yajie; Yu, Yonghao; McKnight, Steven L.

    2016-01-01

    SUMMARY Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein. PMID:26544936

  15. Charge ordering, ferroelectric, and magnetic domains in LuFe{sub 2}O{sub 4} observed by scanning probe microscopy

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

    Yang, I. K.; Jeong, Y. H., E-mail: yhj@postech.ac.kr; Kim, Jeehoon

    2015-04-13

    LuFe{sub 2}O{sub 4} is a multiferroic system which exhibits charge order, ferroelectricity, and ferrimagnetism simultaneously below ∼230 K. The ferroelectric/charge order domains of LuFe{sub 2}O{sub 4} are imaged with both piezoresponse force microscopy (PFM) and electrostatic force microscopy (EFM), while the magnetic domains are characterized by magnetic force microscopy (MFM). Comparison of PFM and EFM results suggests that the proposed ferroelectricity in LuFe{sub 2}O{sub 4} is not of usual displacive type but of electronic origin. Simultaneous characterization of ferroelectric/charge order and magnetic domains by EFM and MFM, respectively, on the same surface of LuFe{sub 2}O{sub 4} reveals that both domains havemore » irregular patterns of similar shape, but the length scales are quite different. The domain size is approximately 100 nm for the ferroelectric domains, while the magnetic domain size is much larger and gets as large as 1 μm. We also demonstrate that the origin of the formation of irregular domains in LuFe{sub 2}O{sub 4} is not extrinsic but intrinsic.« less

  16. A Logic for Inclusion of Administrative Domains and Administrators in Multi-domain Authorization

    NASA Astrophysics Data System (ADS)

    Iranmanesh, Zeinab; Amini, Morteza; Jalili, Rasool

    Authorization policies for an administrative domain or a composition of multiple domains in multi-domain environments are determined by either one administrator or multiple administrators' cooperation. Several logic-based models for multi-domain environments' authorization have been proposed; however, they have not considered administrators and administrative domains in policies' representation. In this paper, we propose the syntax, proof theory, and semantics of a logic for multi-domain authorization policies including administrators and administrative domains. Considering administrators in policies provides the possibility of presenting composite administration having applicability in many collaborative applications. Indeed, administrators and administrative domains stated in policies can be used in authorization. The presented logic is based on modal logic and utilizes two calculi named the calculus of administrative domains and the calculus of administrators. It is also proved that the logic is sound. A case study is presented signifying the logic application in practical projects.

  17. 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

  18. Atomic resolution structure of the E. coli YajR transporter YAM domain.

    PubMed

    Jiang, Daohua; Zhao, Yan; Fan, Junping; Liu, Xuehui; Wu, Yan; Feng, Wei; Zhang, Xuejun C

    2014-07-25

    YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Multiple nonlinear Bragg diffraction of femtosecond laser pulses in a {\\chi^{(2)}} photonic lattice with hexagonal domains

    NASA Astrophysics Data System (ADS)

    Vyunishev, A. M.; Arkhipkin, V. G.; Baturin, I. S.; Akhmatkhanov, A. R.; Shur, V. Ya; Chirkin, A. S.

    2018-04-01

    The frequency doubling of femtosecond laser pulses in a two-dimensional (2D) rectangular nonlinear photonic lattice with hexagonal domains is studied experimentally and theoretically. The broad fundamental spectrum enables frequency conversion under nonlinear Bragg diffraction for a series of transverse orders at a fixed longitudinal quasi-phase-matching order. The consistent nonstationary theory of the frequency doubling of femtosecond laser pulses is developed using the representation based on the reciprocal lattice of the structure. The calculated spatial distribution of the second-harmonic spectral intensity agrees well with the experimental data. The condition for multiple nonlinear Bragg diffraction in a 2D nonlinear photonic lattice is offered. The hexagonal shape of the domains contributes to multibeam second harmonic excitation. The maximum conversion efficiency for a series of transverse orders in the range 0.01%-0.03% is obtained.

  20. Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

    PubMed

    D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele

    2018-05-01

    Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.